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Proportion of sperm and eggs for maximal in vitro fertilization in haliotis asinina and the chronology of early development.

ABSTRACT To obtain the highest yield during in vitro fertilization of tropical abalone Haliotis asinina, optimal proportion of the gametes, the timing of sperm egg interaction, and subsequent development were investigated. The highest yield of fertilization (75%) with fewest abnormal eggs was obtained when incubating eggs and sperm at the ratio of 1:100 in seawater with a salinity of 27.5 ppt, a pH of 7.8, and a temperature range from 27 29[degrees]C. After incubation, sperm swim through the egg jelly coat and become bound to the vitelline envelope within 30 sec, followed by an acrosomal reaction at 1 min. The fertilized egg extrudes the first and second polar bodies at 8-10 min, and then the zygote begins cleavage at 15-20 min. This is followed by the second cleavage, and development through the stages of blastula, gastrula, trochophore, veliger, and early creeping larvae, which were completed within 3 days. Noticeably, occurrence of egg jelly condensation after penetration of the first sperm would not allow other sperm bind to the egg jelly and to penetrate through its vitelline envelope. This event is thought to be a weak blocking against polyspermy, because the classic cortical reaction initiated by cortical granule exocytosis could not be observed in this species.

KEY WORDS: abalone, Haliotis asinina, sperm, egg, fertilization, early development


Haliotis asinina is a tropical abalone that has become endangered as a result of overfishing (Singhagraiwan & Doi 1993). To meet commercial demands, it is now necessary to produce the abalone by aquaculture. To obtain high yields of larvae for culture, in vitro fertilization is a technique that must be developed and optimized. Generally, sperm concentrations for optimal fertilization in temperate abalone species H. rufescens, H. tuberculata, and subtropical abalone H. diversicolor supertexa range from 1 x [10.sup.5] to 1 x [10.sup.6] sperm/mL per [10.sup.3] eggs (Leighton & Lewis 1982). It has been reported in H. asinina that 5 x [10.sup.3] to 1 X [10.sup.5] sperm/mL is required for maximal fertilization and normal trochophore development (Encena et al. 1998). Eggs can still be fertilized 2 h after spawning, but thereafter both fertilization and development rate decrease with time. In H. asinina, there has been 1 report on the duration between egg fertilization and the beginning of the cleavage stage (Jarayabhand & Nittharatana 1996), but there is still no report on the exact timing of fertilization, cleavage, and subsequent development. Furthermore, the fertilization success of abalone may require other factors, such as density of the gametes during spawning, chemotaxis, and sites where sperm enter eggs, all of which affect the number of sperm reaching eggs (Gould & Stephano 2003). To gain efficiency in fertilization, as well as to reduce abnormality of larvae, it is important to obtain an optimum ratio of eggs and sperm, and to understand the sperm egg binding steps that are crucial to successful fertilization. Therefore, we set out to gain knowledge on conditions leading to optimum fertilization and early development, and to produce a timetable of these events that could be exploited to increase H. asinina production, for example, in polyploidy induction.


Aquaculture and Gamete Collection

Male and female abalone broodstock (>24 mo) were used at the land-based aquaculture system at the Coastal Aquaculture Research and Development Center, Department of Fisheries, Prachaubkirikhun Province, Thailand. The abalone were kept in concrete tanks, housed in the shade, and were well flushed with mechanically circulated filtered seawater and an air delivery system to maintain a stable, controlled environment at a salinity range from 27.5-32.5 ppt and a temperature range of 24-26[degrees]C. Abalone broodstock were fed with Gracilaria spp., supplemented with artificial feed. During spawning, animals of each sex were placed in separate tanks in a room with a controlled dark/light period (12 h:12 h), reversing the natural day and night periods. Females and males spawned into filtered seawater during the dark period. Debris was filtered by pouring eggs and sperm repeatedly through nylon mesh. The gametes were suspended in filtered seawater within separate tanks. Sperm and egg samples were "pipetted" into Eppendorf tubes (Sorenson Bioscience, Inc., Salt Lake City, UT) and fixed in 1% paraformaldehyde. The number of sperm and eggs in each tank was estimated using a hemocytometer.

Determination of Optimal Fertilization

Aliquots of seawater (salinity, 27.5 ppt; pH, 7.8), each containing [10.sup.3] spawned eggs, were added to wells of a 16-well plate and kept at 27 29[degrees]C. Subsequently, aliquots of sperm suspensions were gently added into each egg suspension so that the final concentrations of sperm were at [10.sup.3], [10.sup.4], [10.sup.5], and [10.sup.6] sperm/mL. Chronological changes of sperm-egg interactions were observed over 20 min. The number of fertilized eggs (i.e., the presence of polar bodies) and abnormal eggs were counted at 8 min, 15 min, and 20 min after gamete mixing. The experiments were repeated 6 times for each concentration of sperm. Data recorded included percentage of fertilization, based on the number of fertilized eggs for each sperm concentration, and percentage of abnormal eggs.


Histological and Ultrastructural Observations of Fertilized Eggs

Fertilized eggs were centrifuged for 5 min at 200g and immediately fixed in a solution containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH, 7.8) overnight at 4[degrees]C. After washing with 0.05 M cacodylate buffer, the samples were postfixed in 1% osmium tetroxide in the same buffer at 4[degrees]C for 1 h. Subsequently, the samples were washed with the same buffer, dehydrated in increasing concentrations of ethanol (from 50-100%), and embedded in Araldite 502 resin. Semithin and thin sections were cut using an ultramicrotome. Semithin sections were stained with 1% methylene blue and then observed under a light microscope. Thin sections were placed on copper grids, stained sequentially with uranyl acetate and lead citrate, and then observed under a Hitachi H-600 transmission electron microscope operating at 75 kV.

Sperm-Egg Interactions and Early Development

Maximum fertilization occurred at a sperm-to-egg ratio of 100:1 (see Results), and this was used for mixing spawned eggs with sperm in a larger quantity. The sequence and timing of the developmental event, starting from the binding of sperm to the egg surface, until the formation of trochophore larvae, were observed under a light microscope at 1-min intervals. Thereafter, development to the veliger stage, and extending to metamorphosed (settled) postlarva, was observed at 1-h intervals. The timing of each step of events was carefully recorded during and after the process of sperm egg binding. Data were recorded as the average of 10 observations of similar samples. For morphological studies, both live and fixed specimens were observed.


Data are presented as mean + SEM. Statistical analyses were carried out by an analysis of variance using an SPSS program. Data with P [is less than or equal to] 0.05 were considered to be statistically significant.


Optimal Ratio of Eggs to Sperm for Yielding Maximal Fertilization

The results of mixing 103 unfertilized eggs with sperm at [10.sup.3]-[10.sup.6] sperm/mL in 1 mL seawater are shown in Figure I and Table 1. Unfertilized eggs (Fig. 2A) and the eggs with attached sperm are shown in Figure 2B, C. An indication of successful fertilization was first demonstrated by the presence of the first polar body, which appeared at 8 9 min after incubation (Fig. 2D; Table 1). At 10 min of incubation, there was an extrusion of the second polar body, with characteristics, size, and location of appearance similar to the first polar body (Fig. 2E). A slight increase in size of the fertilized eggs was found when compared with unfertilized eggs. Using 106 sperm to incubate with 103 spawned eggs generated the largest proportions of fertilized eggs: 66.5 % within 8 min, 69.1% at 15 min, and 68.1% at 20 min of incubation. These were significantly different when compared with the other sperm concentrations (P [ greater than or equal to] 0.05). The use of 106 sperm also produced the largest numbers of abnormal eggs (25.8% at 8 min, 22.1% at 15 min, and 22.6% at 20 min of incubation; Fig. 1), which were also significantly different when compared with the other sperm concentrations (P [is less than or equal to] 0.05). Abnormal eggs were characterized by the tearing of vitelline envelopes or rupture of yolk matrices into perivitelline spaces. These eggs did not undergo cleavage. Using [10.sup.5] sperm to [10.sup.3] eggs, a 10-fold reduction of sperm resulted in 35.5% fertilized eggs at 8 min (less than that for 106 sperm), but 73.8% at 15 min and 76% at 20 min (both greater than for [10.sup.6] sperm, but the differences were not significant). In addition, fewer abnormal eggs were observed with the use of [10.sup.5] sperm (4.1% at 8 min, 3.8% at 15 min, and 6.6% at 20 min; P [less than or equal to] 0.05; Fig. 1). The use of only [10.sup.3] and [10.sup.4] sperm to 103 eggs generated low numbers of fertilization, ranging from 5 20%. From this experiment, the optimal ratio to obtain maximal fertilization rate was estimated to be [10.sup.5] sperm mixed with [10.sup.3] eggs (i.e., a ratio of spawned sperm to egg of 100:1). This 100:1 ratio also gave fewer abnormal eggs, similar to values obtained using [10.sup.3] and [10.sup.4] sperm per 103 eggs (with no significant difference).

Observations of the fertilized eggs, at the ratio of 100 sperm:1 egg, showed a maximum value of larvae developing to first cleavage as 74%. The percent hatching and undergoing embryo development to gastrulation decreased slightly to 67 %. The percent of larvae surviving at trochophore stage was close to the percent hatching (64%).

Sperm-Egg Interaction and Development During Subsequent Stages

The optimal sperm-to-egg ratio of 100:1 for fertilization was used to study the sequence and timing of events during fertilization and subsequent development. The results are given in Table 1.

The first contact of sperm with an unfertilized egg (Fig. 2A) was observed when freshly spawned sperm came to surround the peripheral jelly coat of the egg (Fig. 2B). Several sperm bound and penetrated this layer. Interestingly, the sperm-bound eggs showed condensation of the jelly coat after being inseminated for 10-15 sec (Fig. 2C). It was found in this study that condensation of the jelly coat was caused by rapid aggregation of its fibers, which made this layer denser, especially at the zone close to the vitelline envelope (Fig. 3A, B).


After passing through the jelly coat, sperm reached the vitelline envelope within 1 min after mixing of the gametes (Fig. 2C). Generally, only 1 sperm reached the vitelline envelope, but in a few cases polyspermy was observed. The sperm acrosoreal vesicle then disappeared and an acrosome reaction was observed at 1 min from the start. Within 1-2 min, the sperm head passed through the thickness of the vitelline envelope and became lodged within the perivitelline space. No obvious change was found when sperm stopped moving at the plasma membrane. There was an extrusion of the first polar body from the egg plasma membrane into the perivitelline space at 8 9 min (Fig. 2D, Table 1). The polar body appeared as a dense mass with the same color and substance as ooplasmic content, but was smaller (Fig. 3C, D). The second polar body began to appear at 10 min at the same site, close to the existing first polar body (Fig. 2E, Table 1). Between 10 15 min after mixing sperm and eggs, a pronucleus formed in the egg cytoplasm (Fig. 3C, E). The pronucleus appeared as a large membrane-bound body containing dense material. Some of the eggs had centrally aligned chromatids with clearly visible spindle fibers, indicating that they were undergoing metaphase 2 (Fig. 3F). Thereafter, the 2-cell stage (first cleavage; Fig. 2F, G) appeared at 15 20 min, followed by the 4-cell stage (second cleavage; Fig. 2H) at 35-40 min, and the 16-cell stage (morula; Fig. 2I) at 45 55 min after mixing the gametes (Table 1). The approximate development times for subsequent stages (i.e., blastula, gastrula, trochophore, veliger, and creeping larvae) are also shown in Table 1.



This study indicates that, in H. asinina, incidences of abnormal eggs and polyspermy can be reduced, while still obtaining a high fertilization rate, by using an optimal ratio of egg to sperm of 1:100. In other species reported so far, the optimal sperm concentration for fertilization with [10.sup.3] eggs ranges from 1 x [10.sup.5] to 1 x [10.sup.6] sperm/mL (i.e., ratios of egg to sperm of 1:100 1:1,000)(Leighton & Lewis 1982). Because abalone gametes, especially the spawned eggs, are sensitive to external conditions (Singhagraiwan & Doi 1993), we recommend that the collection of eggs, fertilization, and early development be done at the following conditions: the pH should be 7.8; salinity, 27.5 ppt; and the temperature range from 27-29[degrees]C. Using too much sperm can increase the number of abnormal eggs, which could be the result of the higher level of proteolytic enzymes released from the acrosome-reacted sperm, which can damage the egg surface.

In abalone there is a rapid sequence of fertilization events starting with sperm swimming toward and making contact with the external coat of the egg until first polar body extrusion. Timing during various steps of fertilization is similar to other species of abalone and bivalves. The first polar body was observed in 3 min in South African abalone H. midae (Septo & Cook 1998), 7-9 min in the blacklip abalone H. rubra (Liu et al. 2004), 15 min in the Pacific abalone H. discus hannai (Li et al. 2000), and 60 min in the surf clam Spisula solidissima (Yi et al. 2002) after the start of egg and sperm incubation. In H. asinina, the first polar body extruded at 84 min after insemination, followed by a quick appearance of the second polar body at 10 min, which was within the time range of other species. Interestingly, the first and second polar bodies were usually extruded at the same site, and both coexisted until the first cleavage. This is different from the situation in mammals, in which polar bodies always undergo degeneration before the fertilized egg reaches the 2-cell division (Wakayama & Yanagimachi 1998), and that the first polar body has a shorter life than the second one (Choi et al. 1996). Knowing the exact timing of events in sperm-egg interaction could yield a practical benefit, such as the induction of polyploidy. An interesting means of obtaining a polyploid population of mature abalone with large size, in a short time, is to treat fertilized eggs with a mitotic blocking agent, cytochalasin B, after the first polar body extrusion (Liu et al. 2004). Inhibition of the second polar body extrusion in blacklip abalone by cytochalasin B results in triploidy chromosomes. This treatment can reduce reproductive output, but the triploid animals tend to have a faster growth rate (Liu et al. 2004). However, inhibiting generation of the first polar body generally results in low yields of triploidy and a low survival rate of larvae (Gerard et al. 1999). Similar experiments remain to be done in H. asinina.

Another notable change after fertilization in H. asinina is the sudden condensation of the egg jelly after insemination. This might be a mechanism that aids in blocking polyspermy, because no other noticeable structural changes occurred at the egg envelope or in the peripheral cytoplasm. Similar changes have been observed in the red abalone H. rufescens (Lewis et al. 1982), the molluscs M. edulis (Humphreys 1967) and S. solidissima (Longo 1976, Longo & Anderson 2005), as well as in the annelid Urechis caupo (Paul 1975) and the cnidarian Actinia fragacea (Larkman & Carter 1984). In the case of the mussel M. edulis, both fast and slow blocking mechanisms have been suggested to ensure monospermic fertilization (Togo & Morisawa 1997). We could not observe other morphological changes of the inseminated egg during abalone fertilization, such as exocytosis of the cortical granules and the elevation of vitelline envelope, as seen in fertilized sea urchin eggs, in which an altered vitelline envelope prevented the binding of other sperm (Carroll & Epel 1975). Thus, the blocking of polyspermy in this abalone is thought to be a rather weak mechanism, such that a high sperm concentration can easily damage the egg coats, causing them to collapse and dissolve, and thereby resulting in a high percentage of abnormal eggs. High sperm densities in the natural environment are unlikely to cause a problem with polyspermy (Babcock & Keesing 1999); however, they are important in abalone culture where in vitro fertilizations are performed.


This study was supported by the Thailand Research Fund (TRF), the Commission on Higher Education, and Mahidol University (to P. S.), and a TRF RGJ PhD scholarship (to W. S.).


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(1) Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; (2) The Coastal Aquaculture Research and Development Center, Department of Fisheries, Klong-wan, Prachuabkirikhan 77000, Thailand

* Corresponding author. E-mail:
Chronological sequence of developmental events that occurred
after fertilization of H. asinina, when [10.sup.5] spawned sperm
were incubated with [10.sup.3] eggs under optimal conditions
(i.e., salinity, 27.5 ppt; pH, 7.8; temperature, 27-29[degrees]C).

Stages                            Diameter          Time

Unfertilized eggs                 180 [micro]m     0 sec
Sperm attached to egg             185 [micro]m   20-30 sec
Sperm acrosome reaction           185 [micro]m     ~1 min
Sperm-egg membrane fusion         180 [micro]m    2-3 min
Appearance of first polar body    185 [micro]m    8-9 min
Appearance of second              185 [micro]m     10 min
  polar body
First cleavage                    200 [micro]m   15-20 min
Second cleavage                   195 [micro]m   35-40 min
Morula                            200 [micro]m   45-55 min
Blastula                          192 [micro]m   75-85 min
Gastrula                          192 [micro]m   85-90 min
Trochophore larvae                210 [micro]m   90-250 min
Early veliger                     225 [micro]m    5-24 h
Late veliger                      228 [micro]m   28-40 h
Early creeping larvae             230 [micro]m   48-72 h
Creeping larvae                   300 [micro]m    3-6 days
Juvenile                            2 [micro]m     30 days
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Article Details
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Author:Suphamungmee, Worawit; Engsusophon, Attakorn; Vanichviriyakit, Rapeepun; Sretarugsa, Prapee; Chavade
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2010
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