INTRASPECIFIC ARTIFICIAL INSEMINATION

Transcription

1 INTRASPECIFIC ARTIFICIAL INSEMINATION

2 7.1 Introduction Hatchery people in breeding shnmps under captivity encounter many problems. Mating failure is the foremost among them (Primavera, 1984). Mating requires a minimum water volume and depth; the success and frequency of mating are limited in small tanks for Penaeus monodon (Prirnavera, 1979; Poernomo and Hamami, 1983). Besides, spermatophore could be more easily dislodged from open thelycum sbps even after successful mating. Low frequency of mating is also frequently observed in both open and closed thelycum species due to the presence of diseased or impotent males (Aiken et al., 1984). Artificial insemination offers solution to these problems. Moreover, artificial insemination is the first step towards hybridization aimed at improving the genetic makeup of shrimps. Experiments related to artificial insemination have been carried out in

3 prawns particularly on Macrobrachiurn rosenbergii (eg. Chow, 1982; Chow et a1,1985 ). Limited works related to artificial insemination in penaeid shrimps (e.g Muthu and Laxminarayana, 1984; Lin and Ting, 1986) prompted to carry out studies on commercially important species, Penaeus monodon and P.remlstrlcattr.~. 7.2 Materials and methods Matured males and females of Pmonodon and Psemisulcatus were collected fiom trawlers and transported to the laboratory in oxygenated polythene bags. Male and female shrimps were separately stocked in large plastic pools containing filtered seawater for acclimation. The temperature, DO, salinity, and ph of seawater averaged to 30.0 f 1.5OC, ml O2 I', %O and 8 1 i 1.9 respectively. Fresh or frozen squid and clam ~neal was provided ad lihrtum twice a day for the animals. Fifty percent of the tank water was changed daily removing sediments. After thorough acclimation, female shnmps were subjected to eyestalk ablation for ovary maturation (Wear, 1975; Wear and Santiago, 1976). For detailed procedure please refer chapter 6. Ablated females were kept without males in the maturation tank (1 mt capacity). About a week after eyestalk ablation, ablated females started molting. Newly molted females were chosen for artificial insemination studies, because the thelycum was soft and favourable for manual transfer of spermatophore

4 (LaubierBonichon and Ponticelli, 1981; Lumare, 1981). Artificial insemination was carried out within hours of molting. Molted females were divided into two groups containing 10 and 20 female shrimps. One group was transplanted with a single spermatophore and the other with a pair of spermatophores. The transplantation operation was completed quickly withm a couple of minutes with the assistance of colleagues. Experimental animal was captured fiom the tank and gently held upside down in a soft towel. Then with the help of nice blunt forceps, the thelycum was gently opened. Then immediately with the help of another forceps a single or a pair of spermatophore was introduced into the seminal receptacle without damaging the thelycurn. Sometimes there was a possibility of rejection of transplanted spermatophore. In order to prevent the loss of spermatophore, the thelycum was temporarily glued by a synthetic adhesive Pcynoacrylate (Chow, 1982). Inseminated females were then kept in a basin with seawater, which was continuously aerated. After recovery from the transplantation shock, experimental animals were returned to the maturation tank. Few cases of mortality were observed. Dead animals were removed and recorded. Percent survival was calculated. Inseminated animals in the maturation tank were continuously observed for maturation of ovary and spawning. Parallel to the experimental group, a control group was maintained. Ablated females without artificial insemination were left along with the

5 males in the ratio of 1: 1 in large sized separate tank for natural mating and transfer of spermatophore by males. They were continuously monitored. Once mating occurred, females with spermatophore were transferred to the maturation tank and observed for spawning. Spawning was identified by aliquot samples of eggs with scum present in the spawning tank. In order to stabilize the water ph and reduce the heavy metallic ions, EDTA (10 ppm), Treflan (O.lppm), Sodium carbonate (10 mg I 200 1) and pinch of Tris buffer were added to the water in the spawning tank (Licop, 1988). Individuals of Prnonodon and Psemistllcatus were separately observed for spawning. Once spawning symptom was noticed, particular individual was separated and allowed to spawn in spawning tank. The number of eggs spawned was counted by talung 100 ml of aliquot sample from the spawning tank. Fertilization was observed under the microscope. Hatching occurred hours after fertilization. The nauplii were harvested in 5 1 bucket utilizing the phototrophc property of nauplii and. then the number of nauplii was calculated. Unfertilized eggs and hatched cases of eggs were found settled at the bottom of the tank. Hatch percent was calculated using the following formula given by Lim et al. (1987). Hatch percent = N /N+E X 100 where N = total number of nauplii E = total number of unhatched eggs

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7 I spawning I1 spawning 111 spawning Fig 7.1: Number of eggs spawned in natural spawning under captivity in P. monodon.... pp El Single spermatophore 1 I3 Pair of spermatophore I I spawning il spawning I Fig 7 2: Effect of single or pair of spermatophore transplantation on spawning in P.monodon

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9 1 601 GI Control 61 Single spermatophore I3 Pair of spermatophore I spawning I1 spawning I11 spawning Fig 7 3 : Percentage of hatching in control, single or double spermatophore transplantation in P. monodon. I spawning I1 spawning I11 spawning Fig 7.4: Number of eggs spawned in natural spawning under captivity in P. semi.sulcat24s

10 7.3 Results Artificial insemination in Penaeus monodon Table7.1 presents data on natural spawning of eye ablated female Penaeus monodon without artificial insemination. Twentyfive shrimps were used for the experiment whose body weight and length averaged to 101 g and 20 cm respectively. Within a period of 2ldays, a shnmp totally produced 66 spawns in three subsequent spawning; the intermolt period increased in subsequent spawning. The average percent fertility of spawn was 70. The total number of eggs produced in three spawning was eggs gving an average of eggs (Fig.7.1). Out of three spawnings, maximum number of eggs (about 50%) was produced in the fust spawning and it gradually decreased in subsequent spawnings. The hatch percent averaged to 46 producing a total number of nauplii. There was not much difference in hatch percent between subsequent spawning. For artificial insemination experiments, 20 and 10 shrimps were used for double and single spermatophore insemination. The survival averaged to 75%(Table7.2).Doubleor single spermatophore transferred shrimps spawned twice or once producing a total of eggs in 22 spawns or eggs in 7spawns respectively (Table7.2)(Fig.7.2). There was no difference in the intermolt period and it averaged to about 10 days in both cases. The nauplii obtained averaged to 19371or 6962 with the hatch percent of 6.5 or 5.4 in double or single spermatophore transferred group respectively (Fig.7.3).

11 7.3.2 Artificial insemination in Penaeus semisulcatus Twenty five Penaeus semisulcatus females were used for natural mating and spawning without artificial insemination. The average body weight and length of shrimps was 29 g and 14 cm respectively. Percent survival was 88 (Table7.3). Psemwulcatus totally. produced eggs in three subsequent spawning in a total number of 66 spawns (Table 7.3) (Fig.7.4). The average fertility was 62%. The fertility gradually decreased in subsequent spawning. Control l?semisulcatus produced a total of nauplii with an average hatch percent of 37. Totally 30 eye ablated Psemzsulcatus females were used for artificial insemination. The percent survival was 70 or 55 in single or double spennatophore implanted animals. Double or single spermatophore implanted animals spawned twice producing a total of or eggs in total spawn of 19 or 7 respectively (Table 7.4)(Fig.7.5). The total nauplii obtained averaged to or 3561 in double or single spennatophore transferred groups respectively. The hatch percent averaged to 5.0 or 4.0 respectively (Table 7.4)(Fig. 7.6). 7.4 DISCUSSION Out of 60 trials, it has been demonstrated that artificial insemination could be successfully adopted in I? monodon and Psemisulcatus. On an average, per female nauplii of Pmonodon and nauplii of

12 Table. 7.3: Details of molting, natural spawning and hatching under captivity in ablated female P.semisitlcutus, Each value is the average (Mean fsd) of 25 observations.for basic data please refer appendix table A * f f f * 5.6 Parameters No of animals treated No of animals dead Percent survival Interrnoult period (days) I 7 Number of spawning I Total Average 7.1 No.of spawns No. spawns hatched Percent fertility No. of eggs f f f f No of fertilized eggs Percent fertilized eggs * * f f * * * No. of nauplii Percent hatching f k f * f * f 87897

13 El Slngle spermatophore 1 3 Pair of spermatophore I spawning IIspawning Fig 75: Effect of single or pair of sperrnatophore transplantation on spawning in P..semi.sulcatus El Control H Single spermatophore III Pair of spermatophore I1 spawning I11 spawning Fig 7.6: Percent hatch in control, single or double spermatophore transplantation in P.semisulcatzts. L

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15 Psemisulcatus were produced in double spermatophore artificial insemination. Many previous workers have tried artificial insemination in fresh water prawns and penaeid shnmps; their works were limited to few trials from which one could not arrive at a conclusion. Artificial insemination was successfully tried in Macrobrachizrn? rosenbergii, but the hatchng success and its practical applications were not fully discussed (Sandifer and Smith, 1979; Sandifer and Lynn, 1980). Chow (1982) and Chow et al. (1985) carried out artificial insemination experiments in Macrobrachium rosenbergii. Ths techtuque was adopted by different researchers in penaeid prawns (LaubierBonichon and Ponticelli, 1981; Lurnare, 1981; Muthu and Laxminaryana, 1984). Artificial insemination using cryopreserved spermatophore had been undertaken in American lobsters, Homarus americanus (KoodaCisco and Talbot, 1983). The success rate by the assessment of nauplii survival in all the above cited works varied widely depending upon the experimental conditions and it points to the need for perfecting the technique before adopting in hatcheries. The survival rate after artificial insemination in Penaeus monodon and l? semisulcatus was over 70% and there was no significant difference in percent survival between shrimps implanted with double or single spermatophore. Ablated control Pmonodon and P.sem~sulcaius without artificial insemination recorded 88% survival and it indicates that survival was not remarkably affected by artificial insemination.

16 One of the interesting observations made in the present experiment was cent percent fertility of spawns shown by both Prnonodon and Psem~sulcatzrs; they when implanted with double or single spermatophore produced 22 or 7 and 19 or 7 viable spawns respectively. On the otherhand, f monodon or Flsemlsulcatz~s without artificial insemination produced 46 or 41 viable spawns out of 66 total number of spawns. Artificial insemination perhaps restricts the total number of spawns produced but enhanced the fertility by producing only viable spawns.. The hatch rate recorded in the present study eventhough was poor, could be comparable with the data of previous workers. P monodon and 19 semlszrlcatus implanted with double or single spermatophore have exhibited 6.5 or 5.4 and 5.0 or 4.0 hatch percent respectively. Muthu and Laxminarayana (1984) reported a low percent hatchability of 2.4 from only one out of 10 artificially inseminated female P monodon. Lumare (1981) obtained a very poor result of 3.3% hatch rate in female I? japonrcus. From the artificial insemination experiment on I? rnonodon, Lin and Ting (1986) reported a range of 9.4 to 16.8 % hatch rates when transplanted with a single spermatophore. LaubierBonichon and Ponticelli (1981) on the other hand, claimed 80% and more fertilization rate for P japonicus; however, the number of animals used for the experiment was only 5 out of which 4 animals retained the spermatophore and one hatched. Eventhough the low hatch percent is a common occurrence in artificial insemination of penaeid

17 shrimps, it is a major point of concern for scientists. Nonviable eggs are frequently produced even by the naturally impregnated spawners collected from the sea (see also Muthu and Laxminarayana, 1984). The hatch percent appears to be improved in double spermatophore implanted animals compared to single spermatophore transferred animds; hatch percent of P monodon and P semisulcatz~s implanted with double or single spermatophore averaged to 6.5 or 5.4 and 5.0 or 4.0 respectively. The hatch rate of females implanted with a pair of spermatophore averaged to 82.3 and 39~ 1% for the first and the subsequent spawning respectively, while from those implanted with only one spermatophore the hatch rate of 71.9 and 13.2 % were obtained (Lin and Ting, 1986). It is advisable to implant two spermatophores during artificial insemination in penaeid shnmps as occurs in natural mating to improve fertility and hatch rate. Handling stress is a major problem, which is likely to reduce success rate in artificial insemination. Handling stress may reduce not only the survival but also affect the spermatophore retention and hatch rate of nauplii. To reduce the handling stress in the present study, the females were anesthetizec1 by lowering the temperature to 18OC for 15 minutes (see also LaubierBonichon and Ponticelli, 198 1). Tave and Brown (1981) used a gill irrigator to minimize the stress to females.

18 The gill irrigator ensured continuous supply of water to the branchal cavity of female. Using gill irrigator and restrain device during the spennatophore transfer, 88% of the females spawned and finally released healthy larvae (Tave and Brown, 1981). Artificial insemination without any device produced only limited success (Lumare, 1981).