Note: Descriptions are shown in the official language in which they were submitted.
-
il'7~8t~6
The present invention relates to a simple, rapld and
speciflc test for recognising the sex of oviparous animals, without
injuring them.
Sex recognition, particularly in fish, is useful to
5 ecologists for determlning the breeding potential of the popu-
lations. Management of the breeding stocks intended
for fish-farming is also optimised thereby.
Outside the period of reproduction, the search for
the sex-determining criterla from morphological features is a
10 long and haræardous operation. On the other hand, it is possible
to envisage a sex-determlning technique based on the bio-
chemical recognition of a protein specific to one of the sexes.
In 1932, Sasaki proposed sex recognltion of chlcken
by using, as sex-determining criterion, the presence of a plas-
15 matic protein precursor of the vitellin reserves of the egg. Morerecently, an equivalent proteln (vltellogenin) has been demon-
strated in salmon and trout by techniques of electrophoresis and
immunodiffusion.
Although these techniques are sufficiently sensitive to
20 enable the vitellogenin to be detected in the course of the annual
cycle, the difficulty of carrying them out in the field precludes
their being considered as being capable of furnishing the potential
users with immediate responses.
The sex-determining process according to the invention
25 consists in detecting the vitellogenin specifically present In the
blood of females at a certain moment of the cycle of reproductior~
i. e. in the course of vitellogenesis, by an lmmunoagglutination
test made on a sample of serum or blood of the animal; thls
sample ls brought into presence with a support sensitized by
30 antlvitellogenin antibodies, and the formation of a precipitate
(positive test) indicates the presence of vitellogenin.
The immunoagglutination test is based on the reaction
between the antigen (vitellogenin in the medium to be tested) and
its specific antibody fixed on a support having a relatively large
35 specific surface such as tanned sheep erythrocytes or particles
of latex.
8~6
--2--
The purpose of the support is to render the antigen-
antlbody reactlon vlsible. Although this always takes place
whatever the ratio oI the antigen-antibody concentrations, it is
vlsible only in a reduced range of the concentration ratios,
5 called "zone of equivalence". In fact, the visualisation of the
reaction corresponds to a precipitation of the antigen-antibody
complex due to the formation of a sufficient density of bridges
between the antlbodies by the antigens. The accompanying
Figure illustrates this phenomenon.
This Figure shows:
- at a), the different products present: 1) the support (latex
balls), 2) the antibody, 3) the antigen;
- at b), the sensitised support, i. e. coated with the antibody;
- at c), if the antigen is in excess: there is saturatlon by the
antigen of the sites of connection (antibody) on the
support;
- at d), if the antibody is in excess: there are only very few sites
of antibody-antigen connection on the support.
In the two cases c) and d) where one of the reagents
20 ls in excess, the density of the bridges formed is too low to
form a precipitant network, and the reaction appears negative
to the observer.
- On the other hand, at e), which shows what occurs in the "zone
of equivalence", the number of bridges formed between the anti-
25 bodles by the antigens is sufficient to provoke a visible preci-
pitation (agglutination): the reaction appears positive.
Consequently, for a given concentration of antibodies
on the support, an apparently negative reaction Inay result from
an absence of antigen, but also from an excess concentratlon of
30 antlgen In the tested medium, In this case, to decide between
these two possibilities (absence or excess of antigen), there is
adcled to the reagents already present,known serum giving a posi-
tive reaction (i. e. containlng antigen): if a precipitation is pro-
duced, there was absence of antigen in the medium tested. In the
35 contrary case, the antigen~vasin excess with respect to the con-
centration of antibody chosen.
8~16
--3--
To carry out the agglutination test, the antiboay specific
of the vitellogenin, with which an adequate support will be sensi-
tised, must be available. Antisera containing antivitellogenin
~'-globulins (anti-VgP) were prepared by injection into
5 rabblts of pure vitellogenin (VgP) isolated from the blood of
oestrogenlsed fish ( particularly of the Salmonidae family)
Obtainln~ of pure vitello~zenin:
In the Salmonidae family, the oestrogenisatlon of the
male and of the emale, whatever the time of the reproductive
10 cycle, induces a hepatic synthesis of vitellogenin and an increase
in the plasmatlc concentration of this protein (Bailey, 1957,
Takashima et al., 1972). This method was applied to female
rainbow trouts which were treated with oestradiol-17/3 by
lntraperitoneal injection for four weeks, at a rate of one injection
15 of 0. 5 mg/kg every other day. At the end of the treatment, the
animals were sacrificed and the blood collected at the caudal
artery. Purification of the vitellogenln is inspired from the tech-
nique of Hara and Hirai (1978): after coagulation, the serum is
separated by centrifugation and dialysed three days against one
20 hundred volumes of distilled water renewed each day. The pre-
cipltate formed is separated by centrifugation (300 g, 4C, one
hour). The sediment is redissolved in a tris 0. 05 M NaCl M
buffer, pH 9. 2. The insoluble matter is eliminated by centrifu-
gation. The supernatant matter is deposited on a column of
25 sepharose 6B (lm x 1. 6 cm)
The vitellogenin in the fractions issuing from chromato-
graphy was recognised by electrophoresls on 5'10 polyacrylamide
gel (14 cm long) according to the technique of Ornstein ancl Davis
(1964) modified by readjusting the pH of the migration bufer to
30 9. 2 by N NaOH. Sera of females in the course of vitellogenesis
and of males are used as reference. The richest fractions are
collected together and dlalysed against distilled water. The pre-
cipltate is taken up in the tris 0 05M NaCl M buffer, at pH 9. 2,
then rechromatographed over sepharose 6B and dialysed under the
35 same conditions as hereinabove.
The concentration of the final solution of vitellogenin
(VgP) was determined by the method of Lowry et al (1951)
,
8~6
--4--
Preparation of the antivitello~enin ~-~lobulins
0. 5 mg of VgP, in the presence of complete Freund
adjuvant, is injected into rabbits by the subcutaneous route, and
this injection is repeated every fifteen days for three months.
5 ~fter this period, the animals are bled, and the anti-VgP
~ -globulins are isolated from the serum by precipitation with
amnlonium sulfate at 33% saturation; the precipitate is centri-
fuged, dialysed against distilled water and lyophilised
Preparation of the sensitised support:
Latex balls (Difco 0. 81) were used, but any other con-
ventional adsorption support may be used. The lyophilised
anti-VgP ^~-globulins are redissolved in a veronal buffer ~0. 05M)
CaC12 (5mM). The antibody is fixed on the latex balls according
to the technique of McCarthy (1975) in which the glycine-NaCl
15 buffer has been replaced by a veronal buffer at pH 8. 2. The tests
were made by sensitising the balls by solutions containing the
anti-VgP ~r -globulin s at concentrations 4 and 9 times higher
than the concentration of the ~r-globulins in the serum.
A~zlutination test:
The agglutlnation test is carried out by bringing into
presence, on a trim~ed glass slide, a drop of the medium to be
tested, a drop of veronal buffer at pH 8~ 2 and a drop of the
sensitised latex ball suspension. The mixture is homogenised
and stirred slowly. The positive reaction, characterised by the
25 appearance of aggregates of balls, is developed in 2 to 3 minutes.
When the reaction ls negative, control positive serum is added and
1 to Z minutes are allowed to lapse to determine whether there
ls an excess (negative response) or an absence of vitellogenln
(positive response),
No difference was observed between the reactions of
the serum and of the plasma of the same animal The whole blood
may also be used to carry out the test. However, the blood
provokes coagulations which may be confused with the specific
agglutination and this difficulty is eliminated by adding heparin
35 (300 UI/ml) to the reaction buffer. For the same résponse, a
quantity of blood one and a half to two times greater than that of
11788~6
-5-
the corresponding sera must be used.
Validation of the test:
-
The validation of the responses to the agglutinatlontest was sought on the one hand by immunodiffusion on 0. 8%
5 agarose gel in veronal buffer 0. 05M, pH 8. 3, and on the other
hand by carrying out the test on the plasma of animals whose
se~ was previously determined by observation of the gonads.
The tests were made on two species:
- fario trouts (Salmo trutta): males and females were killed
10 monthly in the course of their first reproductive cycle. The
females were characterised either from the gonadosomatic
ratios (RCS) or from the precise stage and the diameter of the
ovocytes in the course of the final phases of the cycle, according
to the criteria defined by Jalabert (1978): end of vitellogenesis
15 (FV), maturing, ovulated. Plasma of each animal was obtained
after blood sampling before slaughter. This plasma is kept
frozen until the test.
- Altantic salmo_: come from catches by sport fishermen
during the whole legal fishing season. The whole blood was fro-
20 zen in situ, the serum being separated by centrifugation only afterit has arrived in the laboratory. The animals were characterised by
by thelr gonado-somatic ratio and by the mean diameter of the
ovocytes calculated from 48 ovocytes taken on six different ovi-
gerous slides.
25 Sensitivitv of the test:
Table I hereinafter shows, for concentrations of vitello-
genin varying from 120 to 0. 05 mg/ml, the responses obtained on
the one hand wlth the lmmunoagglutination test according to the
lnvention using two concentrations of ~-globulins for sensitising
30 the latex balls, and on the other hand wlth the immunodiffusion
te st.
The table shows that two different ranges of concentra-
tion of vitellogenin leading to positive responses correspond to the
two concentrations of sensitisation anti-VgP (9 times and 4 times
35 greater than that of the whole antiserum). The sensitivity of res-
ponse for media with low concentration of vitellogenin is increased
8~6
-b -
by reducing the quantity of sensitlsing antibodies.
On the two sides of the positive response range, a
negative reaction may be due either to an excess or to a lack of
vitellogenin; hence the necessity,in the case of a negative reaction,
5 of the addition to the reactlon medium of a known positive serum,
to decide between the two hypotheses.
The agglutination test according to the invention was
applied to the detection of the vitellogenin in the course of the
reproductive cycle of the two species of fish indicated hereinabove.
10 Fario trout: The results obtained are shown in Table II herein-
after,
Any any time of the cycle, no positive reaction was
demonstrated Irom the male sera.
On the other hand, in the female, the appearance of
15 vitellogenln may be detected in a few animals from April 12 and
becomes general on May 26, whilst the phase of rapid growth aof
the gonad and the ovocytes has not begun. During a period of one
or two post-ov~ation months, no positive response was obtained.
On a larger sample, the immunodiffusion test confirmed that
Z0 all the females possess circulating vitellogenin around mid-May.
Atlantic salmon Catches were spread out from the end of March
to the end of June, at which period no secondary sexual character
enabled the sexes to be differentiated
The vitellogenin is detected In all females by immuno-
25 agglutination, whilst the RGS's are low and the diameters of theovocytes do not vary substantially during the fishing season. None
of the males responded positively.
- Thus, the study carried out on a complete cycle in the
farlo trout shows that the sensitivity of the lmmunoagglutinatlon
30 test allows a qualltatlve detection of the vltellogenin from the
month of April and constitutes a sufficiently reliable process for
sex determination as frorn the month of May. The use of the process
according to the invention therefore enables stocks of breeding
females to be constituted in Iish Iarms several months before the
35 app~ ance of the morphologlcal features generally used for recog-
nising the sexes,
8~36
~ or the Atlantic salmon, earlier studies indicate the
presence of vitellogenin in the females at dlfferent stages at
ascent and on spa~hning grounds. The study carried out with the
agglutination test gives the same results: all the females fished
5 between the beginning of March and mid-June respond positively;
vitellogenesis is apparently therefore started at the moment of
entry into fresh water. The carrying out of the process according
to the invention at the traps is therefore suitable for determining
the sex ratio and reproductive potential of a population of salmons.
The concentrations of sensitising antibodies have
enabled detection limits to be obtained, compatible with the rates of
circulating vitellogenin in the majority of the animals starting
vitellogenesis. A greater sensitivity of the test may be obtained
by reducing the quantil;y of antibodies fixed on the balls. These
lS condltions should ~make lt possible to extend the ranges o
positlve responses towards the low concentrations of vitellogenin,
and to increase the percentages of sex determination at the be-
ginning of cycle. However, lt should be specified that, the more
the test ls sensitive, tne more the response times increase and
20 the less clear is read-out. Thus, the definition of the optimal
sensitising antib~dy/support ratios must be determined in each
pa rti cula r ca s e .
11 J~86
E
Q, ,~, o ~
~ ~ ~ X ~ o~ ~ ~ E ,~
``-" 11'-ib~886
_ ~_
~
CL, h ~ ~ ~ ~ ~ ~
O
V ¢ O + + + + + +
h 5 ~ _
~ ~o ~
_ ~ X O h
I ~ ~ ~ _ +
~ ~0 ~ ~ ~
~ O ,L h ~ U~
O '~- ~n + + + + + + + + + + + + + _ ~
~ . cX Iq~,~ ~ ^J ~.
. ~ O ~ -- + ' + + + + h
~1~ I ~ ? D ,C o ~ D ¦
~ , _ ~ ~0
l ~ 0~ C~^ O^ O- 0 0~ 0- O^ 0~ O^ C
b + ~ + ~ + ~ + ~ + ~ + ~ + ~ + ~ + ~ + ~ o
. l ~ 1 ~ ~_ O ~;~. V V ~/ V v V c~ o ~ ~ ~ ~ h
. l `$ i~ ~ ~ ~ C`l ~`1 ~) ~ `;t O
I _ _ t~
~ ~ ~ C~ o oo ~ U~ o o U~
_ ~ O ~ ~ ~ ~ u~ ~n
~c~l~OOOOOOOO~ Q)
Q~ - ~ -1-1 +1 +1 +1 +~ +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 ~c ~.
. C~ +1 ~ O ~ o ~ C
~ ~ ., h ~d 'V
~ ~ ~ D ~1 0 0 0 0 ~ ~1 ~ ~ O ~ r~ r~ h ` C
~ I ~ ,U ~ 0~
CJ ~ r~ r~ r~r~ r~ r~ I~ ~V ~
u r~ r~ r~ r~ r~ r~ I~ r~ r~I~ r~ r~ r~ r~ r~ r~ 3 ~ O r
i ~1 ~ r-l r~l ~1 _I r-l ~1 ~1~1 ~1 _I ~I r-l ~ r~l O O h ,_~
~r O O O O O O O O O O ~1
a c~i ~D o ~ ~ o co ~ o r~O r~o ~ ~ rr~ C ~
O ~ ~ ~ ~ ~ ~ ~ ~ ~r ~ ~ ~ ~ ~ ~ h o ~ C,
~ D ~ 'V
~ o r~ rJ~ rl~ rr\ r~ r~ rl~ rl~ ~r~ ~ ~ C ~ IC D
