AnimalNet Dec. 2/03
Reversibly-sterile
fish via transgenesis
Environmental
charges laid in fish kill case
A report
from the AgTech Centre: Research sets performance bar for liquid manure
injectors
Two cattle
herds near Manitoba's Riding Mountain park quarantined for TB
2004 Omnibus
appropriations and COOL
Expect COOL
final rule as scheduled
CCA
confident US COOL will remain voluntary
Australian
sells bottled water for dogs
how to subscribe
Reversibly-sterile
fish via transgenesis
December 2003
ISB News Report
Norman Maclean, Gyulin Hwang, Alfredo Molina, Tom Ashton, Marc Muller, M. Aziz
Rahman, and Arati Iyengar
http://www.isb.vt.edu/news/2003/news03.dec.html#dec0302
Sterile fish are of interest in aquaculture both to help maximise growth and,
when necessary, to ensure reproductive containment. The latter aspect is now of
particular interest in connection with the possible exploitation of GM fish,
since growth enhanced GM salmon (Salmo salar) and tilapia (Oreochromis niloticus)
have been produced and tested1,2. Traditionally sterile fish have been produced
by triploid induction3, but in species such as tilapia this is quite difficult
and the "all-female" triploid technology used with success in salmon
is less satisfactory, since in tilapia the male grows substantially faster and
larger than the female and is the more marketable fish.
If fish are to be made transgenically sterile, then it becomes crucial to
include a reversible element to ensure the availability of brood stock for
future production. Targeting the gene for gonadotropin releasing hormone (GnRH)
is attractive in this regard since this polypeptide monomer controls
gonadotropin production and its subsequent role in reproductive development. If
the GnRH gene were genetically ablated, then fertile brood stock could
theoretically be recovered by treatment of steriles with exogenous GnRH. The
hypogonadal mouse4 is a model example of this approach.
However GnRH genes in fish are not without their problems in that multiple
copies are present in the genome. Numbers vary in different fish species, but
there are at least three distinct GnRH genes in tilapia, and identification of
the "gonadotropin-releasing form" is still somewhat uncertain5. It is
currently suggested that type I (serine 8 or "sea bream" type) is the
relevant "releasing" form. However another form, type III (tryptophan
7 leucine 8 or "salmon" type), may also be implicated in regulating
gonadotropin release. The sites of synthesis of the different GnRH types are
also complex. Type I is made chiefly in brain and spleen and type III in brain
and testis, at least in Haplochromis5, but not all tissue sites have been
tested.
As well as the GnRH genes, the genes coding for gonado-tropin itself make
possible targets having a "reversible" physiological effect. There are
two types of gonadotropin in fish, gonadotropin II (GtH II) otherwise known as
luteinizing hormone (LH) and gonadotropin I (GtH I) otherwise known as follicle
stimulating hormone (FSH). Each is comprised of two subunits, alpha and beta. As
will be clear later, the work in Southampton has focused on targeting genes for
both types and forms of GnRH and the gene for beta subunit of LH.
There are two distinct levels at which gene function can theoretically be
ablated, namely the gene itself or the specific gene message. These are often
referred to as gene knockout and gene knockdown. Knockout requires recognition
and replacement of the gene sequence by a defective copy via homologous
recombination. Since there is still no up-and-running equivalent to the mouse
embryonic stem cell (ES cell) system in fish, gene knockout in fish is currently
difficult or impossible. There is some future likelihood that by using Rad51, a
eukaryotic equivalent of the bacterial RecA, homologous recombination in fish
could be made feasible, but the system remains to be developed and proven. So
unfortunately there seems to be no present technology whereby gene knockout in
fish can be accomplished.
This leaves gene knockdown through targeting of specific messenger RNA species.
Three possible approaches to gene knockdown are RNA interference (RNAi),
ribozyme, or antisense. Although RNAi has proved to be effective in Drosophila6
and mice7, there is as yet only negative evidence of its effectiveness in fish8.
In the Southampton laboratory we have attempted to use RNAi and ribozyme to
ablate reporter gene expression in lines of tilapia expressing lacZ, but neither
have proved effective.
However, some success has been achieved with the third technology, antisense.
The antisense approach consists in designing an artificial DNA sequence which,
when transcribed, will make a messenger RNA that is directly complementary in
base sequence to the message being targeted. If both the messenger RNA and the
antisense against it are transcribed in the same nucleus, they bind together,
thus inactivating the message. It is clear that the efficiency with which
knockdown of the specific gene expression is achieved will depend both on the
quality of message made and the amount of antisense made. If the message is very
abundant or the antisense promoter weak, then knockdown is likely to be partial
or even undetectable. The antisense technology has of course been widely used
with success in a range of plants and animals.
Antisense constructs have been prepared and used in tilapia as follows9.
(a) tilapia beta actin promoter/ tilapia LH beta subunit gene antisense
(b) carp beta actin promoter/ tilapia type III (salmon type) GnRH gene antisense
(c) tilapia beta actin promoter/ tilapia type I (sea bream type) GnRH gene
antisense
(d) tilapia histone 3 promoter/ tilapia type I (sea bream type) GnRH gene
antisense
(e) tilapia type I (sea bream type) GnRH promoter/ tilapia type I (sea bream
type) GnRH gene antisense
Thus far, lines of transgenic tilapia have been produced expressing constructs
(a) to (d) and the promoter isolated to allow production of construct (e). In
preliminary studies, sterility has been tested in adult (seven months or older)
tilapia expressing these constructs. No sterility has been detected to date with
fish expressing constructs (a), (c), or (d). However some of the fish expressing
constructs (b) are completely sterile or of very low fertility, suggesting an
impairment of gonadal development or maturation.
It was initially surprising that no sterility could be detected in fish
expressing construct (c), and equally surprising that some sterility was
detected in fish expressing construct (b). A possible interpretation is that the
antisense is proving effective not through production in the brain but through
production in gonad, since type III GnRH is known to be synthesized in the
testis.
The following caveats must be stressed. (1) Only male fish have so far proved
sterile or partially sterile. (2) Fish have not as yet been successfully
reversed to fertility, although this should not prove a difficult hurdle. (3)
The observation of sterility in fish expressing antisense against type III GnRH
is an observation of a correlation. Much more evidence is needed to demonstrate
that the antisense is causing the sterility, although this looks likely.
Two further positive aspects are noteworthy. One is that, in the male fish which
are partially or completely sterile, impaired sperm motility is evident and
there appears to be some correlation between the degree of reproductive
sterility and the degree of sperm immobility. Another is that all the fish so
far studied are hemizygous for the antisense transgene. Populations of fish have
been produced which are the progeny of crosses within the transgenic lines, and
therefore 25% of these fish should be homozygous transgenics for the antisense
gene. Sterility in such fish should be even more pronounced than in the
hemizygous transgenics.
Interestingly, similar work to this has been carried out with rainbow trout (Oncorhyncus
mykiss)10 and again sterility has been observed in fish expressing antisense
against type III GnRH.
It is now our intention to measure levels of antisense mRNA and GnRH type III
mRNA in gonads and other tissues of hemizygous and homozygous transgenics as
well as control fish. This should help to establish the apparent correlation
between transgenic status and sterility.
This work was partly supported by a grant from the DFID Fish Genetics Programme,
and also by a grant from the EC.
References
1. Hew CL et al. (1995) Transgenic salmon: tailoring the genome for food
production. Journal of Fish Biology (Supplement A) 47: 1-19.
2. Rahman MA et al. (2001) Growth and nutritional trials of transgenic Nile
tilapia containing an exogenous fish growth hormone gene. Journal of Fish
Biology 59: 62-78.
3. Benfey TJ (1996) Use of all-female and triploid salmonids for aquaculture in
Canada. Bull Aquac Assoc Can 96-2: 6-8.
4. Seeburg PH et al. (1987) The mammalian GnRH gene and its pivotal role in
reproduction. Recent Prog Hormone Research 43: 69-98.
5. White RB and Fernald RD (1998) Genomic structure and expression sites of
three gonadotropin-releasing hormone genes in one species. General and
Comparative Endocrinology 112: 17-25.
6. Misquitta L and Paterson B (1999) Targeted disruption of gene function in
Drosophila by RNA interference (RNA-i). Proc Natl Acad Sci USA 96: 1451-1456.
7. Wianny F and Zernicka-Goetz M (2000) Specific interference with gene function
by double-stranded RNA in early mouse development. Nature Cell Biology 2: 70-75.
8. Zhao Z et al. (2001) Double-stranded RNA injection produces nonspecific
defects in zebrafish. Developmental Biology 229: 215-223.
9. Maclean N et al. (2002) Transgenic tilapia and the tilapia genome. Gene 295:
267-277.
10. Uzbekova S et al. (2000) Transgenic rainbow trout expressed sGnRH-antisense
RNA under the control of sGnRH promoter of Atlantic salmon. Journal of Molecular
Endocrinology 25: 337-350.
Norman Macleana, Gyulin Hwanga, Alfredo Molinab, Tom Ashtona, Marc Mullerb, M.
Aziz Rahmana, and Arati Iyengara
a School of Biological Sciences, University of Southampton, England, UK
b Molecular Biology and Genetics Laboratory, University of Liege, Belgium
Environmental
charges laid in fish kill case
December 2, 2003
From a press release
SUMMERSIDE, PE - A potato grower from Kelvin Grove, Prince Edward Island, has
been charged under subsection 36(3) of Canada's Fisheries Act.
Mr. Caseley will appear in Summerside Provincial Court for a plea on December
11. The charges are the result of an investigation by Environment Canada into a
fish kill on the Wilmot River on July 10, 2002. The charges allege that Mr.
Caseley allowed pesticide contaminated soil and water run-off to enter the
Wilmot River at Norboro, Prince Edward Island. About 4,500 dead trout were
recovered from the Wilmot River after the incident.
Under Canada's Fisheries Act, it is an offence to deposit, or permit the
deposition of, a deleterious substance into water frequented by fish.
Environment Canada's Enforcement officers investigate potential pollution
offences under the Canadian Environmental Protection Act, 1999 (CEPA 99) and
Canada's Fisheries Act. They help ensure that companies, government employees
and the general public comply with legislation and regulations that protect
Canada's environment.
A
report from the AgTech Centre: Research sets performance bar for liquid manure
injectors
December 3, 2003
Meristem Land and Science
www.meristem.com
Lethbridge, Alta. -- Studies to develop standardized tests for liquid manure
injectors will provide livestock and crop producers with a buyer's checklist of
performance considerations for four of the most popular models, says Alberta
Agriculture, Food and Rural Development (AAFRD) Project Manager, Brian Sexton.
Liquid manure injection is a more effective means of controlling odour and
nutrient placement is more precise than broadcasting, says Sexton. More
producers are considering injection, but there has been a lack of evaluation
information to help producers determine which model is best for their operation.
"This study is focused on developing standard test procedures for manure
injectors. It will give us evaluation guidelines to help western farmers assess
their injection options," he says.
Researchers are testing four different injectors to determine odour control
performance, the amount of pooling over a range of injection rates and soil
conditions, as well as a comparison of the effects of draft or pounds pull for
each injector. The tests will also measure crop residue and soil disturbance
with respect to each manure injector.
"After manure is injected, we collect odour samples in bags and send the
samples to an olfactometry lab in Edmonton, where a specialized odour panel
-with expertise in detecting smell - determine the intensity of the odour.
These lab results will help us develop a standard measurement to show how well
each injector works at controlling odour," says Sexton.
Injectors will be run at application rates ranging between 3,000 and 13,000
gallons per acre to determine at what range the manure starts to leave the
furrow and begins pooling on the field. "The point is to get the manure
below the surface. You should be able to walk across a field after application
and come out with clean boots," says Sexton.
The pooling tests will also help determine if it's the rate of injection that
causes the pooling, or the equipment itself. For example, modifications made to
openers to allow fluid delivery may cause uneven fluid distribution.
"There's no question that rate plays a role in pooling," says Sexton.
"Some of the problems, though, are with outdated or modified
equipment."
Soil condition also affects how well injectors work. Wet soil often contributes
to pooling, as the manure is not easily absorbed under these conditions.
Soil disturbance is another issue driving the development of the standardized
testing. "A big reason why some producers have been reluctant to adopt
injection is their concern about soil disturbance in a conservation practices
system," says Sexton. "The fact is, there are injectors that can go on
pasture and forage land and cause little soil disturbance. Our study should help
determine which ones work best in these conditions."
The study will also look at how draft impacts injector performance. Sexton says
having the right size tractor and knowing how much fuel it's going to use is a
huge factor when considering the operational costs of injection equipment.
Although some producers with minimum till and pasture land continue to
broadcast, this may not be the best choice. "If producers are interested in
controlling odour, retaining nutrients and treating manure as a resource, then
injection is the way to go," says Sexton.
The liquid manure injection project is part of AAFRD's ongoing commitment to
support sustainable agriculture. Field testing is scheduled to wrap-up at the
end of 2003 and reports are to be published in 2004. The AgTech Centre is part
of the AAFRD Engineering Branch.
Two
cattle herds near Manitoba's Riding Mountain park quarantined for TB
December 1, 2003
CP Wire
ROSSBURN, Man. - Two cattle herds near Riding Mountain National Park have,
according to this story, been quarantined after one cow from each farm reacted
to bovine tuberculosis tests.
The story says that more than 240 cattle in the Rural Municipality of Hillsburg
were quarantined last week after one cow in the herd reacted to a test; the
affected cow has been slaughtered but the herd remains in quarantine until tests
on the carcass are completed.
Dr. Larry Delver, a veterinarian with the Canadian Food Inspection Agency, was
quoted as saying, "When she was slaughtered they found no lesions. It could
be avian TB,," a form of tuberculosis common in wild birds and harmless in
cattle.
An abscess found under the cow's jaw after being slaughtered is more typical to
foot rot than tuberculosis, he added.
2004
Omnibus appropriations and COOL
December 1, 2003
Herd On The Hill
Edited by Kiran Kernellu
Congressional Quarterly Today reported last Tuesday that Republican
appropriators refined the seven-bill Fiscal 2004 Omnibus Appropriations Bill.
The omnibus, HR 2673 (HR 2673; H Rept 108-401), was filed in the House during a
pro forma session held open for that purpose. Members are slated to vote on the
measure Dec. 8, but a Senate vote is not expected until January. (Congress
reconvenes January 20, 2004.) Consequently, most federal agencies will be
operating at Fiscal 2003 levels until then.
Cattle Buyers Weekly reported today that a new independent study of COOL's
impact might be made as part of a deal worked out by the White House and
Congressional Republicans for the 2004 Omnibus Appropriations Bill and other
spending legislation. Reportedly, Republicans might ask President Bush to call
the Senate into session prior to Jan. 20 to vote on the omnibus, now at an
estimated $820 billion.
The COOL moratorium provision in the 2004 Omnibus Appropriations Bill, subject
to a vote in both the House of Representatives and the Senate, is as follows:
"SEC. 749, Section 285 of the Agricultural Marketing Act of 1946 (16 U.S.C.
1638d et seq.) is amended by striking '2004' and inserting in lieu thereof
'2006,' except for 'farm-raised fish' and 'wild fish' which shall be September
30, 2004."
Already livestock, meat and retail organizations are working together to
develop the infrastructure that this huge industry will need to implement a
mandated country of origin declaration for covered commodities. Indeed, they
have been working together for some years on a voluntary program, and it would
seem appropriate that those supporting a mandate join this effort and work
cooperatively with the rest of the industry to identify and devise ways to
overcome the obstacles.
Expect
COOL final rule as scheduled
December 1, 2003
Herd On The Hill
Edited by Kiran Kernellu
Cattle Buyers Weekly (CBW) reported today that USDA will continue to accept
comments on its proposed final rule for COOL through the comment period that
ends later this month. The comment period might be extended, though no known
requests have been made. CBW expects that the Agency will then issue its final
rule, even if a moratorium is included for all products covered by COOL, except
farm-raised and wild fish.
CCA
confident US COOL will remain voluntary
December 2, 2003
Farmscape (Episode 1396)
The President of the Canadian Cattlemen's Association is confident US will
ultimately abandon its efforts to make Country of Origin Labelling legislation
mandatory. A joint Senate-House conference committee has recommended delaying
the introduction of mandatory Country of Origin legislation for most products
until 2006. CCA President Neil Jahnke says US groups that had been opposed to
mandatory COOL are firming up their positions and many that had supported the
provision are reconsidering that support.
Clip-Neil Jahnke-Canadian Cattlemen's Association
Since the USDA released their findings on the cost of mandatory Country of
Origin Labelling, which amounts to something like three billion dollars a year,
there has been some pushback from industry. A lot of the industry down there,
and NCBA in particular, supported a voluntary country of origin not mandatory
and, with a three billion dollar price tag, we're seeing more pushback on
mandatory. I personally believe that, when the dust all settles, that they will
stick with voluntary country of origin. There is the added bit on there that
poultry is not included in mandatory. If it's going to cost the beef and the
pork industry three billion dollars a year and their major competitor is exempt,
we believe that there will be a big push back and hopefully mandatory Country of
Origin Labelling will not become a reality.
Jahnke says voluntary Country of Origin Labelling will serve the same purpose
without disrupting the market. He says CCA has no reservations with a voluntary
system, be it in Canada, Japan, the US or any other country but mandatory will
mean additional costs. He doubts the consumer will want to pay more or that
producers will want to take less for products to cover those costs. For
Farmscape.Ca, I'm Bruce Cochrane.
*Farmscape is a presentation of Sask Pork and Manitoba Pork Council
Australian
sells bottled water for dogs
December 1, 2003
Associated Press
CANBERRA -- Australian Andrew Larkey has, according to this story, launched the
latest accessory for pampered pooches Tuesday -- bottled water in flavors like
chicken and corn, liver and bacon, or beef.
The story says that the purified Dog Plus K-9 water went on sale in pet stores
at $2.10 for 1.3 pint plastic bottle.
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