The demand for semen from bulls of high genetic merit has improved developing and refining
storage technologies for cattle semen. In previous
decades, there have been many improvements in
the methodological approach of cooling and freezing of spermatozoa. Due to the routine incorporation of egg yolk or milk with glycerol as agents to
protect the spermatozoa during cooling and freezing procedures, some progress has been achieved
regarding biochemical environment and physical
conditions in order to improve sperm cryopreservation (
Cooling and freezing procedures result in
a number of physical changes in the external
environment of cell suspension such as water
and solute movement (
The extent of membrane damage may include a
variety of different conditions, like changes in the
organization, fluidity, permeability, lipid composition of the membrane bilayer, and total membrane
Phospholipid composition of sperm, in particular
its high content in omega-3 polyunsaturated fatty
acids (Ω-3 PUFAs), maintained plasma membrane
fluidity and integrity, crucial factors for sperm fertility (
The role of Ω-3 PUFA on sperm resistance to
cooling and storage is controversial (
In bull, spermatogenesis lasts for about 2
This experimental study included ejaculates from five proven fertile bulls, allocated to control and four experimental groups. For group 1, polyethylene glycol (PEG) as a solvent was added alone to the extender, while for groups 2, 3 and 4, different concentration of 1, 2.5 and 5%, respectively, of omega-3 PUFAs (WN pharmaceutical Ltd., BC, Canada) in combination with PEG were added to the semen extender. The basic extender (BX) used in the experiments contained 2.91% sodium citrate, 20% egg yolk and 7% glycerol. In order to make a homogeneous extender and to introduce Ω-3 PUFAs to semen extenders, 5% polyethylene glycol (PEG) had to be added as a solvent. Media were then sonicated for six minutes.
The Ethical Committee of Ferdwosi University
of Mashhad approved all procedures used in this study. Semen was collected by artificial vagina
from five fertile Holstein bulls by conventional
method of sampling at the Khorasan Breeding
Center, Mashhad, Iran. Immediately after collection, ejaculates were transferred to a water bath at
37˚C and examined for semen volume, color, pH,
sperm motility and sperm concentration. Only the
ejaculates with more than 70 % motility were selected for further processing. The selected semen
samples were divided into five parts and diluted
sperms/ml) at 37˚C by different media
in order to achieve the proper concentrations for
control and treatments groups (
Characteristics of diluting media used for control and four treatment groups
|Group||Composition of diluting medium|
|Basic extender (BX)|
|BX + 5% PEG|
|BX + 5% PEG + 1% Ω-3 PUFA|
|BX + 5% PEG + 2.5% Ω-3 PUFA|
|BX + 5% PEG + 5% Ω-3 PUFA|
In experiment 1, the effect of different levels of Ω-3 PUFAs on sperm quality was assessed during storage at 5˚C for 24 and 48 hours, respectively. Sperm aliquots were taken from refrigerator and incubated for five minutes at 37˚C before sperm analyses.
In this experiment, the effect of different levels of Ω-3 PUFAs on frozen-thawed sperm quality was assessed. Straws from each sample were thawed at 37˚C for 30 seconds in a water bath and examined.
Computer aided sperm analysis (HFT-CASA V6.50, Hooshmand Fanavar Tehran Co., Iran) was used for assessment of motility parameters. For evaluation, a 10 μl drop of sample (further diluted to 1×107 spermatozoa/ml with BX) was placed onto a pre-warmed slide, covered with a cover slip of 22×22 mm and studied using a negative contrast-phase optical microscope (×100) (Olympus, Germany) at 37˚C. Five fields of each drop were recorded and processed. This CASA system is based upon the analysis of 25 consecutive digital images obtained from a single field using a camera (Olympus, Germany). Approximately, 200 cells were evaluated per field. Total and progressive motility, different motility classes (A: rapid progressive, B: slow progressive and C: non- progressive), static class (D), curvilinear velocity (VCL), linearity (LIN), average path velocity (VAP), straight-line velocity (VSL) and amplitude of lateral head displacement (ALH) were determined.
To assess sperm viability, 10 μl of sperm suspension were mixed with 10 μl of eosin solution (0.5 %). Immediately, uncolored sperms were counted under a phase contrast microscope Olympus, Germany to calculate the percentage of sperm viability. Two hundred spermatozoa were evaluated to determine viability.
Sperm morphology was examined in smears stained with eosin and nigrosin. The staining solution contained 0.67% eosin Y and 10% nigrosin dissolved in 0.9% sodium chloride in distilled water. Fifty microliter of diluted sperm was mixed with 50 μl of stain and incubated for five minutes. Smears were made on slides and allowed to dry. Slides were mounted and observed under ×400 objective lens of a phase contrast microscope. For each preparation, 200 cells were counted, and the percentages of various defects were enumerated. The morphological defects of acrosome, head, mid-piece and tail were evaluated.
Each experiment was replicated five times. The statistical analysis was performed using SPSS statistical software version 16 (SPSS Inc., Chicago, IL, USA). Repeated measures ANOVA followed by Bonferroni post-hoc test were conducted to investigate the effects of different levels of Ω-3 PUFAs on sperm quality during the study period. P<0.05 were regarded as statistically significant.
Sperm quality parameters of fresh semen are presented as mean and standard deviation (SD) in table 2. Static class (D) was increased, while all other parameters were decreased over the liquid preservation period in all groups including control. After 24 hours of liquid cold storage, significant decreases (p<0.05) were observed in most quality parameters when compared with fresh semen. Although parameters were decreased during the next 24 hours of preservation, they were not significant.
Sperm quality parameters of fresh semen collected from five bulls. Data are presented as Mean ± SD
|Variable||Mean ± SD|
|81.22 ± 4.87|
|67.85 ± 7.26|
|43.84 ± 9.20|
|24.01 ± 2.90|
|13.37 ± 4.14|
|24.77 ± 12.13|
|89.12 ± 27.67|
|39.22 ± 4.93|
|54.57 ± 13.35|
|42.16 ± 10.67|
|6.30 ± 1.64|
|96.60 ± 3.71|
|93.40 ± 1.95|
Different concentrations of omega-3 supplementation did not improve morphology and motility
parameters, significantly, during the liquid preservation period. Furthermore, average of viability in
group 4 (BX plus PEG and 5% PUFAs) was significantly decreased as compared to control group
(p=0.001), which indicates an adverse effect on
Percentage of viability of bull sperm in fresh, 24 and 48 hours after storage in refrigerator for five treatment groups. During the study period, the percentage of viability in the control was significantly greater than HIGH group (p<0.05).
Static class (D) was increased, while other quality parameters were decreased significantly after one month cryopreservation within all studied groups including control as compared with fresh semen (p<0.001).
motility, progressive motility, motility
classes (B), static class (D), linearity and viability in control group were significantly better
than treatment groups (Figs
Percentage of total motility in fresh and frozenthawed semen for five treatment groups. At post-thaw, total motility was significantly greater in the control than all other groups (p<0.05).
Percentage of progressive motility in fresh and frozenthawed semen for five different treatment groups. In post-thaw samples, percentage of progressive motility was significantly greater in the control than all other groups (p<0.05).
Percentage of motility class (B) in fresh and frozenthawed semen for five treatment groups. At post-thaw, motility class (B) was significantly greater in the control than all other groups (p<0.05).
Percentage of linearity in fresh and frozen-thawed semen for five treatment groups. In the post-thaw samples, percentage of linearity in control was significantly greater than all other groups (p<0.05).
ntage of viability in fresh and frozen-thawed semen for five treatment groups. At post-thaw, Percentage of viability in control was significantly greater than all other groups (p<0.05).
As expected from previous studies (
The role of Ω-3 PUFA, eicosapentaneoic
acid (EPA) and docosahexaneoic acid (DHA)
on sperm resistance to cooling and freezing
procedures is controversial, and may be related to the type of long chain PUFA content
As PUFAs are hydrophobic, a suitable solvent
was required. Many industrial products can be
categorized as oil-in-water (O/W) emulsions,
which consist of small lipid droplets dispersed
in an aqueous medium. Polyethylene glycol has
the ability to dissolve hydrophobic materials at
the lowest concentration. Furthermore, it was
the least detrimental effect to sperm motility
compared to other solvents such as ethanol and
dimethyl sulfoxide (DMSO) (
Results showed that the detrimental effects of cryopreservation were greater than the effects of cold liquid storage on sperm parameters. No improvement of sperm quality was observed after cryopreservation of bull semen following the addition of PUFAs to semen extender.
Addition of DHA enriched egg yolk to the
boar semen diluent before freezing did not
improve quality of frozen-thawed boar sperm.
Various conditions are associated with the low
quality of bull sperm after long term cooling
storage and cryopreservation (
Based on our data, it seems that the addition of Ω-3 PUFAs directly to semen extenders
is not effective in protecting the sperm membrane. Instead, they might be supplemented to
the diet of bulls in order to modify the fatty
acid compositions of sperm and to perform
their preventive properties. Several studies
have shown that the addition of PUFAs to
the diet can influence biosynthetic pathways
of both prostaglandin synthesis and steroidogenesis, which are important in reproductive
This study was conducted to investigate possible protective effects of different levels of Ω-3 PUFA in extender on bovine chilled as well as frozen sperm. In contrast to our hypothesis, supplementation of semen extenders with Ω-3 PUFAs did not significantly improve sperm resistance to cooling, especially to cryopreservation. So, it reveals that the addition of Ω-3 PUFAs in bull extender is not a useful method for improvement of bull sperm, and further investigations should be conducted with diet supplementation of different sources of PUFAs in order to convey their protective properties on sperm membranes.