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Morphological alterations in cryopreserved spermatozoa of scallop Argopecten purpuratus/Alteraciones morfologicas en espermatozoides criopreservados de concha de abanico Argopecten purpuratus.


One of the critical points for the development of aquaculture of native species is the reproduction in captivity, which needs a suitable management of breeding condition and incorporation of assisted reproductive techniques such as hormonal induction of spawning (Patino, 1997; Zohar & Mylonas, 2001) and gametes cryopreservation (Caffey & Tiersch, 2000; Espinoza & Dupre, 2001). One of the reproductive problems of fish in captivity is asynchrony of gametes release, which can leads to failure of the effort to obtain brood, since ovulation does not happen at the expulsion sperm moment, ovulated eggs become "overripe" and cannot be fertilized (Zohar & Mylonas, 2001). One alternative to solve this problem is the use of induction methods for spawning, thermal shock for shellfish (Robert & Gerard, 1999) and hormonal induction for fish (Zohay & Mylonas, 2001), complemented with preservation at low temperatures techniques, since sperm freezed in liquid nitrogen could be used anytime and anywhere.

However, to optimize cryopreservation techniques, it is important to know and understand the effects that freezing-thawing has on sperm structure to, thus, design suitable freezing methodologies that allow us to minimize the percentage of sperm injured. It is known that during freezing and thawing, cells suffer osmotic stress because constant chemical potential changes between intra and extracellular medium caused by ice formation (Mazur, 1984). Damages in sperm might be different according to the structure affected, since sperm plasmatic membrane possesses three different domains, one in front region head, other in back region head and other in tail; which have different both lipids and membrane proteins (Alberts et al., 2000), doing that freezing phenomena cause different deficiencies in motility and fertilizing capacity.

In previous investigation it has been reported different alterations in sperm as a result of freezing and thawing; being the most common, axonem structure alteration, folds formation in head plasmatic membrane (Billard, 1983), lysed cells and mitochondrias (Mediavilla et al., 1995), damage in plasmatic membrane (Curry & Watson, 1992), acrosome eversion and broken tail (Bury & Olive, 1993). However, these alterations have not been quantified to correlate neither with motility nor fertilizing capacity of sperm.

The present work identified and quantified morphologic alterations caused on Argopecten purpuratus sperm by cryopreservation process with a system of mechanical freezer, designed by Dupre & Espinoza (2004). The goal is to recognize the injuries caused by freezing-thawing in sperm of this species to optimize an easy handling and lower cost methodology that could be used for preservation of other shellfish and fish in the field.


Ripe scallops were obtained from cultures of La Herradura Bay in Coquimbo, Chile, transported to hatchery at the Universidad Catolica del Norte and placed in 500 L tanks with constant aeration and water flow for conditioning. Then, gonads were dissected and the male portion was cut into pieces and placed into 10 mL Petri dishes containing filtered and sterilized sea water (SSW) for 10 min to obtain a spermatic suspension.

Spermatic suspension was mixed with cryoprotective solution (1:3) into cryotubes to be equilibrated for 5 min at 5[degrees]C. The composition of the cryoprotective solution used is shown in Table 1. Freezing was performed in a portable mechanical freezer, cooling rate used was -8[degrees]C [min.sup.-1] (to -100[degrees]C) and finally, the cryotubes were plunged into liquid nitrogen according to the methodology designed by Dupre & Espinoza (2004). After 24 h, thawing was performed by immersing cryotubes in a 50[degrees]C water bath by 20 s and other in a room-temperature water bath until the last ice crystal had melted. Thawed samples were transferred into Petri dishes to evaluate spermatic motility and morphology (at 5, 30, 60, 110 and 150 min) and fertilization trials.

Sperm motility was observed under optic microscope (250x) and recorded as percentage of motile spermatozoa in a determinate area of hematocytometer. Thus, motility was evaluated in sperm without treatment (CTR), spermatozoa incubated in cryoprotective solution but not freezed (ICS) and freezed-thawed spermatozoa (FTS).

To analyze spermatic extern morphology, samples were fixed in glutaraldehyde 3% (diluted in SSW), dehydrated through a series of ethanol baths and dried in a critical point drying apparatus (Sandri-780 Tousimis) using liquid C[O.sub.2]. Subsequently, samples were placed on bronze porta-samples and sputter-coated with gold using fine-coat Ion Sputter JFC-1100, JEOL. Samples were examined in a scanning electron microscope JEOL-T300. They were tested 100 spermatozoa by sample and there were identified spermatozoa that had malformations in acrosome, head, middle piece and tail.

Oocytes obtained by artificially induced spawning and frozen-thawed sperm were placed in 60 mL Petri dishes and gently mixed together. Were used 1200 oocytes [mL.sup.-1] and 15 spermatozoa per oocyte. After being washed and hydrated, eggs were transferred to a 250 mL bowl. Fertilization rates were determined by percentage of mobile trochophore larvae, 24 h after fertilization. The control was fertilized with fresh spermatozoa and a control of self-fertilization was also tested.

Data were presented in mean [+ or -] SE. Data normality was tested using Lilliefors test and variances homogeneity with Cochran test. When it was necessary, data were converted using arc-sine transformation. To determine significant differences in motility percentage, repeated measurement ANOVA and Tukey test were used for comparisons among time in the same treatment, and t-Student test with Bonferroni adjustment to compare among treatments of the same period. To determine correlation between i) injuries-fertilization, ii) injuries-motility and iii) fertilization-motility, Pearson was used. We employed Systat 8.0 or SPSS 8.0 for Windows.


Sperm motility

Frozen-thawed sperm (FTS) showed significant decrease in motility compared with control (CTR) and spermatozoa incubated in cryoprotective solution (ICS) (p < 0.001). No significant differences were found among CTR (81.1 [+ or -] 2.3%) and ICS (76.6 [+ or -] 4.2%) (p > 0.05) to 30 min. Motility duration in FTS also was considerably lower than ICS and CTR. Motility in ICS stayed high without significant changes during 110 min (72.3 [+ or -] 5.5%) (p > 0.05), whereas in CTR this time was 30 min (81.1 [+ or -] 2.3%) and in FTS was 60 min (45.2 [+ or -] 4.9%) (Fig. 1).

Alterations in the external morphology of sperm

Typical sperm of A. purpuratus is differentiated in an acrosome in the anterior part of the head, an oval head, the middle piece conformed by four mitochondrias (being evident as convex small projections) and a posterior tail (Fig. 2a). Considering these characteristics, morphological alterations observed in each of the spermatic structures were: head (HE): plasmatic membrane folded or broken, deformed, swollen or lysed (smooth), (Fig. 2b); acrosome (AC): acrosomic reaction (Fig. 2c); middle piece (MP): mitochondrias out of its normal position or absent (Figs. 2d-2e); tail (TA): it was the most affected structure, broked, rigid or loss of linear structure (Figs. 2f, 2g, 2h).


In control group, 87.7 [+ or -] 3.4% of sperm were undamaged, being significantly different of injured sperm percentages (p < 0.001). The 79.0 [+ or -] 3.0% of ICS were undamaged (p < 0.05), percentages of damage in tail (13.8 [+ or -] 2.0%) and in head (12.4 [+ or -] 1.3%) were significantly more than those observed in the middle piece (2.8 [+ or -] 1.4%) and acrosome (2.1 [+ or -] 1.2%) (p < 0.05). Only 14.2 [+ or -] 2.8% of FTS were undamaged, and structures more injured were the tail (77.0 [+ or -] 3.0%) and head (55.1 [+ or -] 7.4%) with regard to acrosome (28.7 [+ or -] 3.3%) and middle piece (23.9 [+ or -] 4.1%) (p < 0.05) (Fig. 3).

Fertilization rate

Fertility trials showed that frozen-thawed sperm could fertilize oocytes. Fertilization rates were 68.3 [+ or -] 6.6%, 67.9 [+ or -] 4.2% and 58.2 [+ or -] 7.3% with fresh sperm (CTR), ICS and FTS respectively; no significant difference was observed among the three values (p > 0.05) (Fig. 4).

Correlation injure vs motility and injure vs fertilization

Correlation indexes between injured percentages and motility were highly significant (p < 0.05), being 0,739 between Mot5 and HE; -0,888 between Mot5 and AC; -0,859 between Mot5 and MP; -0,882 between Mot5 and TA (Tabla 2). Nevertheless, there was no significant correlation between injures percentages and fertilization rates (p > 0.05). Significant correlation was observed between fertilization rates and spermatic motility evaluated to 30 min (0,668; p < 0.05), but not between fertilization rates and spermatic motility to 5 min (0,650; p > 0.05) (Table 2).


Motility observed in ICS was higher than in CTR. It suggests few toxicity of cryoprotective solution during equilibration time. Even more, it has a positive effect on the spermatic motility, since major time of activity was observed in ICS (Fig. 1). Osmolarity of cryoprotective solution was higher than SSW (Table 1); such factor would have been influencing motility and time of activity of A. purpuratus sperm like it was observed in other species (Morisawa & Suzuki, 1980; Chambeyron & Zohar, 1990; Ohta et al., 1997). Minimal or no toxicity of the cryoprotective solution on A. purpuratus sperm could have been favored by inclusion of cryoaditives (sucrose and egg hen yolk), since spermatic motility in tests performed with only [ME.sub.2]SO, was always less than fresh sperm (Dupre et al., 1999).



Previous works have also reported better results in sperm cryopreservation of other species using cryoaditives as sucrose (Babiak et al., 1998; Usuki et al., 1997), egg hen yolk (Wheeler & Thorgaard, 1991) and lyophilized milk (Leung, 1987; Sztein, et al., 2001) than those that only use cryoprotective. Cryoaditives would have fulfilled different functions of protection during cryopreservation process, complementing the action of permeable cryoprotective, such as decreasing the melting point (Rana, 1995), to serve like osmotic buffers during freezing and thawing (McWilliams et al., 1995), or possibly to strengthen the cellular membrane. However, the effect of cryoaditive could be species-specific, since there are cases in which they do not fulfill the desired cryoprotective function; for example, good results were obtained with egg hen yolk in cryopreservation of brown trout (Piironen, 1993) and rainbow trout (Wheeler & Thorgaard, 1991) sperm, but not in cyprinid Aspius aspius sperm (Babiak et al., 1998).

The significant decrease of spermatic motility in treatment FTS and not in ICS indicates that damages that affected spermatic motility was occasioned by ice formation during freezing; more than by the osmotic processes and toxicity during equilibrate time.


Although motility of frozen-thawed sperm diminished considerably with regard to CTR, fertilization rates obtained with these sperm did not change in the same proportion (Fig. 4). It was observed low correlation between motility and fertilization percentages of freezed-thawed sperm of A. purpuratus. Similar results were brought for Pleuronectes ferrugineus (Richardson et al., 1999), they observed motility percentages of 80.0% and 43.3% with fresh and cryopreserved sperm respectively, and fertilization rates of 61.5% and 56.1% respectively. In the same way, Ritar (1999) observed lower percentages of motility in cryopreserved sperm than in fresh sperm in Latris lineate, but fertilization rates were not statistically different (82.7% and 71.2% respectively). To note such differences, probably, it is necessary to decrease sperm/oocytes ratio.

In this experiment, we observed that tail was the most vulnerable structure, owed to its fragility and thin shape, and it seems also that cellular membrane in tail is more vulnerable than in head or acrosomic region. In spite of microscope observations and video analysis, it was observed that sperm with broken tail had motility and it fertilized oocytes without much difficulty. It seems that energy offered to mitochondrias would play an important role in motility and fertilization capacity.

Sperm with rigid tail had slow motility; this rigidity could be owned to the loose of capacity of slide between adjacent doublets of microtubules of the axoneme; which makes us suppose that molecules of dineine are affected. Something similar would be happening with the sperm that presented circular motility in samples FTS. Possibly, they have had some injury in dineine arms that prevents the correct slide of microtubules of the axoneme towards one of the sides. However, this injury would be slightly frequent due to the scarce quantity of sperm with this characteristic.

Deformed heads of the sperm could be caused by swollen or injuries by crystal growth of ice. Of the sperm with head injuries, only 4.5% was lysed, the rest (51.7%) were swollen (possibly a state previous to lysis) owned to osmotic processes caused by ice crystal formations during freezing and thawing (Mazur, 1984). In contrast, when Mediavilla et al. (1995) cryopreserved sperm of common trout, it was observed that biggest alteration of frozen-thawed sperm was the cellular lysis (between 60 and 80%). Higher percentages of sperm with reacted acrosome in FTS samples compared to ICS and CTR were observed; this because stress during freezing and thawing produces changes in the superficial polarity of the cell (Fernandez et al., 2000), it start the acrosomic reaction (Longo, 1987).

In general, injuries in whatever spermatic structures are related to the cellular membrane, in this way, lysis of the head is due to the rupture of membrane, acrosomic reaction also involves alterations in membrane as well as expulsion of mitochondrias and discontinuity of the tail. This fact is because the membrane is a barrier between intra and extracellular matrix (which is highly changeable) and because it regulates osmotic processes caused by changes of chemical potential intra and extracellular during frozen of the matrix. These changes of concentration generate water flow and solutes towards both sides of the membrane, which change cell volume constantly. In this sense, the life of the cell depends on the structure of the membrane and, basically, of the fluidity and permeability that its components give.

Our results indicate that sperm incubation in cryoprotective solution used does not affect significantly the spermatic morphology, being freezing-thawing the cause of injuries in cryopreservation process.

Although major interrelation was observed between structures injured with motility than structure injured with fertilization, our results show that injury in any spermatic structure has equal incidence on its viability. Possibly, the main cause of the decrease of fertilizing capacity of sperm would be ultrastructural alterations than external morphology. In this sense, it would be necessary to continue investigating the effects of cryopreservation of sperm cells at ultra-structural and genetic level. It would be also more interesting to continue growing larvae obtained with cryopreserved sperm, since finding malformations and high mortalities during the larval development might indicate some damage in the spermatic-ADN.

DOI: 10.3856/vol38-issue1-fulltext-11


The authors gratedully acknowledge the support of General Direction of Scientific Research of the Universidad Catolica del Norte (DGI-UCN) and FONDEF project D05 I-10246. The authors also thank Blg. Betsy Buitron for their assistance with the manuscript translation.


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Received: 14 January 2009; Accepted: 14 December 2009

Carlos Espinoza (1), Martha Valdivia (2) & Enrique Dupre (3)

(1) Laboratorio de Biologia Experimental, Instituto del Mar del Peru Esquina Gamarra y General Valle s/n, Chucuito Callao, Peru

(2) Laboratorio de Fisiologia de la Reproduccion Animal, Universidad Nacional Mayor de San Marcos, Peru

(3) Laboratorio de Criopreservacion, Universidad Catolica del Norte, Coquimbo, Chile

Corresponding author: Carlos Espinoza (
Tabla 1. Composicion de la solucion crioprotectora
utilizada para congelar espermatozoides de A.

Table 1. Composition of cryoprotective solution
used to freeze sperm of A. purpuratus.

                 Cryoprotective   Sterilized
                    solution      sea water

[ME.sup.2]SO       10 % (v/v)
Sucrose              125 mM
Egg hens           10 % (v/v)
Distilled          80 % (v/v)
Osmolarity            2003           1387

Tabla 2. Correlacion de Pearson entre estructuras
lesionadas en espermatozoides con la motilidad o con
tasas de fecundante en Argopecten purpuratus. Los
porcentajes de espermatozoides lesionados en cada
estructura fueron correlacionados con la motilidad a 5
min debido a que fue el momento en que las muestras
fueron colectadas para el analisis morfologico.
indican correlacion significativas. Mot 5: motilidad
cuantificada a 5 min post-dilucion en agua de mar; Mot
30: motilidad cuantificada a 30 min post-dilucion en
agua de mar; Fer: porcentaje de fecundacion.
lesionados en cabeza (HE), acrosoma (AC),
pieza media (MP) o flajelo (TA).

Table 2. Pearson correlation indexes of injured sperm
structures whit motility or fertilization rates in
purpuratus. Percentages of sperm injured in every
structure was correlated whit motility evaluated to 5
because it was when samples took for morphologic
analysis. Asterisks indicate significant correlation.
5: motility quantified to 5 min post-dilution in sea
Mot 30: motility quantified to 30 min post-dilution in
water; Fer: fertilization percentage. Injured sperm in
head (HE), acrosome (AC), middle piece (MP) or tail

                Pearson index   Significance

Mot 5 vs HE        -0.739          0.023 *
Mot 5 vs AC        -0.888          0.000 *
Mot 5 vs MP        -0.859          0.003 *
Mot 5 vs TA        -0.882          0.002 *
Fer vs HE          -0.432          0.245
Fer vs AC          -0.556          0.120
Fer vs MP          -0.651          0.058
Fer vs TA          -0.653          0.057
Mot 5 vs Fer        0.650          0.058
Mot 30 vs Fer       0.668          0.049 *
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Title Annotation:Research Article
Author:Espinoza, Carlos; Valdivia, Martha; Dupre, Enrique
Publication:Latin American Journal of Aquatic Research
Article Type:Report
Date:Mar 1, 2010
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