efficacy of lactobacillus acidophilus on the sperm quality...

International Journal of Chemical and Analytical Science ISSN: 0976-1206

Research Article

www.ijcas.info

Corresponding Author: Somnath Chakraborty, Faculty of Marine Sciences, Centre of Advanced Study in Marine Biology, Annamalai University,

Parangipettai – 608502, Tamil Nadu, India Received 22-11-2010; Accepted 21-07-2011

September, 2011 International Journal of Chemical and Analytical Science, 2011, 2(9), 1178-1185 1178

Efficacy of Lactobacillus acidophilus on the sperm quality and regeneration of spermatophores in Penaeus monodon (Fabricius, 1798)

MA Badhul Haq, R Manimurugan, AR Nazar, R Shalini, Upasana Ghosh, Somnath Chakraborty* Faculty of Marine Sciences, Center of Advanced Study in Marine Biology, Annamalai University, Parangipettai – 608 502, Tamil Nadu, India

INTRODUCTION

The annual global production of farmed shrimp has grown at

the rate of 8% since 1989. In 1998, the production reac hed

1,113,887 mt, representing around 32% of world shrimp and

prawn catches [1]. T he industry has expanded mostly through

Southeast Asia and Latin America, where diverse penaeid

species are cultured. The most important species in Latin America is Penaeus (Litopenaeus) vannamei, followed by P.

(Litopenaeus) stylirostris and P. (Litopenaeus) schmitii. In Asia P. monodon, P. chinesis, P. (Marsupenaeus) japonicus, P. pencillatus,

P. merguiensis, and P. indicus are cultured [2]. The traditional

dependency on wild stock for naupii production will thus

change towards a more controlled production of genetically

improved animals, selected for impo rtant aquaculture traits

like growth rate and dis ease resistance [3]. However, t he

genetic improvement of shrimp requires the application of

breeding programs, which relay to excellent gamete quality,

controlled fertilization, and, if possible, cryogenic storage. For

this a proper understanding of basic shrimp reproductive

biology, and the developm ent of in vitro fertilization

techniques and cryogenic p rotocols for gametes, embryos,

and larvae are needed.

Female maturation is achieved by u nilateral eyestalk ablation.

This technique is applied to each female at intermolt, selecting

one of t he eyestalks. Few days after the p rocedu re, ovaries

will start to grow and first spawning will occu rs within a

week, with an increasing rate in t he subsequent days. Each

female generates various spawns, but eventually t he

spawning activity decreases, until around 2 to 3 months after

ablation, when t hey are rep laced by new females.

Eyestalk ablation affects all aspects of shrimp physiology [4]

and over time it has a deleterious effect on spawn quality and

quantity, as well as on survival [5, 6, 7]. This dependency on

eyestalk ablation is a major bottleneck for the adva nce of

controlled reproduction, and major research activities are

dedicated to develop hormonal treatments for controlled maturation. The reproductive cycle of the genus Penaeus,

consisting of two phases was desc ribed [8], as in the first

phase, pre-adults migrate from estuarine waters to a

reproduction zone in deeper a nd higher saline waters. After

spawning occurs, the postlarvae migrate to estuarine waters. This pattern is fol lowed by various species: P. duorarum, P. brasiliensis, P. aztecus, P. schmitii, P. stylirostris, P. vannamei, P.

setiferus and P. occidentalis [9 ]. The quality of wild

spermatophores during natural mating has been partially

documented. This type of analysis has been reported for

males under cu lture conditions (controlled reproduction) and wild P. setiferus broodstock [10, 1 1, 12].

The evaluations of reproductive quality in male penaeid

shrimp was first repo rted by resea rchers [13] who measured

spermatophores weight, sperm count, and p ercentages of live

and abnormal sperm for evaluating the effect of eyestalk ablation in P. vannamei. Using the same parameters, pioneer

researchers [10 ] studied a decline of sperm quality in P. setiferus

held in captivity a nd others [14] used t he same approach to evaluate the effect of water t emperature, EDTA, and Vibrio

bacterin o n sperm quality in captive P. setiferus. The effects of

dietary rancidity and Vitamin E on gonadal maturation of P.

setiferus were assessed [11].

In the present research, Penaeus monodon broodstock was collected f rom a Shrimp hatchery. After their acclimatization healthy

male P. monodon were selected based on size, general appearance, p resence of spermatophores from the culture tanks for the

probiotic test. Shrimp brooders were disinfected with formalin and maintained in the tanks for 3 days before beginning

experiments. All experimental animals were tra nsferred into epoxy coated rectangula r tank along with the control tank

maintained at identical relevant stocking density after treating t hem with intramuscular injection, containing Lactobacillus

acidophilus in different quantities (0.5, 1.0, 1.5 and 2.0 ml containing 3 × 109 cells/ml) into lateral su rface of the p receding segment of male brooder. Gonopores of male P. monodon were washed with sterile seawater and 70% ethanol prior to

collection of spermatophores. Manual ejaculation of spermatophores was carried out using elect ric stimulus on all males. Two

spermatophores were obtained from each male that were weighed and the sperm quality was examined morphologically

using light microscope. The length a nd weight of the shrimps were measured after subsequent ejaculation. Percentage of

viable sperm was calculated in triplicate from counting of a minimum number of 250 sperm cells under 1000-fold

magnification on each slide. Rep roductive routine was evaluated in the experimental animals inj ected on t he zeroth day of

initiation of the experiment with 0.5, 1.0, 1.5 and 2.0µl/g body weights of shrimp. During the four different treatments in 1.0µl

injected males performed differently. In the I extrusion, mean sperm counts in GM ranged from left vs right spermatophore

was 0.842, 0.912, 0.940 and 0.972; in II extrusion 0.521, 0.422, 0.988 and 0.591, in III extrusion 0.711,0.451, 0.910 and 0.740 and in

the IV extrusion 0.421, 0.422, 0.945 and 0.431 in the successive intervals respectively. Higher counts were recorded in the right halve of spermatophore of shrimps treated with 0.1µl. Among all the L. acidophilus treated broodstock shrimps, only 0.1µl/g

of body weight performed better after 7 days of administration. Therefore, this research depicts that the application of L.

acidophilus enha nces sperm quality and regenerates the spermatophores in male P. monodon brooders.

Key words – Penaeus Monodon, Spermatophores, Probiotics, Lactobacillus Acidophilus, Gonopores, Electric Stimulus

M. A. Badhul Haq, et al.: Efficacy of Lactobacillus acidophilus on the sperm quality and regeneration of spermatophores in Penaeus monodon

(Fabricius, 1798)


Researchers [10] evaluated spermatophores redevelopment time in wild P. stylirostris, P. vannamei, P. setiferus; and also

studied the effect of ascorbic acid on reproductive quality in P. vannamei using the previous mentioned quality indicato rs.

Eminent workers measured spermatophores weight, sperm

count, and percentage of abnormalities for evaluating

nutritional variations on t he onset of the Male Reproductive Tract Degenerative Syndrome (MRTDS) of P. setiferus [15].

Spermatophores of p enaeid s hrimp may vary considerably in

quality and have been evaluated in terms of spermatophores

weight, sperm count, and t he percentage incidence of

abnormalities [11, 13]. Recently the activation of an acrosome

reaction by the egg - water technique was developed [16] and

has been evaluated as a more accurate measure of sperm

quality [17, 18 ]. A new approach of inducing gonadal

maturation and spawning was defined [19]. This approach

relies in the roles t hat neurotransmitters play in regulating

gonadal maturation. Workers [20] established that 5-

hydroxtryptamine (5-HT, serotonin) induces ovarian maturation invivo and invitro, in Procambarus clarkia, by

acting to stimulate release of a gonad-stimulating hormone

(GSH) from t he brain and thoracic ga nglia; GSH is an abstract

entity, which has not been identified nor measured nor measured directly. The effect of Bacillus spp. used as

probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus was proved [21]. Microbial flora of spermatophores from black tiger s hrimp

(Penaeus monodon) declines over long-term cryostorage was

also showed [22].

Serotonin also stimulates the release of other neurohormones,

including the crustacean hyp erglycaemic hormone (CHH),

red pigment-dispers ing hormone (RPDH), neurodepressing

hormone (NDH) and molt-inhibiting hormone (MIH) [20].

Methyl farnesoate (MF) is a factors that stimulates gonadal

maturation [23] and it is inhibited by 5-HT; on the other hand,

RPCH stimulates MF synthesis by the mandibular organ;

other roles played by 5- HT are migration of the proximal

retinal pigment, pericardial organ neurohormone,

stomatogastric ganglion neuromodu lator or nerohormone,

behavioural responses, osmoregulation, and

mechanoreception [19] as a effort to develop alternatives for

the comm ercial rep roduction of penaeid shrimp, this study

was conducted to evaluate the effect of two doses of 5-HT on P. vannamei maturation and spawning quality, compared to

unilateral eyestalk ablation.

The aim of the present investigation is to assess the effect of Lactobacillus acidophilus (different quantities) on the sperm

quality, and there was four different extrusions were

performed in 7 days intervals. Besides quality and quantity

present between the extrusions, an emphasis is also paid to

evaluate the regeneration interval of spermatophores.

OBJECTIVES 1. To study the efficacy of probiotics Lactobacillus acidophilus

on broodstock Penaeus monodon.

2. To find out the effectiveness of sp erm quality and regeneration using probiotics bacteria L. acidophilus in

different level dosage.

3. To determine the probiotics profiles in the different level of

ejaculations.

MATERIAL AND METHODS Penaeus monodon broodstock was collected from a Shrimp

hatchery collection centre in Marakkanam, Pondicherry. The

present study was carried out at the Faculty of Marine Sc ience

laboratory, CAS in Marine Biology, Annamalai University,

Tamil Nadu, India. The collection area is depicted in Fig.1. Experimental design

Fig. 1: Location of the shrimp hatchery in Pondicherry

After acclimatization at the laboratory for 5 days, uniform

size brooders were selected for probiotic testing (F ig. 2).

Fig. 2: P. monodon broodstock in experimental tank

Healthy male P. monodon were selected based on size, general

appearance, presence of spermatophores from the culture

tanks with the ABW of 65 – 80 g weight were stocked at the

density of 10 shrimps per tank (Fig. 3).


(Fabricius, 1798)


Fig. 3: View of length and weight measurement of P. monodon

Before stocking, the shrimps were disinfected with 20 ppm

formalin for 20 minutes. Initially the animals were stocked in

three different one tonned capacity annular synthetic tanks.

Shrimp were maintained in the tanks for 3 days before

beginning experiments. 100% water tune was done daily and

initial stocking of 40 brooders. After intramuscular injection,

all experimental animals were t ransferred in to 25 l capacity

epoxy coated rectangular tank (Fig. 4). Control tank were

maintained at identical relevant stocking density. Three

replicates were used for a total of 120 broodstock per

treatment.

Fig. 4: The view of experimental set up

Probiotic (Lactobacillus acidophilus) challenge test

Bacterial inoculums: L. acidophilus probiotic inocu lum was

prepared for int ramuscular injection in different dosage to the experimental animal P. monodon broodstock.

Microorganism: Among commonly used p robiotic organisms

in endothermic animals, only few can be compared to the

wealth of information available on probiotic bacteria; most

studies demonstrate the use of the same in aquaculture.

Consequently for t he p resent study, one benefic ial probionts

was commonly encountered as Lactobacillus acidophilus

strain (Fig. 5), that was obtained from the Marine

Microbiology Laboratory, Faculty of Marine Science, where it

was used as the probiotic organism. The authenticity of the

culture was verified by sub culturing and identification based

on t heir morphological and biochemical characteristics

(Bergey’s Manual of Systematic Bacteriology).

Fig. 5: Slant culture of Lactobacillus acidophilus

Production of bacterial biomass

L. acidophilus was grown aseptically in 10 ml of nutrient broth

for 24 hr at room temperature (28 ± 2 °C). 5 ml of log phase

culture was then transferred under aseptic conditions into 250

ml of MRS broth and p laced on a rotary shaker at 150 rpm for 24 hr at 28 ± 2°C. The cells of L. acidophilus strain were

harvested separately by cent rifuging at 10, 000 rpm under

sterile conditions for 15 min at 4°C. The liquid supernatant

was then discarded, and the pellet was suspended in sterile

saline solution. This process was rep eated again, and the cell

concentration in t he suspension was adjusted to an optical

density of 1.00 at 610 nm in a spectrophotometer (model DR-

2000; Hach, Loveland, Colo.). To estimate the bacterial

concentration achieved, the suspension was serially diluted in

sterile saline and spread plated in nutrient agar.

Brood stock conditioning and intramuscular injection

All the brood stock shrimps were injected the probiotic Lactobacillus acidophilus with different quantities (0.5, 1.0, 1.5

and 2.0 ml containing 3 × 109 cells/ml) into lateral surface of

the preceding segment of male brooder two days after

stocking into t he experimental tanks (Fig. 6) and (Table. 1 - 4).

Each experimental tank were stocked with 10 shrimps.

Experimental animals were fed with fresh frozen and

chopped squid, oyster and polycheate worms at 8:00, 13:00

and 18:30 hrs daily. Uneaten food was removed 3 hrs after

feeding. Feeds were given at the rate of 20% of the body

weight per day. Water quality remained constant between t he

treatments. No females were stocked, eliminating t he

possibilities of intervention. During stocking, the males were

marked with synthetic coloured tags.

Fig. 6: L. acidophilus for intramuscular injection


(Fabricius, 1798)


Spermatophores collection

Male P. monodon were washed with sterile seawater and 70%

ethanol was sprayed a round t he gonopores prior to collection

of spermatophores. Manual ejaculation of spermatophores

was performed on all males by stripping around the base of

the fifth pairs of walking legs (pereiopods) with forceps and

aseptic technique. Two spermatophores were obtained from

each male. The length and weight of the shrimps were

measured and t he shrimps were released into the tanks

initially and also after subsequent ejaculation.

Evaluation of sperm viability

Spermatophores were removed using a technique [24], in

which electrical stimulus was applied to the coxae of fifth pair

of pereiopods. The spermatophores were lifted from t he

gonop hores using forceps, dipped in Ca free saline solution

(NaCl- 21.63g; KCl-1.12g; H3BO3-0.53g; NaOH-0.19g and

MgSO4.7H2O-4.93g the pH of which was adjusted to7.4 with

1N HCl). The samples were prot ected from cross-

contamination.

All the extruded spermatophores were weighted to t he

nearest 0.01mg on an electronic bala nce, before

homogenization in 1 ml of Ca free saline as suggested [10], and

the sperm suspensions were prepared uniformly. Quality of

sperm was assayed using the met hod as described [18].

Percentage of morphologically normal sperm was determined

by recording the number of normal sp erm with a single spike

that project from an oral body or nucleus and abnormal

sperm with malformed bod y or bent or missing spike.

A sample of sperm suspension (50μl) was transferred to a

slide, mixed with 50 μL of Haematoxylin and Eosin, dried

under flame and examined using a light microscope (Fig. 7).

Live sp erm was unstained against the pink background of

Hematoxylin, while dead sperm appeared red. All sperm

examinations were made under p hase-contrast microscope

using haematoxylin and eosin stain. Percentage of viable

sperm was calculated in triplicate from counting of a

minimum number of 250 sperm cells under 1000-fold

magnification on each slide. The data collected at initial day

(Day 0) were used as t he baseline for comparisons with t hose

obtained during t he experiment.

Fig. 7: Micrograph showing sperm smear at different

resolutions

RESULTS AND DISCUSSION

Statistical analyses were conducted to examine the

relationships between body weight (BW), spermatophore

weight (SW) a nd sperm count (SC). In the I ext rusion, t he

body weight showed a significant correlation with

spermatophore count (Table.1 - 4).At the beginning of the

experiment, the mea n, standard deviation (SD) and range for

sperm quality assays tested were evaluated for each male. An

evaluation was made for each half of the compound

spermatophore including spermatophore weight, sperm

count, and percent morphologically normal sperm using

haematoxylin and eosin biostain assay. During stocking, no

significa nt differences were found b etween the males. At the

Fig. 8: Comparison of sperm quality after administering L.

acidophilus into P. monodon

Fig. 9: Changes of normal sperm (%) between different doses

of L. acidophilus in P. monodon

In the II and III extrusion, body weight showed significant

correlation with spermatophore weight and sperm cou nt.

Over t he experimental period in different feed combinations

there was a drop off spermatophore weight upto the III

extrusion a nd it increased in the IV extrusion. However, t he

sperm count generally increased upto the III extrusion and

then d eclined in the IV (Fig.8).


(Fabricius, 1798)


Table 1. Average cha nges (+standard deviation) in body weight, spermatophore weight, sperm c ount and sperm quality assay

result at the I extrusion of male P. monodon


result at the II extrusion of male P. monodon


result at the III extrusion of male P. monodon

Variable or assay Dosage (ml)

0.5 1 1.5 2

Ave. body weig ht (g)

61 .79 + 6.55 58 .90 ± 8.88 60 .94 ± 8.81 65.04 ± 8.25

Spermatophore w eight (mg) 11 .19 ± 2.79 13 .1 ± 2.95 13 .08 ± 1.9 14.62 ± 1.8

Sperm count (X 1,000)

871.9 ± 81.70 961.4 ± 330.41 965.4 ± 325.40 1050 ± 451.12

Gross sperm morphology (% normal)

42 .17+18.55 36 .19 ± 20.20 35 .15 ± 25.03 41.65 ± 23.04


result at the IV extrusion of male P. monodon


0.5 1 1.5 2


61 .29 ± 7.18 55 .51 ± 6.20 58 .22 ± 10.63 57.41 ± 8.67

Spermatophore w eight (mg)

12 .40 ± 2.90 13 .71 ± 1.60 13 .90 ± 1.70 14.39 ± 2.31

Sperm count (X 1,000) 914.2 ± 281.50 952.7 ± 371.10 1123.9 ± 436.42 1271.3 ± 123.71


38 .61 ± 11.11 31 .51 ± 9.60 34 .17 ± 14.95 34.79 ± 12.54


0.5 1 1.5 2


64 .26 ± 4.51 56 .91 ± 3.17 61 .02 ± 5.93 61.14 ± 4.46



1254.2 ± 341.50 1042.7 ± 181.56 1205.7 ± 164.42 1291.3 ± 112.07 Gross sperm morphology (% normal)

39 .25 ± 8.22 24 .1 ± 17.61 46 .01 ± 10.11 47.34 ± 15.11


0.5 1 1.5 2 Ave. body weight (g)

60 .99 + 3.50 61 .79 ± 4.88 61 .90 ± 8.83 66.10 ± 9.12



961.9 ± 81.70 1222.3 ± 310.21 1365.4 ± 25.0 1390 ± 11.1


47 .11 + 32.06 46 .11 ± 10.59 36 .15 ± 21.01 42.95 ± 13.01


(Fabricius, 1798)


Differences between spermatophores from right and left

terminal ampoules: The average changes in body weight,

spermatophore weight, sperm count and sperm quality in

different treatment, differed significa ntly in the tanks,

administered with 1.0 µl (/g body weight of broodstock shrimp) of L. acidophilus and a detail of different extrusions

are given in Table. 1-4 and Figure.9. The body weight,

spermatophore weight, gross However in the (1.0 ml containing 3 × 109 cells/ml) of L. acidophilus treated shrimps,

significa nt differences were there, between right and left with

respect to gross morphology (II and III extrusions) (F ig. 10). Initially al l males of different treatments with (L. acidophilus)

group’s spermatophores were present in the terminal

ampoules of the vas deference’s and no discoloration was

apparent.

In the same way [25], the juveniles of Penaeus indicus (average

weight 0.985 ± 0.1 g) on administering orally a moist feed base

containing 5 × 106 cells/g of the four LAB probionts for a

period of four weeks showed better survival (56 to 72%) when challenged with V. alginolyticus by intramuscular injection of

0.1 ml containing 3 × 109 cells/ml; were studied. Paired-

comparison t-tests were conducted by poo ling the data from

all the tim e periods for all the four different administrations

(0.5, 1.0, 1.5 and 2.0ml containing 3x109/cells/ml), the

differences between sp ermatophores from each of the terminal ampoules of male P.monodon were analyzed

(Figure.9). A significa nt increase in normal sperm was found

in subsequent extrusions from t he II extrusion and p ercentage

of abnormal sperm (primarily malformed heads or spikeless

cells) decreased. No significant differences in size and quality

were found between spermatophore from right and left sides

of the terminal ampoules generally (Table.5-8).

Table 5. Paired comparison between (t) spermatophore from

both sides of terminal ampoules in four different extrusions of male P. monodon administered with L. a cidophilus (0.5 µl)

Variable or assay

N

Extrusions

I II III IV Left vs Right

Left vs Right

Left vs Right

Left vs Right

Spermatophore weight

10 1.231 0.230 0.620 0.582

Sperm count 10 0.842 0.521 0.711 0.421

Gross sperm morphology

(H&E)

10 2.321 2.295 0.260 0.362

However in the (1.0 ml containing 3 × 109 cells/ml) of L.

acidophilus treated shrimps, signif icant differences were there,

between right and left with respect to gross morphology (II

and III extrusions) (Fig. 10). Initially all males of different treatments with (L. acidophilus) group’s spermatophores were

present in the terminal ampoules of the vas deference’s and

no discoloration was apparent.



Variable or assay

N

Extrusions


Left vs Right

Left vs Right

Left vs Right

Spermatophore weight

10 1.244 0.336 0.628 0.572

Sperm count 10 0.912 0.422 0.451 0.422 Gross sperm

morphology (H&E)

10 2.422 2.395 0.264 0.365

During the first 15 days, all males appeared in excellent

condition with spermatophores exhibiting good morphology

and colour. By the III extrusions, however, evidence of

external sp ermatophore det erioration was apparent. This

degradation ca n be desc ribed as a crumbling deterioration,

initially affecting the perip heral po rtions of the

spermatophore i.e., the wings and outer ma rgins of t he

germinate body, t he flange areas with wings generally falling

away first. In the III and IV extrusions, all spermatophores

sampled exhibited extensive perip heral damage and

characteristic thick white colouration when compared with

the pervious extrusions. In t he I extrusion, mean sperm

counts in GM ranged from left vs right spermatophore 0.842,

0.912, 0.940 and 0.972; In II extrusion 0.521, 0.422, 0.988 and

0.591 In III extrusion 0.711,0.451, 0.910 and 0.740 and in the IV

extrusion 0.421, 0.422, 0.945 and 0.431, in the subsequent

intervals respectively (Figure.8). All the sperm count is

presented in thousands. Higher cou nts were recorded in t he

right halve of spermatophore of shrimps administered with 1.0 ml containing 3 × 109 cells/ml. Among all the L. acidophilus

treated broodstock shrimps, only 1.0 ml containing 3 × 109

cells/ml performed better after 7 days of administration to

the shrimps. Through trial a nd error, it was found t hat

optimal conditions for electro-ejaculation had a frequency of

160-172 cps. The duration of 7.5-10.5v was for 0.5 to 3.8 secs

and (Fig. 11). Higher voltage produced greater

spermatophore extrusion success but at the same time caused

mild injury and higher mo rtality.



Variable or assay

N

Extrusions


Left vs Right

Left vs Right

Left vs Right

Spermatophore

weight

10 1.280 0.330 0.850 0.781

Sperm count 10 0.940 0.988 0.910 0.945 Gross sperm morphology

(H&E)

10 2.321 2.295 0.260 0.462

There have been several related studies on weather, age, size,

or culture conditions influencing the reproductive quality of

male penaeids over the decades [12, 26 , 1 7, 1 8, 27, 28, 29]. Only petite

information is available concerning the formation and


(Fabricius, 1798)


development of spermatophores. Resea rchers [12] reported that pond-reared Penaeus stylirost ris were found to p roduce

spermatophores in individuals, larger that 23.6g, and

spermatophores were s ignificantly lower in weight for the 20-

30g shrimp that the 30-40 g shrimp. Similar scenario is noticed

in the p resent investigation.



Variable or assay

N

Extrusions


Left vs Right

Left vs Right

Left vs Right

Spermatophore

weight

10 1.801 0.563 0.712 0.621

Sperm count 10 0.974 0.591 0.740 0.431 Gross sperm morphology

(H&E)

10 2.321 2.295 0.260 0.362

Fig. 10: Ejaculation of spermatophores with electric stimulus

at different time interval

Fig. 11: Electric stimulus applied on the spermatophore

The earliest time of spermatophore formation was different.

The age and size of males influ ence spermatophore

formation. The difference may be due to the growth rate and

culture conditions t hat differed for the t wo environments.

Similar result was repo rted for Penaeus merguiensis [30]; but

they suggested that the primary sexual maturity of prawns

depends upon size rather than age.

CONCLUSION

Reproductive performance was evaluated in sexually mature wild male P. monodon administered on the zeroth day of

initiation of the experiment with 0.5, 1.0, 1.5 and 2.0 µl /g

body weight of shrimp. Among the four d ifferent treatments

in 1.0 µl administered males p erformed in signif icantly

different.mannner. In the I extrusion, mean sperm counts in

GM ranged from left vs right spermatophore 0.842, 0.912,

0.940 and 0.972; in II extrusion 0.521, 0.422, 0.988 and 0.591, in

III extrusion 0.711,0.451, 0.910 and 0.740 and in the IV

extrusion 0.421, 0.422, 0.945 and 0.431 in the subsequent

intervals respectively. All the sperm counts are present ed in

thousands. Higher counts were recorded in the right halve of

spermatophore of shrimps administered with 0.1 µl. Among all the L. acidophilus treated broodstock s hrimps, only 0.1 µl/g

of body weight performed better after 7 days of

administration to t he shrimps. Invigoration of male for

ensuring effective fertilization and thereby p erfect hatching should also be attempted. Application of L. acidophilus in

enhancing sperm quality assay from this investigation would

immensely go to a longer way in maximizing the production

of shrimp seeds in hatchery facility which exists both east and

west coast of India.

ACKNOWLEDG MENT

The authors are tha nkful to the authorities of Annamalai

University, for providing t he facilities and support. Many

sincere t hanks to the fis hermen society for p roviding live

animals and their kind moral support towards the completion

of our research work.

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Source of support: Nil, Conflict of interest: None Declared

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