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Seasonal evolution of gonadal maturation, gamete quality, and fertilizability of Pacific oyster (Crassostrea gigas) on the Western Mediterranean Coast.


The development of reproductive technologies in the Pacific oyster (Crassostrea gigas) requires gamete availability, but the reproductive seasonality of the species (Utting & Millican 1997) limits this. The problem can be avoided by subjecting oysters to artificial conditioning in a controlled area. This option makes it possible to obtain gametes regularly (almost) all year round (Chaez-Villalba et al. 2002), but oyster conditioning requires high water volumes and renewal rates (Utting & Millican 1997, Helm & Bourne 2004), with seawater availability/proximity and quality as another important limitation. Moreover, the artificial feeding of oysters during their conditioning period becomes one of the major costs (Helm & Bourne 2004).

One option to ensure both gametic and embryonic material regularly throughout the year is the cryobanking of gametes (both sperm and oocytes) and embryos, as proposed several times (Smith et al. 2001, Adams et al. 2004, Tervit et al. 2005). Whatever the case, to facilitate the study and development of laboratory techniques that require Pacific oyster gametes, the study of the natural period of complete gonad development is essential.

Therefore, the study of availability and quality of gametes obtained from animals throughout the year in western Mediterranean climatic conditions was the aim of the current work, because, to our knowledge, only one work has studied the gonadal maturation evolution of C. gigas in the Mediterranean Sea, in Tunisia (Dridi et al. 2007).


Oyster Source

The experiment was carried out from October 2007 to November 2008. Two 3 year old Pacific oysters (C. gigas) were provided weekly from a commercial longline culture system (ACUIMA S.L. Burriana, Valencian Community, Spain), located in Burriana, the Valencian region, Spain, with an annual temperature range at 10 m in depth (oyster culture beginning at 8 m) of 11.2[degrees]C in December to 26[degrees]C between July and August. After oyster collection, they were transported to our laboratory, located 60 km from the company, where they were carefully cleaned without carrying out depuration treatments and stored "dry" for a maximum of 2 days in a refrigerator at 4-5[degrees]C until gonad evaluation.

Gonad Maturation Assessments

The apparent gonad maturation degree was assessed by visual inspection after removing the flatter shell valve. Each oyster gonad was ascribed to 1 of the 5 maturation degrees established and described as follows: NO, empty gonad; EA, early gonad activity, white ramifications in the gonad; BF, gonad begins to fill, digestive glandule perfectly seen; PD, gonad poorly developed, digestive glandule partially seen; D, gonad developed, perfectly covers digestive gland; and TD, gonad totally developed, stands voluminously. These gametic developmental stadia are a modification of those previously described by Marteil in 1976.

Gonads from all oysters except those classified as NO were lacerated with a sterile hypodermic needle and gametes were removed through this opening by exerting gentle suction with a sterile Pasteur pipette inserted beneath the overlying gonad epithelium (Helm & Bourne 2004). After this procedure, sex recognition was assessed by the presence of spermatozoa or oocytes under a 100x magnification microscope (Davenel et al. 2006).

Fertilization Assays

Fertilization assays were only performed when both sperm and oocytes were obtained in the same session, and were replicated thrice. They were carried out independently of the apparent gonad maturation degree.

Males were only selected when sperm had a minimum of 70% motility with a high degree of lineal motility. Motility was estimated visually at 200x magnification and was expressed as the percent of spermatozoa actively moving in a forward direction. Sperm vibrating in place were not considered to be motile (Dong et al. 2005). In the selected females, the viable oocytes (defined as fertilizable oocytes) were around 30-60 [micro]m in diameter (Lango-Reynoso et al. 2000) and rounded out completely some minutes after their first contact with seawater, thus observing the germinal vesicle breakdown at 100x magnification (Dong et al. 2005, Eudeline et al. 2000a, Eudeline et al. 2000b).

Recovered sperm was diluted with microfiltered seawater (0.22 [micro]m Millex GP Filter Unit; Millipor Express) to make a "cloudy suspension" (Helm & Bourne 2004). The procedure carried out to clean oocytes consisted of introducing them into a 20-mL sterile vial (8 cm of water column) with microfiltered seawater (0.22 [micro]m Millex GP Filter Unit) and replacing the supernatant with microfiltered seawater after the main part of the amount of oocytes dropped to the recipient bottom (around 10 min). After 2-3 washes using this procedure, oocytes looked clean and well rounded.

Fertilization took place in 3.5-cm Petri dishes (Corning, Sigma) filled with 5 mL microfiltered seawater at room temperature. In each plate, around 50 oocytes were deposited and sperm was added, achieving a final concentration of 1-10 sperm/oocyte (Chao et al. 1997, Eudeline et al. 2000a, Cardona-Costa et al. 2010). After the 1st and 2nd polar body (PB) expulsions, fertilized oocytes were incubated at 28[degrees]C (Chao et al. 1997, Lin et al. 1999, Cardona-Costa et al. 2010) for 24 h, ensuring a good culture density lower than 10 D-larvae/mL (Helm & Bourne 2004). Then, the presence or absence of D-larvae (either normal or abnormal) in plates was noted.

Experimental Design

Obtained data were grouped and presented bimonthly to avoid excessive data dispersion, so 6 time periods were established: January to February, March to April, May to June, July to August, September to October, and November to December. The effect of these different annual periods was analyzed separately in both males and females on the following aspects: the number of both identified as males and females from the total; the number of both males and females showing maximum apparent gonad maturation degree (D + TD) from the total number of both recognized males and females; the number of selected males with more than 70% motile sperm with high lineal motility, and the number of differentiated females with fertilizable oocytes. Finally, and in relation to the availability of gametes to perform fertilization assays, those sessions during which only sperm, only oocytes, or both gametes were obtained were recorded, as well as their temporary unavailability throughout the year. Also, when both gametes were present, fertilization assays were carried out, and the number of assays, where either normal (D-shaped larvae after 24 h of culture) or abnormal larvae were obtained, was recorded. The effect of apparent gonad maturation degree on males and females with viable gametes was also studied.

Results were analyzed with the chi-square test. When a single degree of freedom was involved, Yates' correction for continuity was performed.


Results obtained for the annual period effect are shown in Table lA. In the percentage of sampled specimens identified as males throughout the different annual periods, an increasing progression was shown from the lowest values registered in January to February (1%) to the maximum values, corresponding to the July to August period, reaching a 62% of total specimens differentiated as males (Table 1A).

Regarding the percentage of sampled specimens identified as females, the same trend was observed as in males, reaching the maximum values of differentiated females also in July to August (32%), although their recorded values were substantially lower than in males for all annual periods assessed. These imbalanced values in favor of males could be explained as a consequence of the fact that many bivalves will mature during their first year of life as males; subsequently, an increasing percentage may switch sex and become females (protandric hermaphroditism (Helm & Bourne 2004)).

The percentage of males with the maximum apparent gonad maturation degree (D + TD) from the total of identified males was significantly higher in July to August (Table 1B). The same statement is given in the case of females with a maximum (D + TD) apparent gonad maturation degree.

The rate of selected males (with a minimum of 70% motile sperm) did not vary significantly throughout all annual periods (Table IC). However, in the case of females, there were significant differences in the rate of selected females (with fertilizable oocytes) in favor of the period from May to October.

In the works reviewed, only one was found that discussed the gametogenic cycle of C. gigas in the Mediterranean area--specifically, in Tunisia (Dridi et al. 2007). In that work, the gonad inactivity phase coincided with that reported here (November to February, Table 1A) as well as the concordance in months related to the higher or lower frequency in the detection of a percentage of sexually active specimens. It appears that climatic conditions not strictly identical do not significantly affect the reproductive phases in this species. In fact, Dridi et al. (2007) proposed food availability and the energy and protein intake ability of oysters as main factors in the seasonal variations of reproductive activity. This argument would explain why the breeding periods established in the current study, and in agreement with those reported by Dridi et al. (2007), also coincided with those described for the French Atlantic coasts by Chavez-Villalba et al. (2002). Other authors (Enriquez-Diaz et al. 2009) agreed on food availability as an important effect on the C. gigas breeding periods, but also detected that breeding periods significantly and directly depended on the variations in climatic conditions.

The percentage of selected males (with a minimum of 70% motile sperm) did not show significant differences depending on the apparent gonad maturation degree (Table 2), varying from 45-50% in groups EA and BF to 63-66% in groups PD, D, and TD. In the females, significant differences in fertilizable oocytes were detected among groups (Table 2). However, a coherent pattern was not established, probably as a result of the lower number of sampled specimens in some groups. Despite this, the highest values regarding these fertilizable oocytes seemed to be finally ascribed to the TD group. The fact that a minimum of 50% females from each group showed fertilizable oocytes is also important.

Many technological assays of interest require both sperm and oocyte availability. For example, cryopreservation of sperm, oocytes, and embryos (Chao et al. 1997, Dong et al. 2005, Tervit et al. 2005); embryo/larvae obtained for toxicology assays (da Cruz et al. 2007, Stachowski-Haberkorn et al. 2008); and embryo transfection and polyploidy induction studies (Cadoret 1992, Guo & Allen 1994, Buchanan et al. 2001, Cardona-Costa et al. 2010), among others. In this respect, in Table 3, obtaining both gametes could only be achieved in only 16 sessions out of 29. Moreover, normal larvae were obtained in 11 sessions out of the 16 mentioned (D-shaped larvae at 24 h), whereas in the 5 remaining sessions, the larvae obtained were abnormal. Abnormal larvae were detected essentially in March to June, and all normal larvae were obtained from July to October. This result reinforces previous observations reported by Massapina et al. (1999), who stated that oocyte quality also determines the quality of larvae obtained. Because of the reduced number of data from each annual period, statistical analysis was not performed; thus, their significance regarding gamete availability from each session or in obtaining normal larvae when both sperm and oocytes were available could not be determined.

The assessment methodology proposed here is based on the apparent gonad maturation degree, the presence or absence of gametes in the gonad and their characteristics regarding sperm movement and oocyte ability to round out after their contact with seawater. In addition, their fertilization and additional normal development to the D-stage were assessed. All these parameters are of minor technical complexity compared with other methodologies, including histological (Massapina et al. 1999, Lango-Reynoso et al. 2000, Chavez-Villalba et al. 2002, Dridi et al. 2007, Enriquez-Diaz et al. 2009) biochemical (Lubet 1959, Massapina et al. 1999, Dridi et al. 2007), and magnetic resonance imaging (Toussaint et al. 2005, Davenel et al. 2006). Moreover, the evaluation methodology does not impede the comparison of both gonad developmental degree and presence of gametes with their final quality assessment by in vitro fertilization and their subsequent obtaining of normal larvae in vitro.

The results achieved supported the possibility of assessing or anticipating the availability of either gametes or embryos for other types of studies, in which the relevant objective is not gametogenic evolution, but the actual gamete availability. In this sense, results indicated that oysters from commercial cultures are an alternative to performing either laboratory studies or assays, but only for a few months--from July to October--as we proved after using C. gigas zygotes obtained by in vitro fertilization in cell electrofusion assays for obtaining tetraploid oysters (Cardona-Costa et al., 2010). These results and methodologies could be of interest to laboratories that do not have suitable facilities or insufficient quality seawater available for conditioning and maintaining C. gigas (Utting & Millican 1997, Chavez-Villalba et al. 2002), although the cryopreservation of gametes is still indispensable if gamete availability is required year-round.


We thank ACUIMA S.L. for supplying all oysters used in this work. We also thank Javier Rubio Rubio for his valuable technical support and Neil Macowan for improving the English of this manuscript.


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(1) Aquaculture and Environmental Research Group (ACUMA), Universidad Politecnica de Valencia, Camino de Vera 14, 46022 Valencia, Spain; (2) Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Universidad Politecnica de Valencia, Camino de Vera 14, 46022 Valencia, Spain

* Corresponding author. E-mail:
Effect of annual period on the percentage of recognized males
and females from all specimens assessed.

No. of                             Annual Period
Specimens   Jan-Feb   Mar-Apr    May-Jun   Jul-Aug   Sep-Oct   Nov-Dec

Males       1%  (a)   14% (bc)   43% (d)   62% (c)   17% (c)   7% (b)
            1/127     20/146     61/141    68/110    44/254    8/109

Females     0% (a)    12% (b)    13% (b)   32% (c)   6% (b)    0% (a)
            0/127     17/146     18/141    35/110    16/254    0/109

Between columns, data with different letters differ statistically
(P < 0.05).

Effect of annual period on the percentage of males and females
that showed maximum "apparent gonad maturation degree" from the
total number of either recognized males or females, respectively.

                                         Annual Period

                                Jan-Feb    Mar-Apr    May-Jun

No. of fully mature males       0% (abc)   35% (ab)   43% (a)
  (D + TD) from total males     0/1        7/20       26/61
No. of fully mature female      --         47% (a)    50% (a)
  (D + TD) from total females   0/0        8/17       9/18

                                         Annual Period

                                Jul-Aug   Sep-Oct   Nov-Dec

No. of fully mature males       88% (c)   14% (b)   O% (ab)
  (D + TD) from total males     60/68     6/44      0/8
No. of fully mature female      92% (b)   44% (a)   --
  (D + TD) from total females   35/38     7/16      0/0

Between columns, data with different letters differ statistically
(P < 0.05). D, gonad developed, perfectly covers digestive gland;
TD, gonad totally developed, stands voluminously.

Effect of annual period on the percentage of selected males
and females with quality gametes from the total number of
either recognized males or females, respectively.

                                  Annual Period

No. of
Specimens   Jan-Feb   Mar-Apr   May-Jun   Jul-Aug   Sep-Oct   Nov-Dec

Selected    100%      70%       62%       53%       64%       25%
Total       1/1       14/20     38/61     36/68     28/44     2/8
Selected    --        6% (a)    72% (b)   92% (b)   100%b     --
Total       0/0       1/17      13/18     35/38     16/16     0/0

Between columns, data with different letters differ statistically
(P < 0.05).

Effect of apparent gonad maturation degree on obtaining both
males and females with quality gametes from the total number
of recognized males or females, respectively.

                         Apparent Gonad Maturation Degree
No. of
Specimens            EA          BF       PD          D        TD

Selected males     45%        50%        63%       66%       63%
Total males        18/40      11/22      26/41     35/53     29/46
Selected females   60% (ab)   83% (ab)   47% (a)   63% (a)   97% (b)
Total females        3/5        5/6       9/19      17/27     31/32

Between columns, data with different letters differ statistically
(P < 0.05). BF, gonad begins to fill, digestive glandule perfectly
seen; D, gonad developed, perfectly covers digestive gland; EA,
early gonad activity, white ramifications in the gonad; NO, empty
gonad; PD, gonad poorly developed, digestive glandule partially seen;
TD, gonad totally developed, stands voluminously.

Effect of annual period on gamete availability from each session
and in obtaining normal D-shaped larvae by in vitro fertilization
when both sperm and oocytes were obtained.

                                             Annual Period

                                 Jan-Feb   Mar-Apr   May-Jun   Jul-Aug

Gamete            Both gametes     0/4       1/4       4/6       5/5
  availability    Only oocytes     0/4       0/4       0/6       0/5
  per session     Only sperm       1/4       2/4       2/6       0/5
                  None             3/4       1/4       0/6       0/5

Larvae (normal    Normal           0/0       0/1       0/4       5/5
  and abnormal)   Abnormal         0/0       1/1       4/4       0/5
  obtained from
  assays (0,1)

                                      Annual Period

                                 Sep-Oct   Nov-Dec   Total

Gamete            Both gametes     6/10      0/4     16/29
  availability    Only oocytes     0/10      0/4      0/29
  per session     Only sperm       4/10      2/4     10/29
                  None             0/10      2/4      3/29

Larvae (normal    Normal           6/6       0/0     11/16
  and abnormal)   Abnormal         0/6       0/0      5/16
  obtained from
  assays (0,1)
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Article Details
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Author:Cardona-Costa, Jose; Garcia-Ximenez, Fernando; Espinos, Francisco J.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:4EUSP
Date:Dec 1, 2010
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