Seminal MAIN FEATURES BLANQUILLO

Sorubim cuspicaudus ( Littmann, Burr & Nass, 2000) INDUCED

Hormonally Ovaprim ®

ARAUJO ALFREDO HÉCTOR-diaz

WISTON ENRIQUE CORDERO BOILER

UNIVERSITY OF CORDOBA
FACULTY OF VETERINARY MEDICINE AND ZOOTECHNICS
AQUACULTURE PROGRAM
HUNTING
2004
Seminal MAIN FEATURES BLANQUILLO

Sorubim cuspicaudus ( Littmann, Burr & Nass, 2000) INDUCED

Hormonally Ovaprim ®

ARAUJO ALFREDO HÉCTOR-diaz

WISTON ENRIQUE CORDERO BOILER

Degree work presented as partial requirement

for the degree of Vocational Aquaculture

director

Prof. CECILIA CLARA RUGELES PINTO, MSc

Co-Director

Prof. VICTOR ATENCIO-GARCIA, MSc

UNIVERSITY OF CORDOBA
FACULTY OF VETERINARY MEDICINE AND ZOOTECHNICS
AQUACULTURE PROGRAM
HUNTING
2004
The jury's work will not be responsible for the ideas put forward by the author (Article 46
Agreement of 29 May 006/1979 of the Superior Council).
 Acceptance note

Foreman

Jury

Jury

Gamekeeping, 2004
God for giving me the opportunity to

to realize my goals. My father

Hector and my dear mother Rebecca

(RIP) for their sacrifices. To me

Ludys wife for their support and patience and

my kids Emanuel and Eliana, by

share causing joy

achieving an achievement.

Hector Alfredo
My parents, my wife and my children

for the support, understanding and patience

throughout the study process and,

particularly, during the performance of this

investigation.

Enrique Wiston
 THANKS

A Professor Clara Cecilia rugeles, MSc by wise guidance and confidence

placed in us during the course of this investigation.

Professor Victor Atencio-García, MSc for their invaluable assistance and guidance

as co-director during the development of this research.

At the University of Córdoba and its Piscícola Research Center (CINPIC) by

its collaboration with infrastructure, equipment and materials that were indispensable

in this investigation.

To our peers and colleagues: Argemiro Cordero, Vicente Pertuz, Rafael Vergara

and Eduardo Kerguelen, among others, for their collaboration in the development of this

study, like the group of workers CINPIC.

 CONTENT

P.

LIST OF TABLES 10

LIST OF FIGURES eleven

LIST OF ATTACHMENTS 12

SUMMARY 13

1. INTRODUCTION 14

2. OBJECTIVES 16

2.1 GENERAL PURPOSE 16

2.2 SPECIFIC OBJECTIVES 16

3. LITERATURE REVIEW 17

3.1 ASPECTS OF BLANQUILLO Bioecological 17

3.2 REPRODUCTION INDUCED pimelodid catfishes twenty-one

Seminal 3.3 EVALUATION teleost 2. 3

3.4 teleost sperm morphology IN 26

28
4. DESIGN METHODOLOGY

4.1 PLACE AND AREA OF STUDY 28

4.2 SAMPLE 28

4.3 Macroscopic 29

4.4 Microscopic characteristics 30

4.5 STATISTICAL ANALYSIS 32

5. RESULTS 33

6. DISCUSSION 37

7. CONCLUSIONS 42

BIBLIOGRAPHY 43

ATTACHMENTS 51
 LIST OF TABLES

Table 1. Characteristics of some teleost Seminal 25

Table 2. Main characteristics seminal blanquillo Sorubim cuspicaudus

(Littmann, Burr & Nass, 2000) hormonally induced with
Ovaprim ® 33

10
 LIST OF FIGURES

P.

Figure 1. Exemplary male blanquillo Sorubim cuspicaudus 17

Figure 2. living and dead spermatozoa by differential staining

eosin-nigrosin 3. 4

Figure 3. Morphology of sperm blanquillo Sorubim cuspicaudus 35

Figure 4. Common abnormalities found in the semen of tilefish

Sorubim cuspicaudus 36

eleven
 LIST OF ATTACHMENTS

P.

Annex 1. Results of the seminal characteristics of tilefish S. cuspicaudus

(Littmann, Burr & Nass, 2000) hormonally induced with Ovaprim 51

12
 SUMMARY

tilefish Sorubim cuspicaudus ( Littmann, Burr & Nass, 2000) is a reofílica species with potential for inland
fish farming due to its high commercial value, excellent meat quality, absence of intramuscular bones,
absence of scales, good adaptation to captivity, resistance to manipulation. One of the problems in
artificial breeding is the low volume of semen produced by males, so it is necessary to induce hormonally.
So far they have not been described seminal characteristics of this species in these circumstances. So at
the Center for Fish Farming Research at the University of Córdoba (CINPIC) during the months of August
to November 2001 and May 2002, with the aim of evaluating the seminal characteristics of tilefish, 24
males three years were selected age, average weight of 0.56 kg, a batch of 110 copies kept in
confinement. All males were selected in the phase spermiation and were treated with 0.75 mL of Ovaprim ®
/ Kg bodyweight in a single dose. After 14 hours of induction hormone applied, with temperature between

27 and 28 ° C semen it was extracted by abdominal pressure in the anteroposterior direction, collected
directly in the glass tube and its volume measured. Semen was maintained at 5 ° C and before the time
characteristics were evaluated as activation time sperm, percentage of living sperm, made by the method
of differential staining eosinanigrosina and mass motility. Sperm concentration was estimated Neubauer
diluted 1: 700. The predominant color of the semen was creamy appearance (95%), the average values ​of
seminal volume and activation time were determined at 1.9 ± 1.0 118.6 ± 34.2 mL and sec respectively, 7 ±
1.2x10 7 sperm cells / mm 3 Y

86.7 ± 9.6%, respectively. The differential staining of eosin-nigrosin showed an average value of 83.9 ±
18% of live spermatozoa, the percentage of normal sperm was 93.9 ± 4.0% and 6.1% of abnormalities in
tail head and midpiece. In general, seminal blanquillo characteristics observed in this study are within the
range described for freshwater teleosts.

Keywords: Sorubim cuspicaudus, tilefish, seminal assessment, semen,

sperm, hormonal induction, catfish, Silurids.

13
 1. INTRODUCTION

Tilefish recently identified as Sorubim cuspicaudus by Littmann,

Burr & Nass (2000) is the most commercially important silurid basin of the Sinu

(Dahl, 1971). It adapts well to captivity, with usable features for

fish farming as the absence of scales and spines Intramuscular and excellent

flavor, although it has been offered as a safe alternative for cultivation,

mainly because still it presents difficulties induced, such as reproduction

low volume of semen and problems in the nursery.

Tilefish appears reported in the Red Book of freshwater species

Colombia, as a species Endangered (Mojica et al., 2002).

In particular, the situation of this fishery resource in the Sinu river has been aggravated by the

Overfishing and the progressive deterioration of the basin and more recently by the impact of the

Urra hydroelectric plant on reofilica species, highlighting the interruption

reproductive migration, loss of maturation and spawning areas, impaired quality

water and loss of reproductive potential (Atencio-García, 2000; Espinosa, 2000)

In Europe and North America there are several studies evaluating the seminal characteristics

in teleosts, particularly of Salmonidae (Billard, & Jalabert Breton, 1971 family;

Fogli da Silveira et al., 1985; Kavamoto et al.1985; 1986; Aas, Refstie & Gjerde, 1991;

Lahnsteiner et al., 1998). In South America, studies in this regard focuses on

Characiforms, highlighting the work of Fogli da Silveira, Kavamoto & Godihno

14
(1985); Kavamoto, Fogli da Silveira & Godihno (1986); Fogli da Silveira et al. (1990);

Cross-Casallas et al. (1999) and Brown-Carrasco et al. (2001). It also highlights

Silurids work of Fogli da Silveira, Kavamoto & Narahara (1981); Kavamoto &

Fogli da Silveira (1986); Pink (2001); Light et al (2001) and Ferreira et al. (2001).

Despite the artificial reproduction of tilefish have been obtained with inducers

as EPC (Yepes, Solano & Lamb, 1994) and Ovaprim ® ( Muñoz & Martínez, 2003) are not

reports found in the literature reviewed on the seminal characteristics of this

species. Therefore, the present study aims primarily to determine the

Seminal characteristics of tilefish through direct assessment techniques as

volume, viscosity, activation time, motility, sperm concentration,

percentage of live and normal.

fifteen
 2. OBJECTIVES

2.1 GENERAL PURPOSE

Determine the main characteristics of seminal white Sorubim

cuspicaudus ( Littmann, Burr & Nass, 2000) hormonally induced with Ovaprim ®.

2.2 SPECIFIC OBJECTIVES

• Determine volume, color and viscosity blanquillo semen.

• Desterminar sperm concentration, motility, activation time, morphology,

and percentage of live sperm tilefish.

16
 3. LITERATURE REVIEW

3.1 ASPECTS OF BLANQUILLO Bioecological Sorubim cuspicaudus

(Littmann, Burr & Nass, 2000)

Figure 1. Exemplary male S. cuspicaudus

Photo: Prof. Charles Olaya Nieto

Tilefish, Sorubim cuspicaudus, It belongs to the order Siluriformes, suborder

Siluroidei and Pimelodidae family. The order includes the so-called siluriforme fish

leather, whose main external characteristic is the absence of scale on the body and

Intramuscular thorns. Barbells have generally a jawbone pair and two pairs

mentonianos and frequently, the first radius of the dorsal and pectoral are

constituted a strong, sharp thorn (Tavares, 1997)

17
 Tilefish is distinguished from other species of the genus Sorubim by having the fin

caudal lobes with two pointy black, a deep groove and on the back of the

supraoccipital bone, broad head, elongated body and reaches sizes above 80

cm, with the largest species of the genus (Littmann, Burr & Nass, 2000). Valenzuela &

Burbano (2001), by cytogenetic characterization with copies of S. cuspicaudus

from the Rio Sinu established important differences between it and the

S. lima typical of the Amazon basin and Parana, ensuring that tilefish is a

different species S. lime. These differences confirm that tilefish was wrongly

identified by several authors, from Miles (1947), Dahl, Medem & Ramos (1963), Dahl

& Medem (1964) to Dahl (1971). It is considered as a new species, the only

the genus that inhabits the west of the Cordillera Oriental Andina, and also in the basin

Lake Maracaibo (Littmann, Burr & Nass, 2000).

It is distributed throughout the magdalenica basin formed by the rivers

Magdalena, Cauca and San Jorge (Miles, 1947; Dahl, Medem & Ramos, 1963; Dahl, 1971;

Mees, 1974; Littmann et al, 2000) in the Sinu River (Dahl & Medem, 1964; Dahl, 1971;

Littmann et al., 2000) and the Rio Atrato (Galvis et al., 1997).

Tilefish is the most important Sinu Basin and the second in silurid

Magdalénica importance in the basin, painted after catfish Pseudoplatistoma

fasciatum ( Dahl, 1971). It is known by several common names according to the region with

names "Whitey", "Bagre", "White Bagre" in the Sinu; "Poor white", "Bagre"
18
"Gallega", "Antiochian" or "Cucharo" in Cauca and Magdalena (Dahl, 1947; Miles, 1947;

Dahl & Medem, 1964; Dahl, Medem & Ramos, 1963).

Tilefish is a reofílica or migratory species and travels great distances in

shoals. Holds two annual recoridos one called subienda or migration

reproductive and another called bajanza or trophic migration (Otero et al., 1986; Garcia-Atencio

et al., 2000). It is a carnivorous fish with fish-eating trend like the big catfish

pimelodids and due to the low diversity of ingested preys (fish and crustaceans) is

It can be considered esepcie of estenofágica diet (Villadiego, Ortiz-Villafane &

Atencio-García, 2003).

The main events in the life cycle of the Sinu reofílicos fish such

as migration and spawning initial development are associated Precipitation regime and

population dynamics of these species depends on the annual migrations and

interactions bogs where feed, grow and recruit, and the river where

reproduce (Atencio-García, 2000). The same author reports that at the end of the period

rainy bogs down level, by affecting the physical and chemical quality

water, and is also a shortage of food is given, encouraging them to start their

spawning migration, seeking areas maturation with low stream velocity,

clearest water and optimal physicochemical quality. Fish in these areas are the

suitable for gonadal maturation and wait for the period conditions

rainy for final maturation and spawning (Atencio-García, 2000).

19
 According to Florez & Solano (2001) tilefish is a fish longevity and rate

means growth, with an average catch size between March / 1999 and February / 2000

39.3 cm standard length. Solano et al. (2003) reported 46.6 cm medium size

maturity and early maturity for this species in the basin of the Sinu.

March / 1997 to February / 2001, tilefish fishery reached 352.8

tons, worth estimated at 1477.3 million pesos constituting the species

which produced higher revenues in artisanal fisheries economy (Valderrama and Ruiz,

1998; & Vejarano Valderrama 2001).

3.2 REPRODUCTION INDUCED pimelodid catfishes

The first trials reported on reproduction induced in silurids

South America were made by Kossowski & De La Madrid (1985) in Venezuela;

who worked with specimens of the genus Pseudoplatystoma fasciatum, using

Pituitary extracts Pirinampus pirinampus, Prochilodus sp Y Pseudoplatystoma

fasciatum, administering five high doses at intervals of 23 hours with a range of

Dosage 0.3 - 0.4 mg / Kg body weight and a final 1.3 - 1.4 mg / Kg.

Subsequently Rodriguez & Nielsen (1990), used carp pituitary extract

(EPC) using two doses at an interval of 8 hours, a first dose of 0.5 mg / kg

live weight and the second 4.5 mg / kg in catfish painted P. fasciatum with a time of

hatch 16 hours and 15 minutes at 25 to 27 ° C.

twenty
 Sato et al. (1997) used in induced breeding EPC wallpaper

Pseudoplatystoma coruscans, for which they administered in a first dose females

0.8 ± 0.1 mg / kg BW and a second dose of 6.1 ± 0.2 mg / Kg. males

They were induced with 2.3 ± 0.2 mg / kg at the time of the second dose female.

Tilefish was played first by Yepes, Solano & Cordero (1994)

using EPC. Both males and females were induced with a first dose of

0.5 mg of EPC / kg body weight, and a second final dose of 6.0 mg of EPC / Kg

alive after 13 hours.

It is important to note that in the first breeding trials

induced catfish Callophysus macropterus, Phractocephalus hemiliopterus

(Rodriguez, 1994) and Sorubim cuspicaudus ( Yepes, Solano & Lamb, 1994) was not released,

in extrusion, sufficient seminal fluid; so that males were sacrificed for

extract their gonads, squeezing on oocytes to achieve fertilization.

Recently Muñoz & Martinez (2003) achieved the induced reproduction

tilefish with Ovaprim ® using doses between 0.25 and 0.75 mL / kg body weight;

the same results on reproductive performance with EPC. The

Ovulation tilefish with Ovaprim ® between 12.8 and 14.0 occurs hours at a temperature between

27 ° C - 28 ° C.

twenty-one
3.3 Seminal evaluation TELEOST

According to Harvey & Hoar (1980) preservation is a process espermazoide

simple, including the preservation of fresh semen on ice or in a refrigerator

temperature between 0 and 10 ° C, considering gas exchange, filling manifold

three-quarters of the bottle leaving it unsealed and avoiding shaking the period

Cooling can vary from a few hours to 5 days.

Christensen & Tiersh (1996) consider that the refrigerated and storage

Sperm is useful for genetic study and artificial fish breeding. He

Storage is important in species such as channel catfish Ictalurus punctatus,

because males do not lose donor sperm storing potential to

a short-term 4 ° C.

Most studies on the regulation of the activation of mobility

fish sperm have been made in the Trout gairgneri psalm, these cells arrive

to be movable as a result of changes in the pH of the plasma membrane and

ionic conductance (Cosson et al., 1999).

Sperm are still in the testis and in most species

the seminal plasma during natural reproduction, mobility is induced after

sperm release of the male genital tract in an aqueous medium. Motility

once activated the sperm is variable, it lasts much longer in fish

22
spawn in brackish water and salt than spawn in fresh water (no more than 2 to 3

minutes). time mobility and activation time is very short in fish being

20 to 25 seconds for trout and slightly higher for tents, between 80 to 90 seconds. And the

Detectable motility disappears after five minutes (Ginzburg, 1972; Harvey &

Hoar, 1980; Billard et al., 1995; Perchec et al., 1998).

Mobility is a useful parameter to determine cell viability

spermatic. In addition the temperature alters the mobility period at low temperatures

It produces a prolonged duration of sperm motility, however, speed

swimming is reduced (Cosson et al., 1999).

Semen collected fish extrusion is frequently contaminated with

urine. From a practical standpoint contamination of semen with urine can be

I avoided; taking the sample collected after the urine through a catheter (Perchec

et al., 1998).

Table 1 seminal characteristics of some species are recorded

teleost reported by different researchers.

2. 3
Table 1. Characteristics of some teleost Seminal

Activation
Alive sperm Count Abnormality
Species Vol (ml) Motility Time (Sec) Reference
(%) (%) (%)

Rainbow trout 15.07 x 10 6 Billard, Breton &

4-8
Psalm gairgneri sperm cells / mm 3 Jalabert (1971)
Rainbow trout 15.98 x 10 6 Fogli da Silveira
Psalm iredeus sperm cells / mm 3 et al (1985)
Rainbow trout 5.01 15.07 and 20.99 x 10 6 Kavamoto et al
55.9 - 72.9 54.43
S. iredeus 12.36 75.75 sperm cells / mm 3 (1985)
Fogli da Silveira,
catfish 55.09 x 10 6 Kavamoto &
1.15 72 76.09
Rhamdia hilari * i sperm cells / mm 3 Narahara (1981;
1985)
Kavamoto & Fogli
Catfish 66.53 x 10 6
0.8 81.9 89.13 da Silveira
Rhamdia hilarii * sperm cells / mm 3
(1986)
Pacu
7.28 x 10 6 Fogli da Silveira et
Piaractus 5.0 90 89.10
sperm cells / mm 3 al. (1990)
mesopotamicus *
Bocahico
Bernal, Uribe &
Prochilodus 1.0 83.8 202
Contreras (1992)
magdalenae *
curimbatá 29.85 x 10 6 Kavamoto et al.
Prochilodus scrofa * sperm cells / mm 3 (1997)
curimbatá Kavamoto et al.
94.2
Prochilodus scrofa * 9.54 (1999)
Spatula 1.1 x 10 9 Brows & Mims
100
paddlefish sperm / mL (1999)
24650000
Jundiá 72
0.41 38950000 Ferreira et al
Rhamdia quelen * 83.3
sperm cells / mm 3 (2001)
Suruvi
190.5 and 120. 1 x 10 6
Steindachneridion 4 to 20 46 -75 Light et al (2001)
sperm cells / mm 3
scripta *

yamú 1.2 x 10 10 Cross-Casallas et

1.82 88 95.39 42.5
Brycon siebenthalae * sperm / ml 6.76 al (1999)

0.85 x 10 10
yamú Pardo-Carrasco et al
6.2 and 1.2 66.1 92.23 36.7 1.79 x 10 10
Brycon siebenthalae * (2001)
sperm / ml

5.22 x 10 9
0.08 93 315
sperm / ml
Captain de la Sabana
Eremophilus mutisii * Pink (2001)
21.4 x 10 9
0.8 98 249.6
sperm / ml

* seminal volume obtained by hormonal induction.

24
3.4 SPERM IN ELEOSTEOS MORFOLOGIA

Cove (1986; 1997) in the captain of the Savannah Eremophilus mutisii; Able Pimelodus

grosskopfii and Zaiden (2000) in the piraputanga Brycon hilarii, They established four stages

in the formation, development and maturity of sperm: stage I formation

spermatogonia, with a predominance of primary spermatogonia and cistos of

secondary spermatogonia. Stage II spermatogenetic process or training

spermatocytes, spermatids and spermatozoa, this stage marks the spermatogenesis or

annual reproductive cycle after fertilization and gonadal involution of the fish. stage III

viable mature sperm, ie the fish are physiologically ready for

reproduction and sperm are libreados pressing the abdomen of the fish. Stadium

IV period of involution, after the reproductive period connective tissue collapses and

the sperm is likely engulfed by phagocytosis.

Zaiden (2000) identified in the testes of piraputanga B. hilarii two

compartments: the seminiferous tube made spermatogenic cells

(Primary spermatogonia, secondary spermatogonia, primary spermatocytes,

side, spermatids and spermatozoa) spermatocytes, somatic cells and

cystic or Sertoli and interstitial compartment consisting of connective tissue,

blood vessels and interstitial Leydig cells.

Kavamoto et al. (1999) considered the case of curimbatá Prochilodus. scrofa,

morphological abnormalities of the middle and the tail cause alterations in the
25
motility, increasing the number of sperm in a circular motion or

oscillatory and consequently decreases the fertilization rate. In addition, they observed

abnormalities increased as the time increased action

hormone

Most teleost fish usually lack acrosome (Ginzburg, 1972;

Atzelius, 1978; Lantnsteiner et al., 1994; Billard et al., 1995; Cosson et al., 1999) .And this

structure is compensated by the presence of the chorion micropyle egg for

sperm penetration.

Lantnsteiner et al. (1995) considered that the sperm of Perca Perch

fluviatilis It is asymmetric and means laterally uniflagelado which is inserted into the core,

lacks an acrosome, it has an ovoid head and a small middle part with a

mitochondrion, and also considers that sperm motility is affected by the

osmolality.

26
 4. METHODOLOGY

4.1 STUDY AREA

The study was conducted during the months of August to November 2001 and May

2002 in Piscícolas Research Center at the University of Córdoba (CINPIC)

It located in the city of Monteria, Department of Córdoba, whose coordinates are 8 °

48 'north latitude and 75 ° 22' W; with a height of 15 m and values

average annual temperature equal to 27.5 ° C, relative humidity of 85% and

precipitation of 1,100 mm respectively.

4.2 MATERIAL AND SAMPLES BIOLOGICO

They were used twenty-four (24) male blanquillo, maintained in

confinement-fed ration of 35% crude protein, once daily,

six times a week. The specimens had mean weight ± 0.56 0.18 Kg and 40

cm total length average three years old, from a batch of 110

animals between males and females. The players were selected according to the

signs of maturation: easy release of semen from the urogenital pore by mild

abdominal pressure (Woynarovich and Horvath, 1983). Then they were induced with 0.75

mL of Ovaprim ® / Kg liveweight, with single dose applied to the base of the fin

pectoral. They were then distributed in circular basins in cement 4 m 3 from

capacity and a usable volume of 2m 3 water, maintained constant flow of 1.0 to 1.5
27
L / min, constant aeration and temperature of 27 ° C. Before the semen collection

Animals were dried with a towel, preventing activation of sperm by

contact with water. Similarly to prevent contamination by feces,

Blood and urine slight pressure was carried out in the previous urogenital papilla expulsion

Semen according Fogli da Silveira et al (1990); Cross-Casallas (2001); Perchec et al

(1999); Pardo-Carrasco & Zaniboni-Filho (2001). Semen of each specimen was obtained

by mild abdominal pressure or massage skull flow (Fogli da Silveira et al.,

1990; Cruz et al., 2000; Pardo-Carrasco & Zaniboni-Filho, 2001) and maintained at

5 ° C during the evaluation period. Before the time were evaluated

features such as sperm activation time, percentages of sperm

alive, by the method of differential-eosin staining nigrosine concentration

spermatic mass motility and estimate.

4.3 Macroscopic

• Color. This characteristic was determined directly when the

semen collection, allowing to know the presence of contaminants semen.

The discoloration is caused by the presence of feces, urine or blood,

these conditions the material must be discarded for evaluation in accordance with

Fogli da Silveira et al (1990)

28
 • Viscosity. It was graded subjectively on a scale of 0 to 4, assigning a value

4 to the higher viscosity observed. The viscosity is related to the

sperm concentration.

• Volume. It was determined directly in the receptacle (graduated tube)

and the amount recorded in milliliters (mL).

4.4 Microscopic characteristics

• sperm motility. 2 uL dilution of semen was used in 200 uL of

solution Sodium Citrate 2.9% (using pipette plunger

Transferpette) which took 1.0 uL. Semen was activated with 10 uL of water

distilled and evaluated by light microscopy (10X - 40X). It took into account

movement together of sperm cells, establishing a percentage

field of 0 to 100% according Salisbury & Vandemark (1964).

• Activation time. It was determined with the help of a stopwatch from

The instant that the water drop semen was added until at least

5% of the sperm movement according to submit Salisbury & Vandemark

(1964); Fogli da Silveira et al (1990); Wuthiphandchai & Zohar (1999); Kavamoto et

al (1999); Cross-Casallas et al (1999); Pardo-Carrasco et al (2001); Tvedt et al (2001).

29
• spermatic concentration. It was determined by counting cells in the chamber

hematimétrica Neubauer, using a 1: 700 with the help of pipette

pasteur. After 1 minute of rest was read using a microscope

(40X). This method is used in cattle (Bearden & Fuquay, 1982; Hafez, 1989)

and in several fish species (Kavamoto et al., 1985; Fogli da Silveira et al., 1985;

Daza, 1987; Cross-Casallas, 1999; Pardo-Carrasco & Zanibono-Filho, 2001).

• Differential staining. To check the percentage of live sperm is

He used the technique of differential staining using Eosin solution (2%) -

Nigrosine (10%). Eosin can not cross the membrane of living cells and

nigrosina makes it possible to see the heads unstained. Both solutions were dissolved

in a buffer of sodium citrate 2.9%. dilution was used

2 uL of semen in 200 pL of solution Sodium Citrate 2.9%. They were mounted

plates by swabbing on lamina carries object and rapidly dried (55-60 ° C)

According to Bearden & Fuquay (1982); Kavamoto et al (1985); Fogli da Silveira et al

(1990); Vuthiphandchai & Zohar (1999) and 100 cells per field (100x) were counted.

• Morphology. For the test, a drop of semen dilution (40 uL of took

sodium citrate: 3 uL of semen) and 3 smears were mounted in lamina carries object

colored with eosin (2%) - Nigrosine (10%) observed microscopically (100X) is

100 cells studied so lamina anatomical description was made

sperm, identifying possible morphological alterations according to

30
 affected part (head, midpiece, tail) expressed in percentage (Daza,

1987; Kavamoto et al., 1999).

4.5 STATISTICAL ANALYSIS

Descriptive statistics were used, expressing the results of the evaluated variables

as mean ± standard deviation.

31
 5. RESULTS

The average value of seminal volume blanquillo induced Ovaprim ®,

after 14 hours of application of the dose was 1.7 ± 0.9 mL with maximum values ​and

minimum of 0.5 and 3.6 mL; It is the average value per unit volume seminal

weight of 3.3 mL / kg bodyweight, as shown in Table 2.

Table 2. Seminal main characteristics of tilefish Sorubim cuspicaudus

(Littmann, Burr & Nass, 2000) hormonally induced with Ovaprim ®.

weight vol Activation Alive normal Sperm Motility mL / Kg Esperm / Kg

Kg mL time (%) (%) concentration
(Sec) (Esperm / mm 3) (%)

Minimum 0.25 0.5 73.0 20.0 80.0 5.6 x 10 6 58.0 0.7 7.5 x 10 6

Average 0.56 1.7 128.1 83.9 93.0 2.2 x 10 7 86.0 3.3 4.2 x 10 7

STDEV 0.18 0.9 33.5 18.5 4.0 1.2 x 10 7 10.9 2.0 2.2 x 10 7

Maximum 0.80 3.6 183.0 99.6 96.0 6.0 x 10 7 95.0 10.0 9.7 x 10 7

n 24 24 24 24 24 24 24 24 24

The color of semen presented creamy appearance by 95% and the viscosity was

considered dense consistency.

The average activation time was 128.1 ± 33.5 sec and maximum values

minimum of 73 and 183 sec.

32
 Test differentiation of living and dead averaged value

83.9 ± 18.5% (Figure 5) of live spermatozoa, ranging between 20 and 99.8%.

to
to

bb
to

Figure 2. Live Sperm and dead by differential staining eosinanigrosina. a) dead sperm. b) live sperm
(100X magnification).

The sperm blanquillo this compound morphologically by: head,

Mitochondria, intermediate piece and flagellum; as shown in Figure 3.

33
 c

my
pi
F

Figure 3. Morphology of sperm blanquillo S. cuspicaudus differential staining. c, head region; my,
mitochondria; pi, intermediate part; f, flagellum. (100X magnification).

The average sperm abnormalities for tilefish was 6.96 ± 3.99%

The most frequent: bent tails, loose heads, midpiece

retroaxial, among others; as shown in Figure 4.

The average value of the sperm concentration was 2.2x10 7 ± 1.18x10 7

sperm cells / mm 3, ranging from 5.63x10 6 and 6.09x10 7 sperm cells / mm 3

It is the average value of sperm concentration per unit weight

4.2x10 7 ± 2.2x10 7 sperm / kg liveweight.

The average value of the mass motility was 86 ± 10.8%, ranging from 58

and 95%
 C
TO

Figure 4. Common Abnormalities found in the semen of blanquillo A) and B) bent tails; C) retroaxial
intermediate part; D) and E) abnormalities in the intermediate part; F) loose heads (100X
magnification).

35
 6. DISCUSSION

The seminal volume is one of the characteristics of importance in

artificial reproductions, particularly when performed fertilizations dry.

One of the problems of induced reproductions tilefish extract

pitituario carp (EPC) was the low volume of semen produced by males.

In the first inductions tilefish with carp pituitary extract (EPC) was

males need to sacrifice for obtaining semen (Yepes, Lamb &

Solano 1994). This paper was obtained that the male induced blanquillo

with Ovaprim ® It yielded a value of 1.7 ± 0.9 mL (n = 24); volume enables

oocyte fertilization without sacrificing males.

There are reports of other seminal volume induced silurids

hormonally. Light et al. (2001) reported seminal volumes between 4 and 20 mL

(N = 5) in the suruví, S scripta teindachneridion, induced EPC, weighing between 1.8

- 4.7 Kg and 2.6 Kg average, equivalent to a volume per unit seminal

liveweight of 4.5 mL / kg. Ferreira et al. (2001) recorded in Jundiá, Rhamdia

quelen, induced EPC, with average weight of 0.15 ± 0.02 kg average volume

0.37 ± 0.41 mL (n = 3). In Rhamdia hilarii induced Chorionic Gonadotropin

Human (HCG) a seminal volume of 1.15 mL was obtained (Fogli da Silveira,

Kavamoto & Narahara, 1981; 1985). Godinho (2000) considers that the volume of

semen produced by fish is variable in the same species and between species

different, depending on the size, age, time and method of collection. In addition, this
36
author suggests that fish leather, as silurids produce greater

semen amounts compared to finfish. However, in the

This study seminal volume registered Blanquillo is in the

I range reported for some characids as Yamu B. siebenthalae, induced

EPC, whose volume ranged from 1.2-1.82 mL (Cruz-Casallas, 1999; Pardo-Carrasco

et al, 2001).; bocachico Prochilodus magdalenae ( 1.0 mL) (Bernal, Uribe &

Contreras, 1998). But less than the volumes reported for Pacú Piaractus

mesopotamicus ( 3.3-5.0 mL) (Fogli da Silveira, Kavamoto & Godinho, 1985; Fogli

da Silveira et al., 1990). Also less than the volumes reported in

Salmonids and rainbow trout, Psalm iredeus ( 5.0 mL) (Kavamoto et al.,

1985); brown trout Salmo gairdneri ( 4 to 8 mL) (Billard, Jalabert & Breton, 1971).

Similarly, the time of activation of sperm motility in

the fish is of great importance in induced breeding; to the extent that

time greater mobility, greater opportunity to have sperm

fertilize oocytes. Billard et al. (1995) reported that the activation time is

very short in fish being between 20 and 25 sec in trout and 80 to 90 sec

the tents. Harvey & Hoar (1980) reported that the activation time

teleosts spawning in freshwater ranging from 120 to 180 seconds. At

This study activation time recorded ranged within this range

(128.1 ± 33.5 sec). It is lower than reported by Bernal et al value. (1999) for the

Bocachico (202 sec) and Captain Savannah (315-276 sec) observed by

Pink (2001). However, the activation time is greater than blanquillo

37
recorded in other silurids as suruví pond water activated (46-75

sec) and sodium (37-56 sec) (Light et al, 2001) bicarbonate...; and yamú (42.5 and 36.8

sec) (Cross-Casallas et al., 1999;. Pardo-Carrasco et al, 2001)..

Although the mass motility test is subjective, it is a good indicator

semen quality should be supplemented by dye test

differential for living and dead. Players generally have analyzed

high mass motility (86 ± 10.8%) which was verified by testing

differential. This value is similar to that found in other silurids as Rhamdia.

hilarii ( 81.5%) (Kavamoto & Fogli da Silveira, 1981); Captain Savannah

Eremophilus mutisii ( 93-98%) (Rose, 2001); Jundiá, and 88.3 ± 2.9%, n = 3) no

induced (72.5 ± 2.7%, n = 6) and induced (88.3 ± 2.9%, n = 3) (Ferreira et al., 2001).

Ferreira et al. (2001) considered that some factors such as age, time of

year, growing conditions can influence on these results. In addition, these

same authors consider that in males of Jundiá induced motility is

most, followed by rapid, sudden and simultaneous stop of sperm

in uninduced, followed by slow, gradual movement. However, in the

This study this behavior was not observed.

Sperm concentration was 2.2x10 blanquillo 7 ± 1.2x10 7

sperm cells / mm 3, being in the range of continental teleost,

although higher than those found in other pimelodids by Luz et al. (2001); Ferreira

et al. (2001); Pink (2001) who recorded values ​between 19.5 and 120.15 × 10 6
38
sperm cells / mm 3 Suruví in male induced EPC; 69.9x10 6 ± 37.7x10 6

sperm cells / mm 3 Jundiá in males and similar to the value recorded for the

Captain Savannah 2.25 and 2.14 x 10 7 sperm cells / mm 3 ( Pink, 2000).

Test for differential staining living and dead in tilefish was

83.9 ± 18.5% of live and 16.1 ± 18.5% of deaths. This test allowed us

complement the outcome of the mass motility found in this study. Which

It allows us to deduce that the semen of male tilefish held in

captivity in CINPIC have good quality. In addition, these results are

similar to those found in other teleost. Among them are the

works by Kavamoto & Fogli da Silveira (1981); Kavamoto, Fogli da

Silveira & Godinho (1981); Fogli da Silveira, Kavamoto & Godinho (1985); Fogli da

Silveira et al. (1990); Cross-Casallas et al. (1999); Bernal, Uribe & Contreras (1999);

Pardo-Carrasco et al. (2001) who evaluated semen characteristics of Bagre,

R. hilarii; curimbatá, P. scrofa; pacu, P. mesopotamicus; pacu, P. mesopotamicus;

yamú, B. siebenthalae; bocachico, P. magdalenae; yamú, B. siebenthalae,

respectively

The percentage of abnormal sperm players blanquillo

analyzed was 6.96 ± 3.99% with morphological defects in the head piece

intermediate and tail. Which can be considered low, when compared with reported

for cattle whose percentage of sperm abnormalities is about 15%

(Blom, 1973). Kavamoto et al. (1999) considered that the number of

39
sperm abnormalities increases as time increases the

hormonal action on individuals. Billard, Dupone & Bernabe (1977) reported that

no semen characteristics constant throughout the period

spermiation being affected by aging of cells more

intensity in the post-reproductive peak period. However, in this study

It found that the percentage of abnormalities was low (6.96 ± 3.99) after 14

hours induction of males with Ovaprim ®.

The sperm morphologically blanquillo is constituted by:

rounded head, middle and the tail. According Billard (1970); Ginzburg (1972)

and Grier (1985) the shape of the sperm head fish this

related to the type of fertilization by the species, stating that there

relationship between the rounded head and external fertilization and between the elongated and

internal fertilization. Furthermore, Ginzburg, (1972); Lahnsteiner et al. (nineteen ninety five); Billard et

to the. (1995) and Cosson et al. (1999) reported that most teleost lack

acrosome, this being compensated by the presence of a micropyle in structure

fish oocyte to sperm penetration. This study found

the sperm has a head blanquillo morphologically

rounded, Typical fish performing external fertilization. In the

dead sperm, a significant increase was observed heads,

possibly due to the tinting solution penetration into the

cytoplasm of the cell (Figure 2).

40
 It suggested for further studies evaluating the seminal characteristics

tilefish induced with other hormonal substances such as pituitary extract

carp, human chorionic gonadotropin, mammalian and analog (LHRH), between

others. The perspectives of these characterization studies should stimulate

creating a cryopreservation technique, evaluating different diluents and

crioprotectantes and promote the establishment of a sperm bank involving

native species in the basin of the Sinu River.

41
 7. CONCLUSIONS

The results obtained in this study allow us to conclude that

features of the semen blanquillo Sorubim cuspicaudus induced

hormonally with Ovaprim • They are within the ranges described for

teleosts regarding semen volume (1.7 ± 0.9 mL, 3.3 mL / kg), time

activation (128.1 ± 33.5 seconds) and sperm concentration (2.2x10 7 ± 1.2x10 7

sperm cells / mm 3).

The seminal quality players tilefish used for this study,

It can be considered viable because of their high values ​of mass motility

(86.0 ± 10.9%), percentage of live (83.9 ± 18.5%) and low percentage of abnormalities

(6.96 ± 3.99%). Among the highlights abnormalities bent tail, loose head and

retroaxial intermediate piece.

42
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51
 ATTACHMENTS

Annex 1. Results of the seminal characteristics of tilefish Sorubim cuspicaudus ( Littmann, Burr &
Nass, 2000) hormonally induced with Ovaprim ®.

No: Weight Vol. Ml Activation Alive normal Sperm Motility mL / kg Esperm / Kg

Kg. time (seconds) (%) (%) concentration
(Esperm / mm 3) (%)

01 0.65 2.5 89.0 2.66x10 7 90 3.85 4.09x10 7

02 0.63 2.5 170 92 95 2.20x10 7 90 3.97 3.57x10 7

03 0.55 2.5 73 40 95 9.57x10 6 70 4.55 1.74x10 7

04 0.75 3.0 90 92 95 5.63x10 6 90 4.00 7.51x10 6

05 0.61 2.5 125 94 95 2.07x10 7 90 4.07 3.37 x10 7

06 0.70 3.8 78 84 95 3.31x10 7 73 5.36 4.73 x10 7

07 0.30 3.0 85 95.6 95 2.03 x10 7 95 10.0 6.77 x10 7

08 0.30 1.5 111 81 95 1.57 x10 7 72 5.00 5.23 x10 7

09 0.30 0.8 100 79 95 1.85 x10 7 58 2.57 6.17 x10 7

10 0.30 0.5 154 79 95 2.16 x10 7 71 1.67 7.20 x10 7

eleven 0.80 0.8 88.8 95.4 90 1.94 x10 7 95 1.00 2.41 x10 7

12 0.45 2.0 158 82.5 95 9.78x10 6 85 4.44 2.17 x10 7

13 0.70 0.5 126 99.8 85 1.02 x10 7 85 0.71 1.46 x10 7

14 0.80 2.5 126 87.6 90 3.93 x10 7 90 3.13 4.91 x10 7

fifteen 0.71 2.0 135 73.3 95 6.09 x10 7 95 2.79 8.49 x10 7

16 0.66 1.0 183 94.5 95 2.52 x10 7 95 1.51 3.80 x10 7

17 0.71 2.0 163.2 97.2 98 2.14 x10 7 90 2.81 3.01 x10 7

18 0.30 1.0 179.4 96.4 95 1.25 x10 7 95 3.33 4.17 x10 7

19 0.25 1.5 97.2 twenty 80 2.42 x10 7 70 5.00 9.58 x10 7

twenty 0.70 1.0 120 88.9 95 1.46 x10 7 90 1.45 2.12 x10 7

0.54
twenty-one 1.0 139.8 84.6 90 1.16 x10 7 90 1.83 2.13 x10 7

22 0.54 1.0 160 97.3 90 1.96 x10 7 95 1.64 3.64 x10 7

2. 3 0.54 1.1 161 97.3 90 1.98 x10 7 95 2.02 3.63 x10 7

24 0.68 1.0 114 126 73.8 95 95 3.53 x10 7 95 1.47 5.20 x10 7

52