LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL.

XLII (2), 2009, TIMIŞOARA

THE EFFICIENTISATION OF THE ASEPTISATION OF THE

SEMINAL MATERIAL AT BOAR BY USING ANTIFUNGI IN THE
DILUTING AGENTS
S.G. CIORNEI1, L. RUNCEANU1, D. DRUGOCIU1, P. ROŞCA1, VIOLETA IGNA2,
2 1
ILINCA FRUNZĂ , CRISTINA PANAITE
1
Faculty of Veterinary Medicine Iasi, M. Sadoveanu Alley No. 8, 700489,
Iasi, Romania
2
Faculty of Veterinary Medicine Timisoara
E-mail: stefan_ciornei@yahoo.com

Summary

A various group of fungi has been identified by doing qualitative mycologic tests of
the diluted seminal material at boar. 98% of these were yeasts. In the diluting agents formula
has been introduced an antimycotic agent for a better aseptisation of the seminal material by
dilution. The quality of the diluted sperm was analyse with C.A.S.A. system to establish the
compatibility. The evaluation of the motility (M%) and progressivity (P%) of the
spermatozoons in the witness and experimental samples was periodically carried out during
the 48 hours in which those doses were preserved at +170 C. The mobility parametres
studied in dynamics have progressively decreased along with the increase of the
preservation duration both of the experimental lots and the witness ones, but to a lower rate.
The results obtained after 12 hours from the dilution demonstrate that the fluconazole of 125
mg‰ has a stimulative effect on the seminal cells, increasing the percentage of those which
have a fertilizing capacity.
Key words: boar, motility, progressivity, diluting agent

The importance of the study factors that may influence the biological value
of the sperm is overwhelming, concerning the quality of the sperm for preservation.
Among these, the presence of the microbial flora has a very important role.
Even the sperm that has been collected taking good care of the aseptic
conditions has bacteria; some authors and L. Runceanu rated the number of
bacteria as between 120 and 270 000 UFC/ml. The numeric appreciation of the
semen germs has a very practical importance, as the semen metabolical changes
can occur not only because of the bacterian quality, but also due to the number of
bacteria per unit volume.
The presence of these bacteria in diluted semen is harmful to
speratozoons viability and represents an infection risk by seeding sows.
Abortions and infecundity in sows correlated with the agglutination and
decreased spermatozoons viability in diluted semen appear by many researchers
due to microbiological contamination dose for sowing.
For a better seminal material aseptisation, we propose and recommend the
introduction in the diluent’s formula of antibiotics and a antimycotic preparation,

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without major change to the longeviability and fecundate capacity of

spermatozoons.
The absence in domestic and international specialized literature of the
contamination data and the possibility of fungal aseptization of boar semen during
preservation has led us to the motivation to try to modify or complete the diluents
formula used for the preservation of boar semen.
Modern livestock units, by biosafety, are free of specific genital
microorganisms, thus making microbiological spermiogram is focused towards
microflora of the association present in semen, which may change the parameters
quality of the insemination doses.

Materials and methods

Research has been performed on samples of semen from boars used for
artificial insemination in the breeding and exploitation units of the swine-intensive
industry system. Zoohygiene conditions, maintenance and operating parameters
were appropriate.
Microbiological determinations were performed and the results confirmed in
the laboratories of Reproduction, Reproduction Pathology, Bacteriology and
Mycology of the Veterinary Medicine Faculty of U.S.A.M.V. from Iasi and in the
Microbiology Laboratory of the Sanitary Veterinary and Food Safety Laboratory
from Iasi. The experiment and it's results, to establish the compatibility degree
between diluent and the semen was performed in the laboratory of Reproduction,
Obstetrics and Gynecology of the Faculty of Veterinary Medicine, from
U.S.A.M.V.B., Timişoara.
For typification the fungal genera were used microenzymatic kits "MINIAPI"
(ID 32C gallery for yeast). To establish the active substances act as large were
used ATB-fungus 3 antifungigrams.
For both better aseptisation semen by dilution, an antimycotic agent has
been introduced into the diluent formula and for determining the concentration the
diluted sperm quality was analyze with the Computer Assisted Sperm Analysis
(CASA) system, of Sperm Analizer IVOS analyzer.
The study was conducted on boars of the same genetic line (PIC), with
ages between 2 and 5 years. After harvesting known protocol and qualitative
analysis of semen, a lot of witness and two experimental lots for each boar have
made (Table. 1).
Table 1
The composition of witness and experimental lots
Witness samples (L.M.) Diluant + semen
Experiment 1 (L.E. 1) Diluant + semen + 250mg Fluconazol/1000ml
Experiment 2 (L.E.2) Diluant + semen + 125mg Fluconazol/1000ml

Samples were worked under strict conditions of isothermal and preserved

at 17 ºC for 48 hours.
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The Computerized Sperm Analysis system (CASA) provides information

about the mobility of each sperm cell, processing electronic images of
spermatozoons, reconstructs the trajectory of each sperm cell, simultaneously and
objectively evaluate each component of the spermatozoon so that minor changes
of spermatozoons mobility may be detected.

Results and discussions

After analyzing the mycologycal qualitative exams of boar semen, after

dilution, a variety group of fungi was identified. In the insemination doses of sows
they found favorable conditions for growth, development and multiplication.
Following the biochemical processes they change the quality parameters of semen,
which is negative reflected on the fecundity and prolificity, and ultimately on
economic efficiency.
An important to note is that among the persistent fungi in diluted semen,
98% of them are yeasts. Being determined in relatively constant way and at the
same titre, we believe that yeasts have an internal origin of contamination.
To know the sensitivity of the yasts strains, they were incubated on ATB
fungus 3 galleries (Figure 1). By using these antifungigram it was able to identify
both antifungi acting on those strains and concentrations which are sensitive at –
by using the Minimum Inhibitory Concentration (MIC) shown on galleries. The
incubation galleries contain several active substances like: (SFC, AMB, FCA, ITR
and VRC). Following the calculations it was observed that the yeasts development
was inhibited by most antifungi and the concentrations they were inhibited ranged
from 0.25 - 4mg/1000 ml. In the specialized literature states that the therapeutic
dose should be greater than the MIC-size to ensure neutralization of all possible
existing strains.
For our studies experiments was chosen Fluconazol used at maximum
concentration of 250 mg/1000 ml diluent, as the antimicotic preparation. We point
out that the MIC was 125 mg/1000 ml, all tested strains were inhibited and it is
hydrosolubile - these are the arguments that formed the basis of choice.

Fig.1. Testing yeasts sensitivity by using ATB fungus Gallery 3

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Today on the market are different diluents for boar semen. They can
preserve semen 2-3 - 5 to 7 days, depending on the ingredients contained. To
conduct our research a diluent was chose, which is used increasingly often in
production farms with notable results.
Adding Fluconazol in the diluant-cocktail was made under isothermal and
strict hygiene conditions. To establish an effective concentration against yeasts,
without altering the fecundant capacity of spermatozoons, were added various
concentrations followed by analysis of semen quality in dynamics during
conservation.
The parameters of semen quality were observe: total spermatozoons
motility (M%) and their progresivity (P%). Progresivity represents the percentage of
spermaozoons able to move in straight line (only those have the ability to fertilize
eggs). These parameters were determined by a computerized analyzer CASA
(Figure 2 and Table 2).
Table 2
The experimental protocol values in the conservation dynamics
T.1 T. 2 T. 3
M% P% M% P% M% P%
L.M. 82 42 70 27 47 14
V1 L.E.1 82 47 73 33 46 17
L.E. 2 84 46 65 27 42 12
L.M. 78 36 69 36 70 36
V2 L.E.1 63 31 61 21 51 22
L.E. 2 78 39 65 33 46 20
L.M. 80 39 69,5 31,5 58,2 25
X L.E.1 72,5 39 67 27 48,5 19,5
L.E. 2 81 42,5 65 30 44 16
L.M. – witness lot. L.E.1 – experimental lot 1 (fluconazol 250 mg‰), L.E.2 –experimental lot
2, (125‰ fluconazol). M% - total motility. P% - progresivity (fecundant capacity ). T 1-3 –
analizing parameters in dynamic ( during preservation the semen at +17˚C, la 12, 24 şi 48 h)

For our protocol research, assessing motility (Figure 3) and progresivity

(Figure 4) of spermatozoons samples was performed regularly during the
conservation of those doses. The isothermal conditions and working methods of
the CASA analyzer were strictly adhered.

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Fig. 2. The appreciation of spermatozoons mobility through the C.A.S.A. system

12 hours after dilution, the samples motility (M%) from group (LM) had
between 82% and 78% with an average of 80%. Among the spermatozoons with
total motility, an average of 39% had foreword movements (Progresivity), with limits
between 42% and 36%.
In the experimental lot 1 (LE 1), where the concentration of Fluconazol was
250 mg/1000 ml diluant, were obtained for M% an average of 72.5%, so by 7.5
percent less than the LM, in contrast, the average P% recorded equal values
(39%). The variation limits were between 47% and 31%.
L.M. L.E.1 L.E.2
T.1. T.2. T.3
90 45
81 42.5
80 80
40 39 39
72.5 69.5
35
70
67
31.5
65 30 30
60 58.2 27
25 25
50 48.5 20 19.5
44
16
40 15
30 10
L.M. L.E.1 L.E.2 T.1. T.2. T.3

Fig. 3. The average of the total motility Fig. 4. The average of the fecundant
percentage in the conservation dynamics capacity in the conservation dynamics

When the concentration of Fluconazol from the diluant was reduced to half,
125 mg/1000 ml in LE 2 (but not below the MIC indicated by the antifungigram), the
average value of M% was 81%. This value is greater than L.M. by 1%. The
progresivity (P%) in this case has the average of 42.5%, registering an increase by
3.5%.

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These values, which are superior to those of L.M. can be explained by

assuming that Fluconazol of 125 mg/100 ml diluant has a stimulating role on the
seminal cells, increasing the percentage of those with rectilinear motion
displacement. Thus these values improves the semen fecundant capacity, at least
in the short term.
According to the protocol, the determinations were made periodically at 24
and 48 hours after dilution, in this time samples were preserved under the specified
conditions for diluant to +17 º C.
The mobility parameters studied in dynamics had been gradually
decreased as increasing the length of preservation on both conservation witnesses
and experimental lots.
Such determinations made 24 hours after dilution (T2) showed average
values of motility in experimental lots (L.E.1 and L.E.2), noticeably lower (67% to
L.E.1 and 65% to L.E.2) than those recorded at L.M. (69.5%).
The determination results made at 48 hours (T3) show lower average
values of progresivity by 5.5% to L.E.1 and by 9.5 % to L.E.2, compared to the
average of 25% recorded by this parameter to L.M.
The decrease of the average values of M% and P% recorded to both L.E.,
both at 24 and 48 hours after dilution, is correlated with the decrease of these
parameters values to L.M., but that decrease is achieved at a lower rate.

Conclusions

For more effective aseptisation of boar semen, including the antifungical

aseptisation, there is necessary, besides ensuring the rules of harvest hygiene, the
introduction in the diluting agents formula of some substances with antifungical
properties.
The experimental protocol aimed to use fluconazol in two concentrations:
125 mg ‰ for L.E.2, and 250 mg ‰ for L.E.1, as antifungal.
The measurements made 24 hours after dilution (T2) showed average
values of motility in experimental lots (L.E.1 and L.E.2), noticeably lower (67% to
L.E.1 and 65% to L.E.2), than those recorded at L.M. (69.5%).
The results of determination made at 48 hours (T3) show lower average
values of progresivity by 5.5% to L.E.1 and by 9.5% to L.E.2, compared to the
average value of 25% recorded for this parameter to L.M.
A concentration of 125 mg ‰ fluconazol, added to the diluant, has
beneficial effects for the semen preservation in a short term, at +17 ˚C.

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