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Effects of gamete concentration on the in vitro fertilization of manually extracted gametes of the oyster (Crassostrea rhizophorae)
Aquaculture ELSEVIER
Aquaculture 123 (1994) 153-162
Effects of gamete concentration on the in vitro fertilization of manually extracted gametes of the oyster ( Crassostrea rhizophorae) Joanne N. Rampersada,*,
John B.R. Agardb, David Ammon$
“Trinidad and Tobago Aquaproducts, 99 Western Main Road, St. James, Trinidad and Tobago “Universityof the WestIndies, Department of Zoology, St. Augustine Campus, St. Augustine, Trinidad and Tobago
(Accepted 3 1 January 1994)
Abstract A number of experiments were undertaken to better understand and characterize the effects of different gamete concentrations on the efficiency of in vitro fertilization in Crassostrea rhizophorae. Maximum fertilization efficiency was achieved with a broad range of sperm concentrations, thus minimizing the importance of sperm concentration in successful in vitro fertilizations. However, a linear and inverse relationship was found between egg concentration and fertilization efficiency. This suggests that egg concentration is an important parameter for successful in vitro fertilizations. No evidence was found to support oxygen depletion or the physical crowding of eggs as the cause for the deleterious effect of high egg concentrations. The deleterious effect was counteracted by continually washing the fertilized eggs, suggesting the presence of a water-soluble substance, originating from the zygote, as the cause of the egg effect. In addition, maximizing the total number of viable larvae/ml was found not to be strictly correlated with fertilization efficiency. Lastly, support is presented for the use of concentrations, instead of ratios, in determining optimal gamete numbers.
1. Introduction Obtaining commercial quantities of oyster larvae on demand can be a fi-ustrating experience. Often broodstock refuse to spawn upon inducement, even though the animals contain viable gametes. By circumventing the unpredictable process *Corresponding author. 0044~8486/94/$07.00
0 1994 Elsevier Science B.V. All rights reserved
SSDZOO44-8486(94)00020-O
154
J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
of inducing natural spawns, in vitro fertilization of manually (surgically) removed gametes offers the oyster culturist a relatively simply means of obtaining commercial quantities of larvae on demand. It has been shown, however, that the conditions under which the gametes are mixed can affect the percentages of viable larvae. For example, increasing amounts of sperm used in the fertilization process have been shown to decrease the percentage of viable larvae (Staeger and Horton, 1976; Dupuy et al., 1977; Alliegro and Wright, 1983; DOS Santos and Nascimento, 1985; Stephano and Gould, 1988), and that polyspermy may be the cause (Alliegro and Wright, 1983; Stephano and Gould, 1988). Similarly, for reasons not totally understood, elevated egg concentrations might also be detrimental to proper larval development. For example, in two other bivalve species, high egg concentration is suggested to detrimentally affect fertilization efficiency of spawned eggs (Loosanoff and Davis, 1963; Gruffydd and Beaumont, 1970). It is conceivable that the deleterious effects of elevated egg and sperm concentrations may be due to the manual extraction of eggs from the gonad. Naturally spawned eggs have for example been shown to be less affected by high sperm concentrations (Stephano and Gould, 1988). These data suggest that in order to ensure maximum larval survival, it is the conditions under which the sperm and egg are mixed that is the important step during in vitro fertilization of manually extracted gametes. In this work, the effect of gamete concentrations on the efficiency of in vitro fertilization for Crassustrea rhizophorae, a close relative of C. virginica (Menzel, 1987), was examined. In doing this we present gamete concentrations that can be used as a guide by individuals to optimize fertilization conditions. Additionally we present results from experiments that explore both the cause of the detrimental effect of high egg concentrations and a possible means to counteract it. Lastly we discuss the questionable use of ratios in describing gamete numbers and the need to standardize the in vitro fertilization technique.
2. Methods
Obtaining gametes Adult Crassostrea rhizophorae were collected from pier pilings located in the Gulf of Paria, north of Port-of-Spain, Trinidad. External pressure was applied to the gonad resulting in the liberation of gametes either through the gonadal pore or a small incision made in the gonad. Both sperm and egg were filtered through a 37 pm screen to remove larger debris. Eggs used in fertilization trial 5 were also retained on a 15 pm screen and rinsed with 1 pm filtered water. Gametes used in the 10 trials came from different individuals, except trials 9 and 10 where sperm from the same male was used. The fullness of the gonad, determined by the percentage of the gonad visibly containing gametes, varied among the parental oysters from approximately 100% to less than 50%.
J.N. Rampersadet al. /Aquaculture123 (1994) 153-162
155
Fertilizations
Gametes were used in fertilization trials 1- 10 respectively within the following time values (no more than 5 min elapsed between the first and last container in eachtrial): eggs- 17,21,21,21,22, 17,21, 14, 18, 11 min; sperm-71, 75,70, 61,6 1, 55,60, 58, 58, 74 min. Temperatures during fertilization ranged from 27 to 28°C. Salinity (determined by a hydrometer) was 28 ppt in trials 2, 3, 4, 24 ppt for trial 5, and 2 1 ppt for trials 6-10. A salinity measurement was not taken for trial 1. Fertilizations were performed in plastic containers except for fertilizations 8, 9 and 10 which utilized glass (Pyrex) Petri dishes. Final fertilization volume was 20 ml with sperm solution contributing less than 1 ml; egg solution ranged from 1 to 13.3 ml depending on final egg concentration and the remaining volume was made up with sea water passed through a 1 ,um rated filter bag. Sperm concentrations tested were 50, 500, 1x 103, 5 x 103, 10x 103, 50 x 103, 100x 103, 500x 103, 1 x lo6 sperm/ml and in trials 3, 4, 5, 6, and 7, 250x lo3 sperm/ml was also tested. Egg concentrations in the sperm experiment were held at 35 eggs/ml. Egg concentrations tested were 15, 30, 40, 50, 70, 90, 110, 130 and 200 eggs/ ml, except for trial 10 where 15 eggs/ml was not tested. The sperm concentration in the egg experiments was 10x lo3 sperm/ml at each egg concentration. Controls for sperm contamination of the egg solution were performed in all cases except trial 10 where it was inadvertently lost. Gamete concentrations and all counts were performed in duplicate. Sperm concentrations were determined with a haemocytometer. Concentrations of both eggs and larvae were determined by counting 1 ml aliquots obtained after thorough mixing. Fertilizations were incubated for between 15 and 18 h; aliquots were then removed to test tubes and placed in an ice bath for subsequent counting. Larvae with a straight hinge at the prodissoconch I (D ) stage were scored as viable larvae; all others were scored as non-viable. Percent larvae were determined according to the total number of eggs used. Effect of spatial proximity of eggs
The effect of spatial proximity on fertilization efficiency was examined. Two methods were used to vary the physical contact between eggs. In Group 1, the bottom surface area of the container was varied since eggs are known to settle. In Group 2, vigorous aeration was used to keep the eggs in suspension. These experiments were done with the same batch of eggs and sperm used in trial 5. In all cases 10 x 1O3sperm/ml were used. Fertilizations resulting in no viable larvae were scored as positive for the detrimental egg effect. Fertilizations resulting in viable larvae were scored as negative for the egg effect. Group 1. Varying the container’s bottom surface area. Case 1. The 50 eggs/ml container from trial 5 was used. A bottom surface area
of 13.2 cm2 and a total volume of 20 ml gave a calculated egg density of 76 eggs/ cm2. (This served as a reference for acceptable crowding. ) Case 2. 70 ml of a 35 eggs/ml solution were fertilized in a glass test tube with a
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J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
bottom surface area of approximately 4.9 cm2. Calculated egg density was 500 eggs/cm2. This represented high egg crowding conditions even though the egg concentration of 35 eggs/ml is low. Case 3. 20 ml of a 200 egg/ml solution were fertilized in a glass Petri dish with a bottom surface area of 54 cm2. Calculated egg density was 74 eggs/cm*. (This represented low egg crowding conditions as in Case 1 even though the egg concentration was high. )
Group 2. Discouraging cell-cell contact by aeration Case 1. 70 ml of a 35 eggs/ml solution were vigorously aerated in a glass test tube as described in Case 2 above. (This represented low egg crowding and low egg concentration, thus serving as a control for possible deleterious effects due to vigorous aeration of gametes.) Case 2. 150 ml of a 200 eggs/ml solution were vigorously aerated in a glass bottle. (This represented high egg concentrations and low crowding conditions.)
Cause of egg effect-potential role of water-soluble substances Experiments were performed to look for a substance responsible for the deleterious effect of high egg concentrations which could presumably have been either removed from the gonad with the eggs or emitted from the eggs. The eggs used in trial 5 were washed on a 15 ,um screen to remove any molecular-size substance that may have been carried over with the stripped eggs. Eggs from the same batch used in trial 5 were also placed in a flow-through system consisting of a 15 pm filter (bottom surface area 21.2 cm2, calculated egg density of 991 eggs/cm2) with approximately 70 ml of egg solution at 300 eggs/ml. The filter was placed in a container that had an overflow which maintained a constant water volume of 5 litres, such that the water volume in the filter was approximately 70 ml. Approximately 15 litres of filtered sea water with a sperm concentration of 10x 103 sperm/ml were passed through the filter over a 3 h and 50 min period. Subsequently, the eggs remained in the filter which was left in the container. D-larvae were counted as before.
Data analysis The number of normal D-larvae occurring at the end of each experiment was standardized as a percentage. The arcsine percent transformed data were then subjected to a one-way analysis of variance. For the sperm concentration experiment, highly significant results (PC 0.005) in the ANOVA were further analyzed using the T-method or Tukey Honestly Significant Difference test between means (Sokal and Rohlf, 198 1). For the egg concentration experiments, examination of the data suggested a linear relationship, so that regression analyses were performed and correlation coefficients calculated.
J.N. Rampersadet al. /Aquaculture 123 (1994) 153-162
157
3. Results
Sperm concentration effe The effects of increasing concentrations of sperm on the viability of in vitro fertilized eggs are shown in Table 1. The ANOVA test indicates that mean percent normal D-larvae occurring is significant (PC 0.05) between the different sperm concentration treatments. The Tukey test also suggests an increase in the percentage of viable larvae with increased sperm concentrations until a plateau (no significant difference (P-C 0.05) between means) is reached (Table 1). After this, there is a decline in the percentage of viable larvae. The plateau is evident between 50 x 1O3and 100 x 1O3sperm/ml where in all 10 trials this relatively large increase in sperm concentration did not result in a significant decrease in the percentage of viable larvae. In 9 of the 10 trials a significant decrease in fertilization efficiency is observed at sperm concentrations exceeding 100 x 1O3 sperm/ ml, thus defining the upper boundary of the plateau. The lower boundary to the plateau is not as sharp, but in the majority of trials (8 of 10) falls at either 5 x 1O3 or 10 x 1O3 sperm/ml. Using the 50 x 1O3to 100 x 1O3 region to define the most Table 1 Mean % normal D-larvae obtained 15- 18 h after fertilisation of 35 C. rhizophorue eggsper ml with different concentrations of sperm Mean % normal larvae f standard error Trial
Sperm.ml-’ 50
500 0 0
0
2 3 4 5 6 I 8 9 10
[12+8 0 0 0 0 [14?10
[22+13 [24? 10 0 17*9 [12f8 [19flO 0 0 0
fc
[3?2
9+3
0 0
1000
5000
10 000
50 000
100 000
[22f9 24i- lo] 31fll [30f9 9+ lo] 14f lo] 29+9] 0 0 17f9
38+12] [48+8 482151 45?13] [47rt12 [52fll [601f-6 [37flO [50f15 24+11]
[46?9 66+ 13 [90? 12 [56&14 531r 10 59+11 74+9 49f9 49f 131 42+12
42f9 85+12 90f7 53f8 58f5 63f12 71+11 602 13 [90flO [90+12
40f9] 70+14] 90f 141 625131 50f 171 59?9] 76+10 53f8] 9Ort8] 70fll]
[35f15 [17&9 22f9 29f8 63f9 _
19+31
66&5]
[33+5
[45f3
58f4
70+5
250 000
500 000 0
1 000 000 0
[17f9 22*9] 26+11] 0 0 54f8] 0 33f15 43flO
12?9] 0 0 0 0 0 0 0 0
20f6
lfl]
ANOVA Source of variation
ss
DF
MS
F
P
Between groups Within groups
61 372.8 15 364.61
9 85
6 819.2 180.76
31.73
Values which do not differ significantly (PcO.05) according to the Tukey HSD test are enclosed in brackets. Each value was arcsine transformed before analysis and is presented with ? 1 standard error. ANOVA is between the average mean % normal D-larvae for each sperm concentration.
58
J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
consistent part of the plateau, trials 9 and 10 show the plateau beginning at 50 x 1O3 sperm/ml. Trials 1, 3 and 4 show the plateau beginning between 5 x lo3 and 10 x lo3 sperm/ml. Lastly, trials 2, 5, 6, 7 and 8 show the plateau beginning between 1 x 1O3and 5 x 1O3sperm/ml. concentration effect The relationship between egg concentration and viable larvae is shown in Table 2. ANOVA suggests highly significant (P~O.005) differences between the mean percentages of viable larvae occurring between different egg concentration treatments in each trial. Linear correlation coeffkients between egg concentration and percent viable larvae are also significant (k 0.05) in all 10 trials. This indicates that larval survivability and egg concentration are inversely proportional throughout the range of egg concentrations tested.
Egg
Effect of spatial proximity on fertilization ef$ciency All experiments designed to test physical contact between eggs as the basis for the egg effect proved negative. Viable larvae were found in all containers at low egg concentrations and not in containers at high egg concentrations, irrespective of crowding conditions. Table 2 Mean % normal D-larvae obtained 15-18 h after fertilisation with different concentrations of eggs
of 10 000 C. rhizophorae sperm per ml
Mean O/anormal larvae II standard error Trial
Linear correlation coefficient
Eggsm-’ 15
30
40
50
70
90
110
130
200
1 2 3 4 5 6 7 8 9 10
48kll 70+9 58k8 39+17 60+9 59k14 56k7 27k15 6529
50+11 81+10 85k8 34+15 55T13 81f12 75kll 44&8 51+12 43klO
51+9 62+9 7Ok8 42+12 55?10 68kll 64T12 44+13 57ilO 44+8
49k6 64+10 63k9 38kll 48+13 55*7 61klO 50f7 58+11 35k8
42k9 61+7 591!16 27+12 45k7 50fll 61k14 40f8 47+6 32k8
41klO 53217 58k8 0 37klO 31k9 49f8 29+9 28+8 32k7
36f9 50+8 27k15 0 37+5 26k8 52k7 27+9 2425 17k9
25f8 44+9 0 0 28+6 0 35k7 2424 1619 20f5
35+10 0 0 18f7 0 27?4 0 0 16f7
x
54f5
60+6
56+3
52+3
46k4
36f5
30f5
19zk.5
lOf4
0
-0.96 -0.93 -0.85 -0.84 -0.98 -0.93 -0.89 -0.83 -0.97 -0.89
ANOVA Source of variation
SS
DF
MS
F
P
Between groups Within groups
24 798.68 15 962.26
8 80
3 099.84 199.53
15.536
Each value was arcsine-transformed before analysis and is presented with i 1 standard error. Linear correlation coeffkients are given for each experimental trial. ANOVA is between the average mean % normal D-larvae for each egg concentration.
J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
Cause of egg effect-potential
159
role of water soluble substances
Table 2 shows that no noticeable change in the manifestation of the egg effect was observed in trial 5 even though eggs were washed free of any deleterious molecules that might have been carried over with the eggs during gamete procurement. However, the eggs in the flow-through system showed a substantial difference. An average of 209 D-larvae/ml were recovered. This translates into a fertilization efficiency of about 70%. 4. Discussion The results indicate that a broad range of sperm concentrations will not significantly affect fertilization efficiency. Table 1 shows that too little sperm (e.g. 50 sperm/ml in 8 of 10 trials) probably fails to fertilize all the eggs while too much sperm ( 1 000 000 sperm/ml in 9 of 10 trials) probably results in overfertilization, polyspermy and death (Stephano and Gould, 1988). Between these extremes is the optimal condition of 50x lo3 to 100x lo3 sperm/ml, giving the highest fertilization efficiency in all 10 trials. This broad range of acceptable sperm concentrations suggests that the threat of polyspermy should not be a major concern when performing in vitro fertilizations. This is supported by reports of successful fertilizations in C. virginica and C. gigas by simply guessing the amount of sperm added (see, for example, Dupuy et al., 1977). Unlike sperm, however, small increases in egg concentration were found to influence the percentage of viable larvae. Table 2 indicates that there is a statistically significant (PC 0.05 ) negative linear correlation between egg concentration and larval viability. However, fertilization efficiency was somewhat erratic in the 15-30 egg/ml range, due possibly to stochiastic effects of low egg numbers. Although the results indicate a significant correlation between gamete concentrations and fertilization efficiency, there were small differences in the correlation coefficient between trials. These variations could be in part due to differences in experimental conditions between fertilization trials (e.g. container type, salinity, and temperature). Additionally there was almost certainly non-specific biological variation among the gametes used in the experiments. For example, it is probable that the biological activity of sperm solutions or the percentage of immature eggs from each parental oysters was not consistent. A negative correlation between high egg concentration and fertilization effrciency should not necessarily deter the culturist from using high egg concentrations. For example, in fertilization trial 5 at concentrations between 15 and 90 eggs/ml there is a decrease in the percentage of viable larvae from 60% to 37%. Over this same range 9-33 viable larvae are recovered per ml. The cost and effort of raising larvae to the D-stage are minimal. Therefore, unless fertilization efficiency specifically needs to be optimized, an argument for using egg concentrations resulting in lower percent survivability, but higher numbers of viable larvae/ml, must be considered for aquacultural applications. The detrimental effect of high egg concentrations may have been the result of
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J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
oxygen depletion, a substance from the oyster that was extracted with the eggs, the eggs themselves through cell-to-cell contacts, or even a deleterious substance originating from the eggs. A series of experiments were therefore performed to test these possibilities. Fertilizations performed with high egg concentrations under heavy aeration resulted in no viable larvae, suggesting that oxygen depletion was not the cause. The high level of aeration showed no signs of itself being detrimental since low concentrations of eggs fertilized under similar levels of aeration resulted in viable larvae. No deviation in the deleterious effect of high egg concentrations was seen in fertilization trial 5 where eggs were washed free of any small substances extracted with them. Cell to cell contact was also not found to influence fertilization efficiency. For example, no deleterious effect was seen in cases of high egg crowding at low egg concentration, nor were deleterious effects lessened when eggs at high concentration were not crowded. The relationship between fertilization efficiency and egg concentration was, however, not observed with the flow-through system. Viable larvae were found under conditions of high crowding and high egg concentrations, thus providing evidence in support of a deleterious water-soluble substance being emitted from the fertilized eggs. The inverse linear relationship found between fertilization efficiency and egg concentration in the 10 trials also is consistent with the release of a water-soluble substance from the fertilized eggs. The release of a deleterious substance from fertilized eggs is not novel. For example, hydrogen peroxide is suggested to be released in sea urchin eggs as a defence against polyspermy (Boldt et al., 198 1). Even a limited review of the literature will reveal that there is an almost universal adaptation of using sperm to egg ratios, instead of concentrations, in describing optimal gamete numbers (Stiles and Longwell, 1973; Alliegro and Wright, 1983; DOS Santos and Nascimento, 1985; Ogle and Beaugez, 1988; Stephano and Gould, 1988). However, gamete numbers based on ratios alone cannot possibly define gamete concentrations nor can they describe the proper fertilization conditions for egg and sperm. For example, if a sperm to egg ratio of 30 was used in an infinitely large volume, the chance that any one sperm would find the egg approaches zero. Conversely in a small volume the chance of the same sperm and egg meeting becomes assured. Ratios only have meaning when volume is considered, because it is only in this manner that ratios represent concentrations. But this roundabout way of determining gamete concentrations leads to two specific problems. Firstly, when reporting data in terms of ratios, the reader must back-track through the methodology used and calculate the gamete concentration in order to understand the ratios being presented. Secondly, mental mistakes can be made by taking the indirect route of ratios when determining gamete concentrations. An example of this last point is the work reported by DOS Santos and Nascimento ( 1985 ) . In determining optimal egg numbers for fertilizations neither sperm nor egg concentrations were fixed to a volume but only in relation to each other through a ratio. As a consequence, when the egg number was increased to ascertain its effect, sperm number and therefore the concentration of both gametes
J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
161
also increased. Their egg experiment was thus confounded by two covariant variables. The common use of ratios for in vitro fertilization serves to underscore the questionable in vitro fertilization methods utilized over the years. Additionally, there is a sense of frustration felt by many researchers due to the lack of standardization in performing and reporting on in vitro fertilizations (Lannan, 1980; Stephano and Gould, 1988 ). It is evident from the work presented here, and that by DOS Santos and Nascimento (1985) and Stephano and Gould (1988), that sufficient information is now available on the in vitro fertilization of oyster gametes that proper techniques and their standardization are possible. We therefore propose that the following be reported when the technique of in vitro fertilization is used: ( 1) gamete concentrations instead of ratios, (2) time elapsed between gamete extraction and fertilization, ( 3 ) temperature and ( 4 ) salinity. Acknowledgements We sincerely thank Harry Rampersad, Ester Rampersad, Rushford Seucharan and Chris Coombs, without whose help this work would never have been possible. We would like also to express our eternal indebtedness to Dr. Sammy Ray, whose energy and extraordinary willingness to help has inspired us in so many ways.
References Alliegro, M.C. and Wright, D.A., 1983. Polyspermy inhibition in the oyster, Crassostrea virginica. J. Exp. Zool., 227: 127-137. Boldt, J., Schuel, H., Schuel, R., Dandekar, P.V. and Troll, W., 1981. Reaction of sperm with eggderived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin. Gamete Res., 4: 365-377. DOS Santos, A.E. and Nascimento, I.A., 1985. Influence of gamete density, salinity and temperature on the normal embryonic development of the mangrove oyster Crassostrea rhizophorae Guilding, 1828. Aquaculture, 47: 335-352. Dupuy, J.L., Windsor, N.T. and Sutton, C., 1977. Manual for the design and operation of an oyster seed hatchery. Special Report in Applied Marine Science and Ocean Engineering No. 142, Virginia Institute of Marine Science, Gloucester Point, VA, 23062, 104 pp. Gruffydd, L1.D. and Beaumont, A.R., 1970. Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximus (Mollusca, Lamellibranchia). Helgoland. Wiss. Meeresunters., 20: 486-497. Lannan, J.E., 1980. Broodstock management of Crassostreagigas. I. Genetic and environmental variation in survival in the larval rearing system. Aquaculture, 21: 323-326. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve molluscs. In: F.S. Russel (Editor), Advances in Marine Biology, Vol. I. Academic Press, New York, pp. l-l 36. Menzel, W., 1987. Hybridization of oysters and clams. In: Proceedings, World Symposium on Selec tion, Hybridization, and Genetic Engineering in Aquaculture, Bordeaux, 27-30 May, 1986, Vol. II. Hienemann, Berlin, pp. 48-59. Ogle, J.T. and Beaugez, K., 1988. Oyster hatcheries on the Gulf Coast: history, current technology and future trends. J. Shellfish Res., 7: 505-509.
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123 (1994) 153-162
Sokal, R.R. and Rohlf, F.J., 198 1. Biometry, 2nd edn. W.H. Freeman and Company, New York, pp. 245-241. Staeger, W.H. and Horton, H.F., 1976. Fertilization method quantifying gamete concentrations and maximizing larvae production in Crassostrea gigas. US Natl. Mar. Fish. Serv. Fish Bull., 74: 698701. Stephano, J.L. and Gould, M., 1988. Avoiding polyspermy in the Oyster (Crassostrea gigas). Aquaculture, 73: 295-307. Stiles, S.S. and Longwell, A.C., 1973. Fertilization, meiosis and cleavage in eggs from large mass spawnings of CrassostreavirginicaGmelin, the Commercial American Oyster. Caryologia, 26: 253262.
Aquaculture 123 (1994) 153-162
Effects of gamete concentration on the in vitro fertilization of manually extracted gametes of the oyster ( Crassostrea rhizophorae) Joanne N. Rampersada,*,
John B.R. Agardb, David Ammon$
“Trinidad and Tobago Aquaproducts, 99 Western Main Road, St. James, Trinidad and Tobago “Universityof the WestIndies, Department of Zoology, St. Augustine Campus, St. Augustine, Trinidad and Tobago
(Accepted 3 1 January 1994)
Abstract A number of experiments were undertaken to better understand and characterize the effects of different gamete concentrations on the efficiency of in vitro fertilization in Crassostrea rhizophorae. Maximum fertilization efficiency was achieved with a broad range of sperm concentrations, thus minimizing the importance of sperm concentration in successful in vitro fertilizations. However, a linear and inverse relationship was found between egg concentration and fertilization efficiency. This suggests that egg concentration is an important parameter for successful in vitro fertilizations. No evidence was found to support oxygen depletion or the physical crowding of eggs as the cause for the deleterious effect of high egg concentrations. The deleterious effect was counteracted by continually washing the fertilized eggs, suggesting the presence of a water-soluble substance, originating from the zygote, as the cause of the egg effect. In addition, maximizing the total number of viable larvae/ml was found not to be strictly correlated with fertilization efficiency. Lastly, support is presented for the use of concentrations, instead of ratios, in determining optimal gamete numbers.
1. Introduction Obtaining commercial quantities of oyster larvae on demand can be a fi-ustrating experience. Often broodstock refuse to spawn upon inducement, even though the animals contain viable gametes. By circumventing the unpredictable process *Corresponding author. 0044~8486/94/$07.00
0 1994 Elsevier Science B.V. All rights reserved
SSDZOO44-8486(94)00020-O
154
J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
of inducing natural spawns, in vitro fertilization of manually (surgically) removed gametes offers the oyster culturist a relatively simply means of obtaining commercial quantities of larvae on demand. It has been shown, however, that the conditions under which the gametes are mixed can affect the percentages of viable larvae. For example, increasing amounts of sperm used in the fertilization process have been shown to decrease the percentage of viable larvae (Staeger and Horton, 1976; Dupuy et al., 1977; Alliegro and Wright, 1983; DOS Santos and Nascimento, 1985; Stephano and Gould, 1988), and that polyspermy may be the cause (Alliegro and Wright, 1983; Stephano and Gould, 1988). Similarly, for reasons not totally understood, elevated egg concentrations might also be detrimental to proper larval development. For example, in two other bivalve species, high egg concentration is suggested to detrimentally affect fertilization efficiency of spawned eggs (Loosanoff and Davis, 1963; Gruffydd and Beaumont, 1970). It is conceivable that the deleterious effects of elevated egg and sperm concentrations may be due to the manual extraction of eggs from the gonad. Naturally spawned eggs have for example been shown to be less affected by high sperm concentrations (Stephano and Gould, 1988). These data suggest that in order to ensure maximum larval survival, it is the conditions under which the sperm and egg are mixed that is the important step during in vitro fertilization of manually extracted gametes. In this work, the effect of gamete concentrations on the efficiency of in vitro fertilization for Crassustrea rhizophorae, a close relative of C. virginica (Menzel, 1987), was examined. In doing this we present gamete concentrations that can be used as a guide by individuals to optimize fertilization conditions. Additionally we present results from experiments that explore both the cause of the detrimental effect of high egg concentrations and a possible means to counteract it. Lastly we discuss the questionable use of ratios in describing gamete numbers and the need to standardize the in vitro fertilization technique.
2. Methods
Obtaining gametes Adult Crassostrea rhizophorae were collected from pier pilings located in the Gulf of Paria, north of Port-of-Spain, Trinidad. External pressure was applied to the gonad resulting in the liberation of gametes either through the gonadal pore or a small incision made in the gonad. Both sperm and egg were filtered through a 37 pm screen to remove larger debris. Eggs used in fertilization trial 5 were also retained on a 15 pm screen and rinsed with 1 pm filtered water. Gametes used in the 10 trials came from different individuals, except trials 9 and 10 where sperm from the same male was used. The fullness of the gonad, determined by the percentage of the gonad visibly containing gametes, varied among the parental oysters from approximately 100% to less than 50%.
J.N. Rampersadet al. /Aquaculture123 (1994) 153-162
155
Fertilizations
Gametes were used in fertilization trials 1- 10 respectively within the following time values (no more than 5 min elapsed between the first and last container in eachtrial): eggs- 17,21,21,21,22, 17,21, 14, 18, 11 min; sperm-71, 75,70, 61,6 1, 55,60, 58, 58, 74 min. Temperatures during fertilization ranged from 27 to 28°C. Salinity (determined by a hydrometer) was 28 ppt in trials 2, 3, 4, 24 ppt for trial 5, and 2 1 ppt for trials 6-10. A salinity measurement was not taken for trial 1. Fertilizations were performed in plastic containers except for fertilizations 8, 9 and 10 which utilized glass (Pyrex) Petri dishes. Final fertilization volume was 20 ml with sperm solution contributing less than 1 ml; egg solution ranged from 1 to 13.3 ml depending on final egg concentration and the remaining volume was made up with sea water passed through a 1 ,um rated filter bag. Sperm concentrations tested were 50, 500, 1x 103, 5 x 103, 10x 103, 50 x 103, 100x 103, 500x 103, 1 x lo6 sperm/ml and in trials 3, 4, 5, 6, and 7, 250x lo3 sperm/ml was also tested. Egg concentrations in the sperm experiment were held at 35 eggs/ml. Egg concentrations tested were 15, 30, 40, 50, 70, 90, 110, 130 and 200 eggs/ ml, except for trial 10 where 15 eggs/ml was not tested. The sperm concentration in the egg experiments was 10x lo3 sperm/ml at each egg concentration. Controls for sperm contamination of the egg solution were performed in all cases except trial 10 where it was inadvertently lost. Gamete concentrations and all counts were performed in duplicate. Sperm concentrations were determined with a haemocytometer. Concentrations of both eggs and larvae were determined by counting 1 ml aliquots obtained after thorough mixing. Fertilizations were incubated for between 15 and 18 h; aliquots were then removed to test tubes and placed in an ice bath for subsequent counting. Larvae with a straight hinge at the prodissoconch I (D ) stage were scored as viable larvae; all others were scored as non-viable. Percent larvae were determined according to the total number of eggs used. Effect of spatial proximity of eggs
The effect of spatial proximity on fertilization efficiency was examined. Two methods were used to vary the physical contact between eggs. In Group 1, the bottom surface area of the container was varied since eggs are known to settle. In Group 2, vigorous aeration was used to keep the eggs in suspension. These experiments were done with the same batch of eggs and sperm used in trial 5. In all cases 10 x 1O3sperm/ml were used. Fertilizations resulting in no viable larvae were scored as positive for the detrimental egg effect. Fertilizations resulting in viable larvae were scored as negative for the egg effect. Group 1. Varying the container’s bottom surface area. Case 1. The 50 eggs/ml container from trial 5 was used. A bottom surface area
of 13.2 cm2 and a total volume of 20 ml gave a calculated egg density of 76 eggs/ cm2. (This served as a reference for acceptable crowding. ) Case 2. 70 ml of a 35 eggs/ml solution were fertilized in a glass test tube with a
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bottom surface area of approximately 4.9 cm2. Calculated egg density was 500 eggs/cm2. This represented high egg crowding conditions even though the egg concentration of 35 eggs/ml is low. Case 3. 20 ml of a 200 egg/ml solution were fertilized in a glass Petri dish with a bottom surface area of 54 cm2. Calculated egg density was 74 eggs/cm*. (This represented low egg crowding conditions as in Case 1 even though the egg concentration was high. )
Group 2. Discouraging cell-cell contact by aeration Case 1. 70 ml of a 35 eggs/ml solution were vigorously aerated in a glass test tube as described in Case 2 above. (This represented low egg crowding and low egg concentration, thus serving as a control for possible deleterious effects due to vigorous aeration of gametes.) Case 2. 150 ml of a 200 eggs/ml solution were vigorously aerated in a glass bottle. (This represented high egg concentrations and low crowding conditions.)
Cause of egg effect-potential role of water-soluble substances Experiments were performed to look for a substance responsible for the deleterious effect of high egg concentrations which could presumably have been either removed from the gonad with the eggs or emitted from the eggs. The eggs used in trial 5 were washed on a 15 ,um screen to remove any molecular-size substance that may have been carried over with the stripped eggs. Eggs from the same batch used in trial 5 were also placed in a flow-through system consisting of a 15 pm filter (bottom surface area 21.2 cm2, calculated egg density of 991 eggs/cm2) with approximately 70 ml of egg solution at 300 eggs/ml. The filter was placed in a container that had an overflow which maintained a constant water volume of 5 litres, such that the water volume in the filter was approximately 70 ml. Approximately 15 litres of filtered sea water with a sperm concentration of 10x 103 sperm/ml were passed through the filter over a 3 h and 50 min period. Subsequently, the eggs remained in the filter which was left in the container. D-larvae were counted as before.
Data analysis The number of normal D-larvae occurring at the end of each experiment was standardized as a percentage. The arcsine percent transformed data were then subjected to a one-way analysis of variance. For the sperm concentration experiment, highly significant results (PC 0.005) in the ANOVA were further analyzed using the T-method or Tukey Honestly Significant Difference test between means (Sokal and Rohlf, 198 1). For the egg concentration experiments, examination of the data suggested a linear relationship, so that regression analyses were performed and correlation coefficients calculated.
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157
3. Results
Sperm concentration effe The effects of increasing concentrations of sperm on the viability of in vitro fertilized eggs are shown in Table 1. The ANOVA test indicates that mean percent normal D-larvae occurring is significant (PC 0.05) between the different sperm concentration treatments. The Tukey test also suggests an increase in the percentage of viable larvae with increased sperm concentrations until a plateau (no significant difference (P-C 0.05) between means) is reached (Table 1). After this, there is a decline in the percentage of viable larvae. The plateau is evident between 50 x 1O3and 100 x 1O3sperm/ml where in all 10 trials this relatively large increase in sperm concentration did not result in a significant decrease in the percentage of viable larvae. In 9 of the 10 trials a significant decrease in fertilization efficiency is observed at sperm concentrations exceeding 100 x 1O3 sperm/ ml, thus defining the upper boundary of the plateau. The lower boundary to the plateau is not as sharp, but in the majority of trials (8 of 10) falls at either 5 x 1O3 or 10 x 1O3 sperm/ml. Using the 50 x 1O3to 100 x 1O3 region to define the most Table 1 Mean % normal D-larvae obtained 15- 18 h after fertilisation of 35 C. rhizophorue eggsper ml with different concentrations of sperm Mean % normal larvae f standard error Trial
Sperm.ml-’ 50
500 0 0
0
2 3 4 5 6 I 8 9 10
[12+8 0 0 0 0 [14?10
[22+13 [24? 10 0 17*9 [12f8 [19flO 0 0 0
fc
[3?2
9+3
0 0
1000
5000
10 000
50 000
100 000
[22f9 24i- lo] 31fll [30f9 9+ lo] 14f lo] 29+9] 0 0 17f9
38+12] [48+8 482151 45?13] [47rt12 [52fll [601f-6 [37flO [50f15 24+11]
[46?9 66+ 13 [90? 12 [56&14 531r 10 59+11 74+9 49f9 49f 131 42+12
42f9 85+12 90f7 53f8 58f5 63f12 71+11 602 13 [90flO [90+12
40f9] 70+14] 90f 141 625131 50f 171 59?9] 76+10 53f8] 9Ort8] 70fll]
[35f15 [17&9 22f9 29f8 63f9 _
19+31
66&5]
[33+5
[45f3
58f4
70+5
250 000
500 000 0
1 000 000 0
[17f9 22*9] 26+11] 0 0 54f8] 0 33f15 43flO
12?9] 0 0 0 0 0 0 0 0
20f6
lfl]
ANOVA Source of variation
ss
DF
MS
F
P
Between groups Within groups
61 372.8 15 364.61
9 85
6 819.2 180.76
31.73
Values which do not differ significantly (PcO.05) according to the Tukey HSD test are enclosed in brackets. Each value was arcsine transformed before analysis and is presented with ? 1 standard error. ANOVA is between the average mean % normal D-larvae for each sperm concentration.
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consistent part of the plateau, trials 9 and 10 show the plateau beginning at 50 x 1O3 sperm/ml. Trials 1, 3 and 4 show the plateau beginning between 5 x lo3 and 10 x lo3 sperm/ml. Lastly, trials 2, 5, 6, 7 and 8 show the plateau beginning between 1 x 1O3and 5 x 1O3sperm/ml. concentration effect The relationship between egg concentration and viable larvae is shown in Table 2. ANOVA suggests highly significant (P~O.005) differences between the mean percentages of viable larvae occurring between different egg concentration treatments in each trial. Linear correlation coeffkients between egg concentration and percent viable larvae are also significant (k 0.05) in all 10 trials. This indicates that larval survivability and egg concentration are inversely proportional throughout the range of egg concentrations tested.
Egg
Effect of spatial proximity on fertilization ef$ciency All experiments designed to test physical contact between eggs as the basis for the egg effect proved negative. Viable larvae were found in all containers at low egg concentrations and not in containers at high egg concentrations, irrespective of crowding conditions. Table 2 Mean % normal D-larvae obtained 15-18 h after fertilisation with different concentrations of eggs
of 10 000 C. rhizophorae sperm per ml
Mean O/anormal larvae II standard error Trial
Linear correlation coefficient
Eggsm-’ 15
30
40
50
70
90
110
130
200
1 2 3 4 5 6 7 8 9 10
48kll 70+9 58k8 39+17 60+9 59k14 56k7 27k15 6529
50+11 81+10 85k8 34+15 55T13 81f12 75kll 44&8 51+12 43klO
51+9 62+9 7Ok8 42+12 55?10 68kll 64T12 44+13 57ilO 44+8
49k6 64+10 63k9 38kll 48+13 55*7 61klO 50f7 58+11 35k8
42k9 61+7 591!16 27+12 45k7 50fll 61k14 40f8 47+6 32k8
41klO 53217 58k8 0 37klO 31k9 49f8 29+9 28+8 32k7
36f9 50+8 27k15 0 37+5 26k8 52k7 27+9 2425 17k9
25f8 44+9 0 0 28+6 0 35k7 2424 1619 20f5
35+10 0 0 18f7 0 27?4 0 0 16f7
x
54f5
60+6
56+3
52+3
46k4
36f5
30f5
19zk.5
lOf4
0
-0.96 -0.93 -0.85 -0.84 -0.98 -0.93 -0.89 -0.83 -0.97 -0.89
ANOVA Source of variation
SS
DF
MS
F
P
Between groups Within groups
24 798.68 15 962.26
8 80
3 099.84 199.53
15.536
Each value was arcsine-transformed before analysis and is presented with i 1 standard error. Linear correlation coeffkients are given for each experimental trial. ANOVA is between the average mean % normal D-larvae for each egg concentration.
J.N. Rampersad et al. /Aquaculture 123 (1994) 153-162
Cause of egg effect-potential
159
role of water soluble substances
Table 2 shows that no noticeable change in the manifestation of the egg effect was observed in trial 5 even though eggs were washed free of any deleterious molecules that might have been carried over with the eggs during gamete procurement. However, the eggs in the flow-through system showed a substantial difference. An average of 209 D-larvae/ml were recovered. This translates into a fertilization efficiency of about 70%. 4. Discussion The results indicate that a broad range of sperm concentrations will not significantly affect fertilization efficiency. Table 1 shows that too little sperm (e.g. 50 sperm/ml in 8 of 10 trials) probably fails to fertilize all the eggs while too much sperm ( 1 000 000 sperm/ml in 9 of 10 trials) probably results in overfertilization, polyspermy and death (Stephano and Gould, 1988). Between these extremes is the optimal condition of 50x lo3 to 100x lo3 sperm/ml, giving the highest fertilization efficiency in all 10 trials. This broad range of acceptable sperm concentrations suggests that the threat of polyspermy should not be a major concern when performing in vitro fertilizations. This is supported by reports of successful fertilizations in C. virginica and C. gigas by simply guessing the amount of sperm added (see, for example, Dupuy et al., 1977). Unlike sperm, however, small increases in egg concentration were found to influence the percentage of viable larvae. Table 2 indicates that there is a statistically significant (PC 0.05 ) negative linear correlation between egg concentration and larval viability. However, fertilization efficiency was somewhat erratic in the 15-30 egg/ml range, due possibly to stochiastic effects of low egg numbers. Although the results indicate a significant correlation between gamete concentrations and fertilization efficiency, there were small differences in the correlation coefficient between trials. These variations could be in part due to differences in experimental conditions between fertilization trials (e.g. container type, salinity, and temperature). Additionally there was almost certainly non-specific biological variation among the gametes used in the experiments. For example, it is probable that the biological activity of sperm solutions or the percentage of immature eggs from each parental oysters was not consistent. A negative correlation between high egg concentration and fertilization effrciency should not necessarily deter the culturist from using high egg concentrations. For example, in fertilization trial 5 at concentrations between 15 and 90 eggs/ml there is a decrease in the percentage of viable larvae from 60% to 37%. Over this same range 9-33 viable larvae are recovered per ml. The cost and effort of raising larvae to the D-stage are minimal. Therefore, unless fertilization efficiency specifically needs to be optimized, an argument for using egg concentrations resulting in lower percent survivability, but higher numbers of viable larvae/ml, must be considered for aquacultural applications. The detrimental effect of high egg concentrations may have been the result of
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oxygen depletion, a substance from the oyster that was extracted with the eggs, the eggs themselves through cell-to-cell contacts, or even a deleterious substance originating from the eggs. A series of experiments were therefore performed to test these possibilities. Fertilizations performed with high egg concentrations under heavy aeration resulted in no viable larvae, suggesting that oxygen depletion was not the cause. The high level of aeration showed no signs of itself being detrimental since low concentrations of eggs fertilized under similar levels of aeration resulted in viable larvae. No deviation in the deleterious effect of high egg concentrations was seen in fertilization trial 5 where eggs were washed free of any small substances extracted with them. Cell to cell contact was also not found to influence fertilization efficiency. For example, no deleterious effect was seen in cases of high egg crowding at low egg concentration, nor were deleterious effects lessened when eggs at high concentration were not crowded. The relationship between fertilization efficiency and egg concentration was, however, not observed with the flow-through system. Viable larvae were found under conditions of high crowding and high egg concentrations, thus providing evidence in support of a deleterious water-soluble substance being emitted from the fertilized eggs. The inverse linear relationship found between fertilization efficiency and egg concentration in the 10 trials also is consistent with the release of a water-soluble substance from the fertilized eggs. The release of a deleterious substance from fertilized eggs is not novel. For example, hydrogen peroxide is suggested to be released in sea urchin eggs as a defence against polyspermy (Boldt et al., 198 1). Even a limited review of the literature will reveal that there is an almost universal adaptation of using sperm to egg ratios, instead of concentrations, in describing optimal gamete numbers (Stiles and Longwell, 1973; Alliegro and Wright, 1983; DOS Santos and Nascimento, 1985; Ogle and Beaugez, 1988; Stephano and Gould, 1988). However, gamete numbers based on ratios alone cannot possibly define gamete concentrations nor can they describe the proper fertilization conditions for egg and sperm. For example, if a sperm to egg ratio of 30 was used in an infinitely large volume, the chance that any one sperm would find the egg approaches zero. Conversely in a small volume the chance of the same sperm and egg meeting becomes assured. Ratios only have meaning when volume is considered, because it is only in this manner that ratios represent concentrations. But this roundabout way of determining gamete concentrations leads to two specific problems. Firstly, when reporting data in terms of ratios, the reader must back-track through the methodology used and calculate the gamete concentration in order to understand the ratios being presented. Secondly, mental mistakes can be made by taking the indirect route of ratios when determining gamete concentrations. An example of this last point is the work reported by DOS Santos and Nascimento ( 1985 ) . In determining optimal egg numbers for fertilizations neither sperm nor egg concentrations were fixed to a volume but only in relation to each other through a ratio. As a consequence, when the egg number was increased to ascertain its effect, sperm number and therefore the concentration of both gametes
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also increased. Their egg experiment was thus confounded by two covariant variables. The common use of ratios for in vitro fertilization serves to underscore the questionable in vitro fertilization methods utilized over the years. Additionally, there is a sense of frustration felt by many researchers due to the lack of standardization in performing and reporting on in vitro fertilizations (Lannan, 1980; Stephano and Gould, 1988 ). It is evident from the work presented here, and that by DOS Santos and Nascimento (1985) and Stephano and Gould (1988), that sufficient information is now available on the in vitro fertilization of oyster gametes that proper techniques and their standardization are possible. We therefore propose that the following be reported when the technique of in vitro fertilization is used: ( 1) gamete concentrations instead of ratios, (2) time elapsed between gamete extraction and fertilization, ( 3 ) temperature and ( 4 ) salinity. Acknowledgements We sincerely thank Harry Rampersad, Ester Rampersad, Rushford Seucharan and Chris Coombs, without whose help this work would never have been possible. We would like also to express our eternal indebtedness to Dr. Sammy Ray, whose energy and extraordinary willingness to help has inspired us in so many ways.
References Alliegro, M.C. and Wright, D.A., 1983. Polyspermy inhibition in the oyster, Crassostrea virginica. J. Exp. Zool., 227: 127-137. Boldt, J., Schuel, H., Schuel, R., Dandekar, P.V. and Troll, W., 1981. Reaction of sperm with eggderived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin. Gamete Res., 4: 365-377. DOS Santos, A.E. and Nascimento, I.A., 1985. Influence of gamete density, salinity and temperature on the normal embryonic development of the mangrove oyster Crassostrea rhizophorae Guilding, 1828. Aquaculture, 47: 335-352. Dupuy, J.L., Windsor, N.T. and Sutton, C., 1977. Manual for the design and operation of an oyster seed hatchery. Special Report in Applied Marine Science and Ocean Engineering No. 142, Virginia Institute of Marine Science, Gloucester Point, VA, 23062, 104 pp. Gruffydd, L1.D. and Beaumont, A.R., 1970. Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximus (Mollusca, Lamellibranchia). Helgoland. Wiss. Meeresunters., 20: 486-497. Lannan, J.E., 1980. Broodstock management of Crassostreagigas. I. Genetic and environmental variation in survival in the larval rearing system. Aquaculture, 21: 323-326. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve molluscs. In: F.S. Russel (Editor), Advances in Marine Biology, Vol. I. Academic Press, New York, pp. l-l 36. Menzel, W., 1987. Hybridization of oysters and clams. In: Proceedings, World Symposium on Selec tion, Hybridization, and Genetic Engineering in Aquaculture, Bordeaux, 27-30 May, 1986, Vol. II. Hienemann, Berlin, pp. 48-59. Ogle, J.T. and Beaugez, K., 1988. Oyster hatcheries on the Gulf Coast: history, current technology and future trends. J. Shellfish Res., 7: 505-509.
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Sokal, R.R. and Rohlf, F.J., 198 1. Biometry, 2nd edn. W.H. Freeman and Company, New York, pp. 245-241. Staeger, W.H. and Horton, H.F., 1976. Fertilization method quantifying gamete concentrations and maximizing larvae production in Crassostrea gigas. US Natl. Mar. Fish. Serv. Fish Bull., 74: 698701. Stephano, J.L. and Gould, M., 1988. Avoiding polyspermy in the Oyster (Crassostrea gigas). Aquaculture, 73: 295-307. Stiles, S.S. and Longwell, A.C., 1973. Fertilization, meiosis and cleavage in eggs from large mass spawnings of CrassostreavirginicaGmelin, the Commercial American Oyster. Caryologia, 26: 253262.