INTRODUCTION - Journal of Apicultural Science

Vol. 55 No. 2 2011
Journal of Apicultural Science
5
MICROSCOPIC IMAGE OF HONEYBEE DRONE SPERMATOZOA
IN THREE DILUENTS
G r z e g o r z B o r s u k 1, K r z y s z t o f O l s z e  s k i 1,
A n e t a S t r a c h e c k a 1, J e r z y P a l e o l o g 1, M a r i u s z G a g o  2,
3
1
Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding,
2
Department of Biophysics, University of Life Sciences in Lublin,
Akademicka 13, 20-950 Lublin, Poland
3
Department of Cell Biology, Institute of Biology and Biotechnology,
Maria Curie Skodowska University, 20-033 Lublin, Poland
e-mail: [email protected]
Received 25 March 2010; Accepted 19 October 2011
S u m m a r y
Microscopic analysis of the behaviour of spermatozoa diluted in diluents was conducted in vitro
and in vivo. Additionally, an attempt was made to determine the factors that may affect semen
coagulation in the instrumental insemination process.
Under a microscope, the following semen smear groups were observed:
1. according to drone age;
2. in commercial diluents: saline, SAFE boar semen diluents;
3. 0.2 l semen with spermathecal uid from 4-6 virgin queen bees;
4. from partially lled spermatheca - we used spermatozoa after “reinsemination”.
Twelve smears in each group were viewed under the OLYMPUS microscope. The microscope
was coupled with a digital camera for lming and taking photographs of the semen.
Motility of spermatozoa/semen was observed in the preparations. A stopwatch was used to
measure the length of time between preparations and the beginning of sperm movement until full
cessation of movement.
Semen from young drones did not coagulate. Semen coagulation most frequently occurred (44%)
when mixing the semen from old and mature drones.
After 12 min, cessation of spermatozoa movement was observed in the semen diluted with saline.
Spermatozoa in the boar semen diluents exhibited motility for 29 min.
After “reinsemination”, spermatozoa displayed circular arrangement.
Keyords: semen smears, semen coagulation, reinsemination, drone, Apis mellifera.
INTRODUCTION
Communities of social insects are
characterised by a polyandric mating
system (S tra s sm an n , 2001). In natural
mating, a queen bee may copulate with
many drones (R u t t n er, 1969; Mo rit z,
et al., 1996; S ch l  n s , et al., 2004).
This is related to polyandry (Tri as k o ,
1951; B o rsu k et al., 2011a,b), which is
most highly organized in bees (F u c h s
and M o rit z, 1999; Ko e n ige r and
Ko en i g er, 2000; P al m er and Ol d ro y d ,
2000). Natural mating occurs in the air,
which means a person cannot control the
choice of the paternal side. Improved
methods of instrumental bee insemination,
however, have provided full control over
the selection of the paternal side during
mating. This significantly contributed to
progress in bee breeding. Still, it is not
known why instrumentally inseminated
queen bees do not always commence
oviposition. It is also not known why queens
who have been instrumentally inseminated,
start egg-laying later than naturallyinseminated queens (Ma ck e n s en , 1951,
1964; J as i  s k i et al., 2005; W o y k e et
al., 2008).
6
In the past, the efficiency of properly
performed instrumental insemination was
affected by semen quality and lack of
drone-specific diluents as well as other
factors. Nowadays though, their analogues
are applied with a common 0.9% saline
solution and boar semen diluents.
However, most diluents or substances are
exposed to semen, reducing the survival
rate of spermatozoa (B i sh o p, 1920).
This explains why an air space in the
insemination needle is necessary during
the instrumental insemination. It prevents
semen contact with the liquid in the needle
and syringe.
Another factor that affects the efficiency
of the properly performed insemination
procedure
is
semen
competition.
Inseminators claim that semen tends to
coagulate in the needle during numerous
insemination processes (Loc personal
information). This coagulation markedly
prolongs the insemination process.
Internal complications may occur after
natural mating between the queen bee and
several drones (Sc h l n s et al., 2004;
An d ere et al., 2011).
The internal complications may be
caused by one drone, which, for instance,
is the first to mate and contributes to
semen coagulation. Semen coagulation
consequently affects further sperm storage
(Pizz ari and F o s t er, 2008). After
the mating, spermatozoa may compete
between ejaculates from different drones
(polyandry), which may induce their
coagulation. The coagulation may be
caused by incompatibility in the sex locus
(Ma ck en se n , 1964) as well as population
selection. If this is so, none of the semen can
be used since it remains in the queen bee’s
reproductive tract (W o jc ie ch o ws k i and
Kr l, 1996).
The survival of the semen stored by the
queen bee is affected by the spermathecal
fluid (V erm a, 1973, 1978) and by
substances secreted by the spermathecal
glands (S ch l n s et al., 2004). The
spermatheca
contains
antioxidative
enzymes protecting the spermatozoa from
oxidative stress (W e iri ch et al., 2002).
It is possible that such stress is induced
by competition between the spermatozoa
(Tr ia sk o , 1951). The competition
involves increased motility, ensuring
sperm precedence in reaching the queen
bee’s spermatheca.
Another reason for lack of oviposition
may be aggregation of spermatozoa
from one drone, which form unbreakable
bundles (Mo ri tz et al., 1996).
After copulation, the continually mixing
spermatozoa are well visible through the
transparent spermathecal wall (R u t tn e r,
1954; W o y k e, 1960).
After insemination with mixed semen
originating from African and European
drones, a higher rate of egg fertilization
by African drones was observed. The
ratio was 9:1 (Gra n d i -Ho ff m an et
al., 2004; S c h nei d er et al ., 2006).
Similar dominance was noted in wild
drones and in those selected in the drone
congregation areas with the progeny
ratio 4:1, respectively (W o y k e, 1971).
This indicates high competition between
spermatozoa (Tofi ls k i et al., 2011).
The introduction sequence of semen
into a queen bee’s reproductive tract also
plays an important role. There is little
probability of progeny from drones that
were the last to be used for insemination
(W o y k e , 1963; Siu d a et al., 2009). This
is because excess semen from the queen
bee’s reproductive tract is removed.
The techniques employed in the
evaluation of the semen include the
macroscopic method and the in vivo
microscopic method. The macroscopic
method uses the consistency estimation
according to Wo y k e (1964). W o y k e
classifies semen into: 1) sparse semen,
which is not suitable for insemination,
2) semen that is optimal for insemination,
and 3) dense semen - also not suitable for
insemination. Semen used in instrumental
bee insemination can be assessed with the
in vivo microscopic method based on the
number of laid eggs (J as i  sk i et al.,
2005; W o y k e et al., 2008).
The aim of the experiment was to
perform an in vitro and in vivo microscopic
Vol. 55 No. 2 2011
Journal of Apicultural Science
analysis of diluted spermatozoa behaviour
in diluents. Additionally, an attempt
was made to determine the factors that
may affect semen coagulation in the
instrumental insemination process.
MATERIALS AND METHODS
The study was conducted in the Breeding
Apiary in Teodorw, Poland. Carniolan
honeybee Apis mellifera carnica drones
and queen bees were investigated.
The following semen smear groups were
observed under the microscope:
1. according to drone age; Three age
groups were distinguished: young drones
up to 12 days old (sparse semen), mature
drones which were from 14 to 27 days
old (optimal semen for instrumental
insemination), and older drones which
were over 30 days old (dense semen);
2. in commercial diluents: saline, SAFE
boar semen diluents;
3. 0.2 l semen with the spermathecal
fluid from 4-6 virgin queen bees;
4. from partially filled spermatheca. The
queen bees were inseminated by one drone
with a dose of approximately 1 l. After
commencement of oviposition the semen
was collected from the spermatheca. Then,
this semen was used for “reinsemination”
of twelve virgin queens using a dose of
approximately 0.5 l. The semen smears
from the partially filled spermatheca and
the spermatozoa after the “reinsemination”,
Fig. 1. Semen from a young drone.
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were prepared to assess the behaviour of
the spermatozoa in the spermatheca.
The semen was mixed with diluents
in a ratio of about 1:4. The smears were
prepared on a Brker’s plate. Twelve
smears were made from each group. The
smears were observed under an OLYMPUS
microscope. The microscope was coupled
with a digital camera to film the observed
semen. Motility of spermatozoa/semen
was observed in the preparations.
A stopwatch was used to measure the
length between the time the preparation was
made and the beginning of spermatozoa/
semen movement - until full cessation of
movement.
The mean time of spermatozoa/semen
movement in the preparations was
calculated with SAS software (Institute,
SAS User’s Guide Version 9.1 ed., 2003)
using univariate ANOVA.
RESULTS AND DISCUSSION
1. Behaviour of the semen in relation
to drone age
Sparse semen from young drones had
a cluster visible in loose spermatozoa
bundles and short mobility duration
(XSD=4.61.5 min, n=12) (Fig. 1).
Similarly, J ay cox (1960), L en s k y and
Schindler (1967), R u t tn e r (1969) and
V erm a (1978) assessed the spermatozoa
after cessation of movement. The optimal
semen for instrumental insemination
8
Fig. 2. Semen from a mature drone.
Fig. 3. Semen from an old drone.
exhibited
long
mobility
duration
(XSD=26.62.3 min, n=12) and was
accompanied by a high cluster mass of
spermatozoa bonds (Fig. 2). Dense semen
sampled from old drones displayed short
mobility duration (XSD=155.7 min,
n=12) in the microscopic picture (Fig. 3).
This dense semen had a very high cluster
mass of spermatozoa and varied mobility
duration. Dense semen demonstrated high
diversity and had a tendency to coagulate.
The tendency to coagulate lowered the
motility of the spermatozoa and induced
their immobility. In the non-coagulated
semen, movements of the spermatozoa
were still visible.
The mixed semen from two mature
drones was very active but this activity
later disappeared. Such reactions may
indicate
competition
between
the
ejaculates (Tr ias k o , 1951). Similar
behaviour was observed in a mixture of
ejaculates sampled from a young and a
mature drone. At first, enhanced motility
was observed. This enhanced motility
may have been induced by an increased
amount of the seminal vesicle fluid and of
the endophallus bulb (F l an d e rs, 1939;
W o y k e, 2008) or proteins (Ki n g et
al., 2011). Sparse semen from immature
drones did not coagulate when combined
with the optimal consistency semen used
Vol. 55 No. 2 2011
Journal of Apicultural Science
for insemination. The lack of semen
coagulation may indicate that there is no
hemolymph and endothelium in the young
drone semen (R u t tn er, 1954; W o y k e ,
2008, 2010).
Dense semen caused rapid loss of
motility and more frequent (44%)
production of coagulates, not motility
bands. The cessation of semen movement
may have been induced by the increased
amount of epithelium entering the semen
(Wo y k e , 2008, 2010). Such an increase
in epithelium may be a result of the
drone aging process (B u rzy  s ki , 2007;
R ho d e s et al., 2010; P al eo l o g et al.,
2011). It may also result from the peeling
9
of considerable amounts of the epithelium
from mucus glands during eversion of the
endophallus.
2. Behaviour of the semen in
commercial diluents
The semen from the young, mature
and old drones was treated with a salinedilution. During the treatment, the
spermatozoa displayed quick movements,
which on average, ceased after
XSD=12.06.2 min, n=12. Movement
cessation was an indication of dying
spermatozoa (Ru tt n er , 1969). Dilution
resulted in disruption of the bonds and the
first characteristic circular arrangement.
Fig. 4. Semen from a mature drone mixed with the spermathecal
uid of a non-inseminated queen bee (0,2 l semen with the
spermathecal uid from 4-6 virgin queen bees).
Fig. 5. Spermatozoa after “reinsemination”.
10
The boar semen diluent also induced
increased
spermatozoa
motility
accompanied
by
almost
complete
disruption of the bonds of the spermatozoa,
which then restored the bonds. On average,
during XSD=29.22.5 min, n=12 period,
single spermatozoa at the sides of the
preparations displayed the highest motility.
3. Behaviour of the semen in
combination ith the spermathecal fluid
Semen sampled from a mature drone
and mixed with the spermathecal liquid
of a non-inseminated queen bee initially
displayed
intensive
motility.
The
mobility declined after a few minutes and
remained at a constant level for average
XSD=50.87.0 min, n=12 (Fig. 4). After
the queen bees were inseminated with
such a mixture and smears from their
spermatheca were prepared, it appeared
that the spermatozoa were arranged in
loose bundles. These loose bundles formed
circles which moved in a counter clockwise
manner (Fig. 4).
Ki n g et al. (2011) argue that sperm
viability is dependent on protein and
fructose found in seminal fluid whereas
Gen e r and K ah y a (2011) state that
sperm viability is dependent on lateral
oviducts and spermathecae substances.
4. Behaviour of the semen sampled
from the partially filled spermatheca
In the partially filled spermatheca,
highly motile spermatozoa arranged in
the form of loose clusters were observed.
Such spermatozoa have the ability of
unconstrained movement and are not
arranged in a cluster mass of spermatozoa
bonds (R u t t n er, 1954; W o y ke, 2008,
2010), therefore their activity is increased.
The higher the cluster of the spermatozoa
in the spermatheca is, the more compact
the bundles are. This means that the
spermatozoa remain in tight clusters
without being able to move.
Characteristic behaviour was observed
in the spermatozoa sampled from the
spermatheca of the queen bees after
“reinsemination”. The spermathecae were
partially filled due to the small amount
of semen sampled from a previously
inseminated ovipositing queen. In all the
smears, the spermatozoa formed more or
less compact circles. The compactness
depended on the amount of semen sampled
from the spermatheca of an ovipositing
queen bee (ca. 0.5 l). All the spermatozoa
were arranged in circles, which displayed
a rotary counter clockwise motion (all
moving in the same direction) (Fig. 5).
The circle arrangement may be related to
the length of the spermatozoa, which is
ca.  mm (R u tt n er, 1954). The circle
arrangement facilitates storage of the long,
circularly arranged spermatozoa in small,
round, 1 mm in diameter spermathecae.
The arrangement of the spermatozoa may
also be related to polyandry. When sampled
from several drones, the spermatozoa form
circular clusters, mix in the queen bee’s
spermatheca (R utt n er, 1954; W o y k e ,
1960), and move into the vas deferens.
Thus, the circular clusters close the vas
deference temporarily until they become
scarce. Other circular clusters then take
their place. This is corroborated by the
fact, that fertilized eggs laid by a queen bee
recurrently descend from a different drone.
A very visible example is in a bee family
with naturally mated dark-coloured bees,
where occasionally light-coloured bees
appear after the queen bee has copulated
with several light-coloured drones.
CONCLUSION
Semen from mature drones coagulates
the earliest. Semen from young drones
does not coagulate.
ACKNOwLEDGEMENTS
We thank Prof. J erz y W o y k e for
critical support and constructive comments
during the preparation of the manuscript.
We also wish to thank Mr. Krz y s zt o f
L o c for his invaluable help in conducting
the experiments.
Vol. 55 No. 2 2011
Journal of Apicultural Science
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Vol. 55 No. 2 2011
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13
MIKROSKOPOwY OBRAZ PLEMNIKw
TRUTNI PSZCZOY MIODNEJ w TRZECH ROZCIECZALNIKACH
Borsuk G., Olszes ki K., Strachecka A.,
Paleolog J., Gago M.
S t r e s z c z e n i e
Celem bada bya mikroskopowa analiza zachowania si plemnikw w rozcieczalnikach in vitro
i in vivo. Dodatkowo prbowano okreli, ktre z czynnikw mog wpywa na koagulacj nasienia
podczas instrumentalnego unasieniania.
Badano trutnie i matki rasy kraiskiej Apis mellifera carnica.
Utworzono cztery grupy dowiadczalne. Nasienie obserwowano w rozmazach mikroskopowych:
1. pobrane od trutni w rnym wieku. Wyodrbniono trzy grupy wiekowe: trutnie mode - do
12-go dnia ycia (dajce rzadkie nasienie), trutnie dojrzae - w przedziale wiekowym 14-27 dni
(dajce nasienie optymalne do instrumentalnego unasieniania), trutnie stare - powyej 30-stu dni
(dajce gste nasienie).
2. z handlowymi rozcieczalnikami (sol zjologiczn oraz z rozcieczalnikiem nasienia knurw
SAFE),
3. z pynem pochodzcym ze zbiorniczka nasiennego dziewiczych matek,
4. ze zbiorniczka nasiennego matki, ktr unasieniano jednym trutniem (dawka ok. 1 l) i czekano,
a zacznie skada jaja nastpnie matk zabijano i pobierano nasienie ze zbiorniczka nasiennego,
ktre wykorzystywano do powtrnego unasieniania dziewiczej matki - „reinseminacja” (dawka ok.
0,5 l). Rozmaz plemnikw ze zbiorniczka nasiennego matki, po „reinseminacji” uyto w celu
stwierdzenia zachowania si plemnikw w zbiorniczku nasiennym innej dziewiczej matki.
Nasienie mieszano z rozcieczalnikami w proporcji 1:4. Rozmazy wykonywano na pytce
Brkera. W kadej grupie wykonano po 12 rozmazw, ktre ogldano w mikroskopie OLYMPUS.
Mikroskop poczono z kamer cyfrow, ktr nagrywano lmy nasienia i wykonano zdjcia.
W preparatach obserwowano ruch plemnikw/nasienia. Stoperem mierzono czas od wykonania
preparatu, pocztek ruchu plemnikw/nasienia w preparacie do cakowitego zaprzestania ruchu.
Nasienie od modych trutni nie powodowao koagulatw. Najczciej do koagulacji nasienia
(44%) dochodzio po zmieszaniu nasienia pochodzcego od starych trutni z nasieniem od dojrzaych
trutni.
Po 12 min obserwowano zaprzestanie ruchu plemnikw w nasieniu rozcieczonym sol
zjologiczn. Plemniki w rozcieczalniku dla knurw wykazyway oznaki ruchu przez 29 min.
Plemniki po „reinseminacji” ukaday si w koa.
Soa
kluczoe: rozmaz nasienia,
Apis mellifera.
koagulacja
nasienia,
reinseminacja,
trutnie,