Introduction
Artificial insemination using fresh diluted semen is used worldwide
in the swine industry. Diluted semen is stored for 3 days to 7
days in certain extenders, eg. BTS, Androhep®, Xcell®.
Viability of spermatozoa in diluted semen depends on several factors,
such as type of extender, type of antibiotics and degree of contamination
in semen. Bacterial contamination in semen is one of the factors
that occur accidentally during the collection process or from
instruments in laboratory. Contamination of diluted semen with
E. coli was reported to reduce motility, fertility
and viability and to severely damage acrosomal membranes. Adding
an appropriate antibiotic could prolong viability and fertility
of sperm. The most common antibiotics used in extender are gentamicin,
neomycin, a combination of penicillin G and streptomycin, amikacin,
lincomycin and ceftiofur.
There is no published study that examined the concentration or
effect of antibiotics in extended semen during storage time. Two
fundamental questions are; does concentration of antibiotics decrease
over period of time and does antibiotics help maintain fertility
of sperm cells for longer storage? The present study was designed
to determine the influence of antibiotic on semen quality and
sperm fertility in minimally-contaminated environment and to study
the "pharmacokinetics" of antibiotic concentration in
extended semen.
Experimental design
Boar semen was collected from 3 boars at the Swine
Educational Unit, NCSU, during July-December 1997. Semen was extended
in Beltsville Thawing Solution (BTS). The diluted semen was divided
into 5 aliquots, 80 ml each with at least 3x109 cells,
to each of which was added one of the following antibiotic solutions,
a combination of penicillin G (1,000 IU/ml) and streptomycin sulfate
(700 IU/ml), amikacin (200 mg/ml), neomycin sulfate (1 mg/ml),
gentamicin sulfate (200 mg/ml) or BTS extender (as a control).
All aliquots were then stored at 17OC.
Sperm motility, acrosomal morphology and sperm morphology were
determined on days 0, 1, 2, 3, 5 and 7. Sperm motility was determined
by warm caffeine coated slides under phase contrast microscope.
Samples of the semen were fixed in formalin buffered saline to
determine sperm morphology and acrosomal morphology. The concentrations
of gentamicin sulfate and amikacin sulfate were determined in
semen samples collected at 0, 6, 12, 24, 36, 48, 72 and 120 hours.
The samples were centrifuged and the supernatant was frozen at
-70OC until assayed.
For the in vitro penetration assay, semen was diluted
as above and store for 5 days. In vitro maturation of oocytes
and in vitro fertilization were conducted with a conventional
protocol. Sperm from day 0 and day 5 diluted semen was used for
in vitro fertilization. Penetrated oocytes were evaluated
with the Orcein staining method. Oocytes with a decondensed sperm
head(s) or male and female pronuclei were considered penetrated
oocytes.
Results
Table 1. Effects of antibiotics on percentage of motility,
morphology and normal acrosomal ridge. Values represent means
for semen samples from 3 boars, evaluated at day 0,1,2,3,5 and
7.
|
| |||
| Values with different superscripts differ at p<0.05 | |||
Table 2. Effect of time on percentage of motility, morphology and normal acrosomal ridge.
Values represent means for semen samples from 3 boars, treated with antibiotics and control.
| morphology (%) | ||
| Values (within column) with different superscripts differ at p<0.05 | |||
* 60% motility was reported as a cut-off point for fertility in
boar semen (Flowers, 1997).
Pharmacokinetics test of concentration of gentamicin and amikacin
sulfate did not change over 5 days of storage.
In vitro penetration assay
From in vitro penetration, there is no effect of treatment
with antibiotics and boar on the percentage of sperm penetration.
However, the penetration rate in day 0 semen was greater than
that of day 5 semen (18.4-35.9 and 2.3-17.2, respectively, p<0.05).
Discussion
The results show that treatment with antibiotics had an effect
on sperm motility only. Semen treated with gentamicin and neomycin
sulfate showed greater percentages of motility than the others.
Treatment with different antibiotics did not affect sperm morphology
and normal acrosomal ridge (Table 1). All semen characteristics
were affected by storage time (Table 2). From Fig 1, all treatments
showed the same trend of slightly decreased motility from day
0 to day 3 of storage. After 3 days of storage, however, gentamicin,
amikacin and neomycin treated groups showed different trends of
decreasing in motility compared to that of the penicillin-streptomycin
and control groups. Semen treated with penicillin-streptomycin
and in the control group showed rapidly decrease in motility after
day 3. At day 7 of storage only semen treated with gentamicin
showed motility at about 60%. Flowers (1997), reported that inseminated
sows with semen that contained lower than 60% of motility would
reduce reproductive performance.
From the in vitro penetration assays, there was no effect
of treatment with antibiotics on sperm penetration rate. We might
conclude that antibiotics did not affect sperm penetration rate.
Antibiotics may only protect sperm cells from detrimental effects
of bacteria or endotoxin but had no role on sperm cells. Auroux
et al (1991), showed the effect of sperm:bacteria ratio on the
impairment of sperm motility. The data showed the motility of
sperm decreased significantly when the bacterial population was
numerous or the sperm count was low. They suggested that bacterial
adherence to sperm was the most likely cause of motility decrease
and sperm death. This observation would be a primary reason for
using the appropriate antibiotics to control bacterial growth
and to protect sperm cells.
Conclusion
From the results we can conclude that a combination of penicillin
and streptomycin was unable to maintain sperm motility at the
same level as gentamicin, amikacin and neomycin sulfate. The antibiotics
in semen extender did not show any effect on sperm penetration
rate. Antibiotics might play only role in control of bacterial
growth in semen, but not on sperm cells. Further studies need
to be done on how antibiotics act on sperm cells. The antibiotics
had the same influence on sperm quality regardless of boar.
The concentrations of gentamicin and amikacin sulfate were maintained
in extended semen for up to 5 days.
Take-Home Message
The degree of contamination of bacteria in semen can be reduced
if we are concerned about the sanitation of semen processing from
collection in the barn to processing in the laboratory. Quality
of antibiotics is another point that should be considered. The
use of a high quality source of antibiotic may help to reduce
of bacterial resistance to antibiotics. The cost of gentamicin
in semen extender is about $5.9 /L, amikacin is $9.46 /L, neomycin
is $1.29 /L and penicillin-streptomycin is about $4.26 /L (all
ingredients are laboratory grade).
References
Auroux, M.R., Jacques, L., Mathieu, D. and Auer, J. 1991. Is the sperm bacterial ratio a determining factor in impairment of sperm motility: an in-vitro study in man with Escherichia coli. Int J Androl 14: 264-270
Flowers, W.L., 1997. Management of boars for efficient semen production. J Reprod Fert (suppl. 52): 67-78
Sone, M., Chikyu, M., Yoshida, M., Bamba, K. and Ogasa, A. 1992.
Prolonged storage of boar semen in liquid form. Jpn J Swine Sci
29(1): 41-50
Suggested reading
Althouse 1997. Comparison of currently used semen extenders in the swine industry. Compend Contin Educ Pract Vet 19(6): 777-782
Gopalkrishnan, K., Joseph, R. and Sheth, A. R. 1994. Alteration of semen characteristics and regulatory factors in human semen with bacterial infection. Arch Androl 32:213-218
Teague, N. S., Boyarsky, S. and Glenn, J. F. 1971. Interference
of human spermatozoa motility by Escherichia coli. Fert
Ster 22(5): 281-285
