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Artificial Insemination and Penaeus Stylirostris

September 28, 2007

 

Michel Alain (alainhenri@aol.com): New Caledonia has had captive Penaeus stylirostris broodstock since 1980.  In fact, there was only one introduction of P. stylirostris into New Caledonia, and those animals are now in their thirtieth generation.  Even though all the stylirostris in New Caledonia may have originated from just a few pairs, no detrimental effects from inbreeding have been recorded.

All the shrimp maturation facilities in New Caledonia use artificial insemination on a routine basis.

Josh Wilkenfeld (josh.wilkenfeld@gmail.com): I’m a bit behind on artificial insemination technology.  I always considered it to be a useful procedure when dealing with relatively small numbers of spawns, but not very practical for large commercial operations.  At Genitech in Mexico, for example, we were dealing with 200-260 spawns a day.  Also, it’s been many years since I used “AI”, but I recall it as being pretty stressful for both the males and the females; not important if it is a one-shot deal for family production, but not so good for continuous commercial settings.

If the intention is to create specific families resulting from the offspring of a known male and known female, clearly the best way to do that is through artificial insemination.  If the idea is to maintain animals of a given line through mass selection using multiple spawns on successive days (reducing or eliminating offspring from spawns involving the same males and females), it doesn’t seem necessary to resort to artificial insemination.

Michel Alain (alainhenri@aol.com): In 1972, the AQUACOP (French Government) team started work on shrimp in Tahiti, French Polynesia, South Pacific (17°40′S, 149°30′W).  At that time, nothing was known about shrimp reproduction in captivity.  Our bosses told us to find out about it and to learn how to farm shrimp!  But we had a problem.  There were no commercial penaeid shrimp in Tahiti.  So we imported several penaeid species to see which worked best.

At that time we were using large maturation tanks (about a hundred square meters) with sand bottoms and water flowing from bottom to top, Kagoshima style.  I dove those tanks almost every day.  I saw the first ovarian development of Penaeus aztecus in a female that we received as a postlarva from Cornelius “Corny” Mock (a famous figure in shrimp farming at that time).  I noticed that this female had only one eye (the other probably cannibalized after a molt).  Some days later I found another female with fully developed ovaries—also with one eye.  Guessing that a missing eye had something to do with sexual maturation, I took ten females that showed no signs of ovarian development and crushed one of their eyestalks.  In a week, all the females were spawning, and we got good nauplii.  In the following years, we used the procedure—eyestalk ablation—with many other penaeids, among them P. vannamei, P. stylirostris and P. monodon.

France Aquaculture, an AQUACOP subsidiary that did private sector consulting work, was chosen by the Morrison Group to build the first shrimp hatchery (Semacua) in Ecuador.  It was followed by the first commercial hatcheries in Indonesia and India, where Aquatic Farms, our main competitor, was building a hatchery.

At the end of the 1970s, around some glasses of beer in Crystal River, Florida, I heard about artificial insemination for the first time from my friend Harvey Persyn, who was working on the famed Ralston Purina project.  I think he was the first person to successfully artificially inseminate shrimp.

In Tahiti, we tried all kinds of ways to get the male spermatophore to stick to the female.  I had my first success using candle wax as glue.  Eventually, however, we found that the simplest technique was to just take the ball of sperm without the wings and stick it on the outlet oviducts.

The quality of male broodstock is important, and in high temperature conditions they have a tendency to develop black spermatophores.  If you keep the sexes apart, it is quite easy to decrease the temperature in the male tanks and maintain the female tanks at a higher temperature for ovarian development.  With good quality males it is really easy to take out the spermatophores by a gentle pressure.  Electric shock also works, but it really stresses the animals.

Josh Wilkenfeld (josh.wilkenfeld@gmail.com): Though we don’t hear much about AQUACOP these days, I remember that when I first started my career in shrimp in 1980 by training with Corny Mock for six months in Galveston, AQUACOP, Ralston Purina and Coca Cola were always mentioned as the innovators of the major advances in shrimp farming.  It’s always nice to hear some of these old recollections from the people involved.

Another interesting coincidence is that in 1988 (I think), I was the biologist assigned to do the first production run of the hatchery designed and constructed by Aquatic Farms for MPEDA in Visaghapatnam, Andhra Pradesh, India, in competition with the hatchery built by AQUACOP in Kerala.  Mike Yates (the Aquatic Farms construction engineer on that project) and I visited the French hatchery, and the French team then visited us in Visag; it was a very friendly exchange.

I remember that not long after I arrived for training at the NMFS Galveston Lab, I was shown the basic techniques of artificial insemination by Ausbon Brown and Doug Tave, but at that time, we were still looking at various attachment methods, including super glue.  I actually constructed a “rack” made of PVC pipe, complete with Velcro straps and a drip irrigation system to keep the female’s gills wet with a constant flow of seawater while we figured out and practiced different techniques for attaching a spermatophore.  It was some time later that either George Chamberlain or Bill Bray (the three of us were with the Texas A&M Shrimp Aquaculture Project at the time) introduced me to the technique of eliminating the wings and just transferring the sticky sperm ball to the thelycum of the females.

I never developed the “touch” for spermatophore extraction with physical pressure; I always had trouble with eversion of the terminal ampoule, which often resulted in the death of the male.  That’s why I was fascinated when the first reports of electro-ejaculation began making the rounds.  I think this work originated in South Carolina, with Paul Sandifer, Al Stokes, Craig Browdy and others.  I don’t know if there has been much progress in this technology, and I haven’t paid that much attention to it, since as I indicated before, I’m not sure that I see a practical reason to do artificial insemination on a commercial scale; however, it’s an important research tool, especially when doing genetic research.

I’ll leave the discussion of problems associated with inbreeding to someone better versed in genetics than I—with one caveat.  As you know, the SuperShrimp hatchery was very successful in Mexico for a few years using P. stylirostris imported from Venezuela, a line that had been developed for RICOA (German Dao) through work done by Henry Clifford and Harvey Persyn (if I remember correctly).  We chose to work with that strain because it had developed resistance to IHHN, and it was susceptible to the less virulent strain of TSV, which originally caused problems in Mexico during the mid-1990s.

What I found most fascinating about the resistance of this line to IHHN and TSV is that apparently, through inbreeding, P. stylirostris had developed the same resistance to these two viruses in three different groups of animals, reared in three completely separate locations—Tahiti, Venezuela and Hawaii/Guam.  As I said earlier, I’m not a geneticist and don’t pretend to understand how this could happen, but it was very interesting that as an accidental result of situations where inbreeding was apparently a necessity (due to mostly to geographically limited availability of genetic variability) rather than a specific choice, three separate groups of stylirostris developed the same resistance to specific disease organisms.

We all know that inbreeding can have significant risks, at least in many advanced species, and I’m not sure that the idea of natural selection by way of low survival rates in the hatchery is a good way to go about a broodstock development program.  I guess I would not be surprised if some broodstock development programs have been or are using inbreeding to develop lines of shrimp that if crossed, will produce good results in maturation, hatchery and growout, but whose offspring cannot be successfully used as a next generation of broodstock animals.  This, of course, would be done to protect the original investment in developing important positive and marketable traits through a genetic selection program.

 

Laurent Ottogalli (ottogalli@lagoon.nc): It’s time for grandpas to tell their stories.  In the early 1980s, when we started to work with stylirostris in New Caledonia, we faced so many problems with maturation and nauplii production that we almost gave up on stylies.  P. monodon, P. vannamei, P. japonicus, P. indicus, P. merguiensis and P. semisulcatus were pretty easy to reproduce in captivity, but not stylirostris.

After observing stylirostris behavior for a year, we learned that stylies only mate once a year.  During that two-month period, one third of the gravid females would mate.  The rest of the year, no matings were observed.

At present all the hatcheries in New Caledonia use artificial insemination.  Males are kept in cooler tanks or captured from a maturation pond on the same day of the insemination, and it works pretty well.

Josh Wilkenfeld (josh.wilkenfeld@gmail.com): It’s interesting to hear of the problem you had getting stylirostris to cooperate in terms of “natural mating”, presumably even with ablation.  Working at just about the same time period (mid-1990s) with the IHHN/TSV resistant stylirostris from Venezuela, we had no problem at all with mating, unless the males flown in from Venezuela had been exposed to high temperatures in the ponds, resulting in black spermatophores.  Most people I have spoken with about stylirostris have commented on how easy they are to mature and mate in captivity compared to other species.  Once we began rearing the Venezuelan strain of stylirostris from our own offspring on site in Mexico, they reproduced like rabbits.

Patrick Wood (patrickjwood@yahoo.com): In the 1980s, from our hatchery in Sinaloa, Mexico, I gave some stylirostris broodstock to Philippe [Riou?], who was part of the France Aquaculture hatchery project in Sonora, Mexico.  I think he’s the one who sent them to Tahiti.

Sources: 1. The Shrimp List (a mailing list for shrimp farmers, “shrimp-subscribe@yahoogroups.com”).  Subject: [shrimp] Re: Mating area for P. vannamei.  September 11-19, 2007. 2. Summarized by Bob Rosenberry, Shrimp News International, September 28, 2007.

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