stress test to assess shrimp postlarval quality

From : Avery Kleon Solco averykleon@yahoo.com
To: shrimp@yahoogroups.com
Sent: 29 June 2003

QUESTION:

Do you know of any stress test for monodon pl’s and at what age should they be tested on? How do I know that the monodon pl’s that I’m getting are good? How fast can the salinity acclimation for monodon pl’s be carried out and starting at what age? I know for vannamei it is 10% change per hour. But what about for monodon?

Avery Kleon Solco
e-mail: averykleon@yahoo.com

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COMMENTS 1 :

For the stress test you can reduce the salinity to 50% in a container and put 100 pl 12-13 in it, then wait for three hours and count the dead one, if more than 15 to 20 it can not pass. You can reduce the salinity every day about 3 to 5 ppt.

Dr. Farshad Shishelchian
PhD Terrestrial and Aquatic Ecology-Shrimp Culture Pond Management
Managing Director for First Pacific Aquaculture Sdn Bhd
Managing Director for Bio-Web Sdn Bhd
e-mail: shrimp@tm.net.my

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COMMENTS 2 :

Here’s another stress test that also works and is a bit quicker. Count 50 PLs into a beaker and then collect them by pouring them onto a small filter. Immediately wash them with freshwater into another beaker containing freshwater. Wait 30 minutes. Collect them again on the small filter and wash them back into a beaker containing the original water. Wait 30 minutes and count any dead. Good PLs that are at least

8 mm long (vannamei or monodon, total length) should pass this test with 80% survival or more. PLs that are 7 mm or less usually do very badly in this test, no matter how healthy they are.

For dropping salinity there should be no problem with PLs that are 7.5 mm or greater. And 10% per hour is fine for monodon as well as vannamei, but I wouldn’t go at this rate for more than 5 hours per day.

Daniel Lee
e-mail : danlee@codetel.net.do

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COMMENTS 3 :

The tests are the same as that for vannamei. When I tried the 30 min saltwater for 0.8 mm long monodon, they all died out after a few minutes in the freshwater.

So, it has got nothing to do with age, but length of PL’s?

Avery Kleon Solco
e-mail: averykleon@yahoo.com

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COMMENTS 4 :

Eight mm monodon are probably only pl 5 or so. Most monodon are harvested from hatcheries at 12-13 mm (from the tip of the telson to the tip of the rostrum) and are pl 15-20 depending on the hatchery. 75% of these will have 5 teeth on the dorsal side of the rostrum.  I did a lot of stress tests using salinity and even formaline. We settled on a 50% shock in salinity (34 to 17 ppt) for 2 hours. 98% survival was acceptable. You can tell if they will do well right from the start. The weak ones will swim against the side of the bowl with their head against the side. Good ones will sit still on the bottom for the first 20 minutes or so. Before you even shock them though, look at the gut to muscle ratio under a scope. If it’s less than 4:1, forget it. Go to another hatchery.

I found that “monos” don’t fair well in this test until they reach that 12 mm, 5 tooth stage, 3 teeth rostrum PL’s do poorly even though they are growing well and healthy.

Hank 
e-mail: Bauman.BAL@starband.net

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COMMENTS 5:

I differ with this view that PL’s of above 12 mm can withstand salinity stress. The size is mostly dependent on the type of feed being used and even a PL 15 could be less than 12 mm but still strong enough to pass the test.

If the PL are fed mostly with Artemia and artificial diet could be lengthy whereas those fed with egg custard are not lengthy but stouter. Irrespective of the size, PL12 or later could pass the stress test if they are healthy. Some times very healthy PL10 pass the test but I will suggest you 12 or later.

Chandrasekar
e-mail: aqua@omanfisheries.com

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COMMENTS 6 :

While reducing salinity, I would like to add here that, it is not how much ppt you drop, but, by how much percentage you dilute the salinity that matters. For example, a 5 ppt drop from 30 ppt to 25 ppt is about 16% drop, but the same 5 ppt drop from let’s say 10 ppt to 5 ppt is a 50% drop in salinity, and this would be far more stressfull to the PLs.

We prefer to reduce salinity at larger PLs (PL12+) normally not exceeding about 25% drop per day, and a resting period of minimum 24 hours before we pack them.

Anil Ghanekar
e-mail: anilghanekar@yahoo.com

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comments 7 :

Ability of the PLs to tolerate extreme and abrupt salinity changes are to my experience with P.vannamei directly correlated to the degree of ramification of the gills.
Age and environment determine gill development and their branching.
The higher degree of ramification of the gills, i.e. secondary and tertiary branching of the gills, which is easily detected under a stereoscope, gives you the certainty that the PLs will perform better under stress, caused by salinity changes.

Michael Stirnberg
e-mail: mstirnberg@oisc.com.om

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comments 8 :

Looking at what Michael observes for vannamei, have you found the same correlation with monodon?

Daniel Lee
e-mail: danlee@codetel.net.do

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Comments 9:

For what it is worth, this is the standard criteria I use for PL selection from hatcheries.

1. Visual Criteria
. Good quality fry should be of the same age,  preferably from the same batch, and should be uniform in size.
. Normal fry range in color from clear to brown, rusty brown, grey to dark grey. Reddish or pinkish colored fry are usually associated with stress due to improper handling, infestation and disease.
. Uropods must be open. closed uropods are indicative
     that the fry are too young for stocking.
. Healthy fry are active swimmers, and respond immediately to external stimuli.  When at rest unhealthy fry usually have arched bodies.  Curled bodies or crooked bodies are usually an indication of a previous drug or antibiotic treatment.
. Strong fry will swim counter to the current when the water in the basin is stirred.  It is not a good sign if many are drawn to the center as the current subsides.

2. Microscopic Criteria
. Healthy fry must have a well developed muscle that completely fills the shell.  This is most evident in the 6th abdominal segment.  The muscle should appear smooth and clear.
. The gut-muscle ratio should be at least 1:4; body length should be 13.5 mm or upwards, with a preference for longer animals at PL20; rostral spine count should be 4-6 dorsal spines.
. The feeding response, and the gut content is a key factor in the evaluation of fry quality.  Healthy PL's will almost always have a full foregut and about the first third of the hindgut full as well, if given the opportunity to eat.  When observing fry at a hatchery, or evaluating fry samples of fry shipped or transported from other locations, keep in mind that empty guts may simply be an indication of a prolonged period without food.  If fry appear otherwise healthy, feed some live Artemia and observe feeding activity.  If the fry are in good condition (after 50% acclimation if shipped cold), guts should fill up after 10-15 minutes.

Alec Forbes
e-mail: docalec@connect.com.fj

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Comments 10 :

While we use the 0 ppt/30 min stress test mentioned by Dan as part of
our analyses, I believe that a multi-part assessment of PL quality is
superior to a stress test; in my experience the stress test provides
very little true information in support of PL quality, at least as far
as vannamei is concerned. A more suitable PL quality analysis should
involve a series of assessments, as Dr. Forbes suggests. The macroscopic and microscopic assessments (including tail muscle to gut ratio factor described by Hank and gill development mentioned by Michael) can be scored and compiled into a multivariate QC index of PL quality. I believe this was presented by Danny Fegan many moons ago in a WAS publication (Danny?). There is way too much at stake to put too much faith in one test that tells us very little other than recent PL nutrition.

David Griffith
e-mail: dgriffith@seafarmsgroup.com

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COMMENTS 11 :

When performing a stress test consider that if the larva has just recently molted it will be far more sensitive to the test.

Francois Brenta
e-mail: fbrenta@hotmail.com

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COMMENTS 12 :

2 ppt/hr from 35 ppt to 25 ppt, 1 ppt/hr from 25 ppt to 15 ppt, 0.5 ppt from 15 to 10 ppt, and under that 0.1 ppt per hr.

Francois Brenta
e-mail: fbrenta@hotmail.com

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comments 13 :

I never looked at gill development. Rostral teeth, body length and gut
to muscle ratio. Then absence of necrosis etc. This was before WSSV.

Hank
e-mail: Bauman.BAL@starband.net

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Comments 14:

Actually I found the opposite. Animals with gut to muscle ratios of 10:1, or just prior to molting, were the first ones in a healthy batch that would die if stressed more than 50%. This may be due to osmosis and the pl taking on water to be able to expand right after shedding the old exoskeleton. Pl’s that had just molted, evident by semi striated muscle tissue (for the first hour after molting) and reduced GMR survived.

Hank
e-mail: Bauman.BAL@starband.net

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Comments 15:

In our paper (Samocha, T. M., Guajardo, H., Lawrence, A. L., Speed, F. M., Castille, F. L., Page, K. I. and McKee, D. A. 1998. A simple stress test for Penaeus vannamei postlarvae. Aquaculture 165:233-242.) we indeed used formalin and salinity to evaluate the performance of PL at different age. We clearly showed increase in PL tolerance with age.

Nevertheless, we did not look into the question whether or not good
performance in stress test is indicative for good performance in the
grow-out phase.

Tzachi Samocha
e-mail: samocha@falcon.tamucc.edu

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Comments 16 :

Now that it has been mentioned, does anybody on this list know about
published data (with statistics) on "the question whether or not good
performance in stress test is indicative for good performance in the
grow-out phase"? Reference is often made to Bauman and Jamandre (1990), but they themselves acknowledge lack of "controlled, replicated grow-out field trials to validate quality assessement of fry at the time of stocking". Typically one of the things that everybody wants to know, but... .
Similarly, I think everybody will agree that multi-part assessment of PL quality (cfr. David Griffith) is in theory safer and better to depend on than a simple stresstest, but it also generates more questions:
- what formula will you use to calculate a representative Condition
Index, i.e. which characteristics and what "weight" do you allocate to
each characteristic?
- how does each characteristic (visual, microscopical, behavioural,
stress tests, ...) correlate to pond survival?
- are there methods to include biochemical data in this formula (a
performing easy field test for lipid analysis?)?
- what is a good value for such a Condition Index?
 
Please share your findings with us. As for the missing links: let's hope
that some research is done on this interesting topic soon.
 
Roeland Wouters
e-mail: r.wouters@inve.be

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Comments 17:

Actually the work carried out at Jamandre by myself was correlated to pond performance. Jamandre Hatcheries had many growers achieving 90% plus survival when stocking fry which passed the microscopic and stress tests in the hatchery prior to sale. This was actually how we came to realize what was important.
    I don't recall acknowledging a "lack of controlled replicated grow out field trials". In fact all our presentations regarding pl quality were based on exactly that. Results from our customers.

Hank
e-mail: Bauman.BAL@starband.net

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Comments 18:

The hatchery is going to do an early acclimation to the monodon Pl’s at 0.7 cm (I think it is only PL 5 or 6). I will be counting and paying when I pick it up, after 4-5 days. Is this good for me? Will it weed out the weaker ones? Or is there a long term consequence (say before harvest)?

Avery Kleon Solco
e-mail: averykleon@yahoo.com

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COMMENTS 19:

Gill development is one of the most important factors.This organ is responsible for the respiratory function. Without a complete development (at least secondary branch) the Pl is not ready for a different environment from where they were born and reared, specially with different salinity, and this will affect directly not only oxygen consumption (which will affect metabolism for sure, and growth) but other physiological processes involved.

Of course, body length and gut to muscle ratio are important also.

DVM Jose Luis Vega
e-mail: ptyczett@yahoo.com

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Comments 20 :

It is true that osmotic stress resistance is very dependent on gill ramification (thus PL age). However, salinity shocks can be used as well to distinguish different qualities in early PL batches (i.e. PLs without gill ramification), thus indicating that general physiological condition (=~ nutritional condition) plays a very important role.
For your info: I have noticed that the use of combined salinity-formalin stress tests on L. vannamei PLs was mentioned repeatedly over  the past years (e.g. Samocha et al. 1998 in Aquaculture 165; Santacruz & Cobo 2001 in Boletín Informativo Quincenal - CENAIM Informa - 15 de julio del 2001). This could be a way around the high variabilities typically seen in osmotic stress tests?

Roeland Wouters
e-mail : r.wouters@inve.be

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comments 21 :

Stress test reflects the healthy condition of Pl’s in a specific moment , statistically there is no relationship between stress test and ponds result.
I agree with you that a Quality Index including development of the PLs through the whole cycle plus the genes backgroung will give a much better idea of the PL quality and its late development in growout ponds.
As there are so many factors involved during a larval rearing cycle a QI number or percentage will give a better and more realistic idea of the animal quality, health and strength. This QI will give you certain idea how these PLs are going to develop in a growout under normal management circunstances and pond conditions.
 
Jaime Baquerizo
e-mail : baquerizojaime@yahoo.com

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Comments 22 :

A reference, surely known to many, that makes a good revision on the
theme of stress tests. It doesn't, however, answer the  question
"whether or not good performance in stress test is indicative for good
performance in the grow-out phase?", as Roeland rightly asked.

Tayamen, A.; Brown, J. (1999) A condition index for evaluating larval
quality of Macrobrachium rosenberghii (de Man 1879). Aquaculture
Research, 30, 917-922.

The authors idea of using an adaptation of the Apgard index
(multivariate ranking index) might work, and it can be easily adapted to shrimp PLs. However, what factors/characteristic should be included, how many levels/ranks for each factor/characteristic and the weight given to each level/rank will certainly be a question of debate.

Any index should include variables assessed both in ordinal and
continuous scales. The simplification to only 3 classification levels
for each factor/characteristic, as used in the Apgard index, is
recommended to avoid subjectiveness. My suggestion is that any index should include assessment of morphological development, osmotic capacity, nutritional condition and presence/absence of pathogens. For the first two there are several quick and practical methods currently used. For biochemical correlates of nutritional condition there is a need to adapt colorimetric rapid diagnostic kits used with humans. As far as I know, Roche is sponsoring research on the field of determining baseline levels of a series of shrimp blood metabolites to determine what is a "normal" concentration. Mind you, this research is being done with adults, but there is no reason why it could not be adapted to PLs. For rapid and simple methods to determine presence/absence of specific pathogens, my guess is that similar diagnostic kits are being developed. All this will certainly increase the price of PLs! And still we haven't answered the earlier question if the PL "quality" is necessarily related to later grow-out performance...

Most will say that a healthy and strong PL will have more chances to
survive than a weak PL, and my view is that in only certain rare
exceptions will this assumption be proved wrong. However, it should be confirmed (with statistics). The use of a complete stress test, despite
increase costs, could be an important point to add to the certification
process if an agreed condition index would be made industry-standard and back-up by some aquaculture institution.

Nuno Simoes
e-mail: nunosimoes@prodigy.net.mx

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comments 23 :

There are formalin (200ppm) induced stress tests and salinity drop induced stress tests.
The survival of pl’s after 1hr is taken into  account to decide the sturdiness of pl.

Biju Narayanan
e-mail: aquabiotechs@yahoo.com

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comments 24 :

Let there be no misunderstanding: the work published on P. monodon
quality assessment by Bauman and Jamandre (1990) was excellent. It
provided plenty of useful information on quality characteristics and
stress tests, and statistically differentiates PLs from distinct
hatcheries. However, I understand from this paper that the link to
subsequent pond performance was based on (probably valuable!)
indications from your customers, but without statistics or experimental
design. In this paper, under "further areas of investigation" the need
of "controlled, replicated grow-out field trials to validate quality
assessment of fry at the time of stocking" is indeed recommended (Page 130).
Anyhow, 13 years after your publication, there does not seem to be extra data published that prove the correlation "PL quality - pond performance " in a statistical manner, although I am convinced that this correlation exists.
 
Roeland Wouters
e-mail: r.wouters@inve.be

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Comments 25 :

The most important quality factor for shrimp PL is not detectable by either visual exam or stress test.  It is whether the PL are free of listed
pathogens (such as WSSV, TSV, IHHNV etc).

Clean PL (i.e. SPF) can be determined two ways:

1.  By PCR screening which is expensive, highly variable and therefore
ultimately unreliable or

2.  Buying from reputable supplier who uses certified SPF broodstock.

Jim Wyban PhD
High Health Aquaculture Inc.
ph/fax: 808.982.9163
e-mail: wyban@gte.net
www.hihealthshrimp.com

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