Document Type : Original Article
Authors
1 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedi- cine, ACECR, Tehran, Iran;Department of Animal Science, Saveh Branch, Islamic Azad University, Saveh, Iran
2 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedi- cine, ACECR, Tehran, Iran
3 Department of Animal Science, Saveh Branch, Islamic Azad University, Saveh, Iran
4 Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran
Abstract
Keywords
The membrane structure of spermatozoa plays a
crucial role in fertilization. The lipids of the spermatozoa
have been suggested to be important for
the viability, maturity, and functions of spermatozoa.
It has also been suggested that the proportion
of unsaturated fatty acids (UFA) may have an influence
over the physical properties of the sperm
membrane including membrane fluidity (
As a basic inception in ruminant, biohydrogenation
(BH) of PUFA is a part of lipid digestion in
the rumen and lipids are extensively altered in the
rumen, resulting in marked differences between
the fatty acid profile of lipids in the diet (mostly
UFA) and lipids leaving the rumen (mostly saturated
fatty acid). It is surprising to note that some
fatty acids can escape from this process and reach
the small intestine and can be found in some organs
such as the mammary gland or testis. Also,
they may stimulate some genes which participate
in FAs metabolism in the organs (
Digestion, absorption, transport and metabolism
of fatty acids in the body, could be affected by the
changes in the structure of fatty acids leaving the rumen,
or the cis to trans configuration switch (
In particular, ram spermatozoa are able to oxidize
C14:0 and C16:0 bound to plasmalogen for energy
production, in this regard C16:0 and C18:1 has
been reported to exist at higher levels in ram sperm
(
Essential fatty acid (EFA) concentration can be altered
by any change in the ratio of UFA n-3 and n-6.
A recent report confirmed that dietary fish oil (FO) increased
the proportion of DHA (C22:6 n-3) in sperm
fatty acid composition (
To our knowledge, no studies to date have focused on sperm FAs persistency or compared the effect of feeding palm with unsaturated fatty acid sources on the ram sperm FAs. Therefore, the objective of this study was to determine the persistence of saturated and unsaturated dietary FAs in ram sperm FA profiles 35 days after removing the FA sources from the diet.
All chemical reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise indicated. This study received the approval of the Ethics Committee of Royan Institute.
This study was carried out from September to December 2009, in the research farm of Saveh University located in Saveh district in central Iran at Latitude: 50° 21´ N, Longitude: 35° 2´ E and an Altitude of 995 m above sea level.
Nine Kalkoohi rams (aged 32 ± 5 months and 65 ± 5 kg average body weights) were used in a completely randomized design and they were assigned to 3 groups (n=3) and different pens in separate groups according to the type of supplementation. Rams were allocated to the treatment groups based on principal component analysis of sperm quality, prior to commencement of the trial in order to balance both high and low quality semen producing rams between treatments.
Experimental groups were offered an isoenergetic
and isonitrogenous ration [metabolizable
energy (ME) was 2.95 Mcal/kg in dry matter
(DM) and crude protein (CP) was 11% in dry matter],
which were given in two equal quantities per
day and rams were allowed to walk freely. Diet
was formulated to meet the nutrient requirements
of AFRC (1995) for rams (
After the trail period, all groups were offered basal diet without any supplementary FA sources for 35 days.
Semen samples from each ram were collected on the last day of feeding on the FA supplemented diet using an artificial vagina (AV). A second semen sample was collected 36 days after excluding fat sources from the diet. Semen from individual rams was transferred from the farm to the laboratory in a portable incubator at 37°C (K-systems G95, Denmark).
The semen was washed twice using an equal
volume of Dulbecco’s Phosphate Buffered Saline
solution (DPBS) (Sigma Aldrich Co.) followed
by vortexing for 10 minutes and centrifugation
(Hettich, EBA 20, Germany) for 20 minutes at
700 × g. Total lipids were extracted from spermatozoa
after homogenization in a suitable excess
of chloroform: methanol (2:1, v:v) (
The data generated from the study were analyzed
by ANOVA (Analysis of variance) using the General
Linear Model (GLM) procedure of SAS Institute
(1999) (
FA composition of fat sources in the diet was analyzed
by GC and has been shown in table 1. At the
end of the experiment (35 days after excluding fatty
acids from the diet) major sperm fatty acids of
the experimental rams consisted of: C14:0, C16:0,
C18:0, C18:1 cis, C18:2 cis and C22:6 (DHA) (Tables
Fatty acid profile of fat sources in experimental diets
| Fatty acids composition (%) | RP10®1 | SO2 (n-6) | FO3 (n-3) |
|---|---|---|---|
| 0.4 | ----- | 4.03 | |
| 3.8 | 0.09 | 1.1 | |
| 0.2 | ----- | 0.2 | |
| 89 | 8.5 | 21.2 | |
| 0.14 | 0.1 | 6.6 | |
| 0.06 | 0.04 | 1.5 | |
| 0.08 | 0.03 | 1.03 | |
| 0.78 | 4.2 | 5.9 | |
| 0.05 | 0.05 | 0.3 | |
| 4.3 | 25.6 | 34.1 | |
| 0.03 | 0.4 | 0.2 | |
| 0.13 | 59.8 | 2 | |
| ----- | ----- | 0.12 | |
| ----- | 0.2 | 1.6 | |
| ----- | 0.3 | 0.3 | |
| ----- | 0.15 | 2.4 | |
| ----- | ----- | 0.3 | |
| ----- | 0.5 | 0.7 | |
| ----- | ----- | 0.5 | |
| ----- | ----- | 5.8 | |
| ----- | ----- | 0.3 | |
| ----- | ----- | 0.8 | |
| ----- | ----- | 8.8 | |
1: RP10®: Provided by Sana Dam Pars Co. (Tehran, Iran), 2: SO: sunflower oil, 3: FO: fish oil, 4: trans, 5: cis.
The concentration of C18:0 in both FO (15.5 %) and SO (14.3 %) treatment groups were higher compared to RP10 (10 %) in samples collected after feeding on FA supplemented diet and basal diet.
DHA concentration increased dramatically with FO at the end of the study (20.3 %) compared to other treatments.
35 days after removing of FA sources C14:0 (p=0.8) and C18:1 cis (p=0.4) showed a similar percentage among the treatments.
The C16:0 percentage was decreased significantly (p < 0.01) as FO was added to the diet (26.18, 27.03 vs. 20.35 % of total FA in RP-10, SO and FO, respectively). The FO treatment had the lowest C16:0 percentage (p < 0.01) along all treatment groups. The C18:0 percentage was significantly different in RP-10 compared with other groups (10 vs. 14.3 and 15.5 % of total sperm FA in RP-10, SO and FO, respectively; p < 0.01).
Fatty acids composition of sperm at the end of the fat supplemented diet feeding (after 12 weeks) (Mean ± SD)
| Fatty acid (% of total fatty acid) | Treatments | P value4 | ||
|---|---|---|---|---|
| RP10®1 | SO2 | FO3 | ||
| 6.3 ± 0.31b | 8.3 ± 0.2b | 13.4 ± 1.4a | ** | |
| 25.5 ± 2.2 | 28.3 ± 2.9 | 28 ± 1.6 | 0.12 | |
| 0.22 ± 0.07b | 0.4 ± .15a | 0.28 ± 0.06b | ** | |
| 0.21 ± 0.5b | 0.5 ± 0.01a | 0.3 ± 0.05b | * | |
| 7.4 ± 0.8 b | 14.8 ± 0.4a | 14.5 ± 0.4a | ** | |
| 30.7 ± 1.9a | 23.5 ± 3b | 10.7 ± 1.4c | ** | |
| 15.2 ± 1.45a | 10.2 ± 0.43b | 4.2 ± 0.15c | ** | |
| 0.44±0.1 | 0.38 ± 0.2 | 0.34 ± 0.17 | 0.8 | |
| 0.29 ± 0.03a | 0.2 ± 0.02b | 0.12 ± 0.02c | ** | |
| 1.4 ± 0.91b | 2.1 ± 0.63b | 3.4 ± 0.09a | ** | |
| 0.21 ± 0.3b | 0.9 ± 0.19a | 0.42 ± 0.28b | * | |
| 0.1 ± 0.04b | 0.5 ± 0.07a | 0.11 ± 0.02b | * | |
| 0.07 ± 0.05b | 0.4 ± 0.22b | 2.3 ± 0.15a | * | |
| 9.9 ± 0.2b | 8.7 ± 0.33c | 20.3 ± 0.95a | ** | |
SD: Standard deviation, 1: RP10®: Provided by Sana Dam Pars Co. (Tehran, Iran), 2: SO: sunflower oil, 3: FO: fish oil, 4: a, b, c: values with differing letters within the same rows are significantly different (*p<0.05; **p<0.01).
Fatty acids composition of sperm lipids measured in semen samples 35 days after excluding FA sources from the diet (Mean ± SD)
| Fatty acid (% of total fatty acid) | Treatments | P value4 | ||
|---|---|---|---|---|
| RP10®1 | SO2 | FO3 | ||
| 10.18 ± 0.5 | 9.98 ± 0.7 | 11.32 ± 1.1 | 0.8 | |
| 26.18 ± 1.3a | 27.03 ± 0.9a | 20.35 ± 1.8b | ** | |
| 0.28 ± 0.9 | 0.3 ± 0.03 | 0.39 ± 0.18 | 0.2 | |
| 0.58 ± 0.06b | 0.71 ± 0.09a | 0.28 ± 0.04c | * | |
| 10 ± 0.58 b | 14.3 ± 1.2a | 15.5 ± 1.3a | ** | |
| 27.7 ± 3.1 | 23.3 ± 1.7 | 21.6 ± 2.5 | 0.14 | |
| 11.9 ± 1.6a | 9.6 ± 1.9ab | 7.7 ± 2a | ** | |
| 0.48 ± 0.04 | 0.49 ± 0.04 | 0.5 ± 0.03 | 0.8 | |
| 0.33 ± 0.1 | 0.47 ± 0.22 | 0.25 ± 0.1 | 0.5 | |
| 1.01 ± 0.8b | 1.26 ± 0.62b | 2.36 ± 0.2a | ** | |
| 0.36 ± 0.04b | 1.45 ± 0.34a | 0.58 ± 0.08b | * | |
| 0.2 ± 0.09 | 0.32 ± 0.06 | 0.47 ± 0.08 | 0.3 | |
| 0.17 ± 0.1b | 0.6 ± 0.44b | 2.75 ± 0.9a | ** | |
| 8.4 ± 0.6b | 8.9 ± 0.82b | 13.6 ± 1.6a | ** | |
SD: Standard deviation, 1: RP10®: Provided by Sana Dam Pars Co. (Tehran, Iran), 2: SO: sunflower oil, 3: FO: fish oil, 4: a, b, c: values with differing letters within the same rows are significantly different (*p<0.05; **p<0.01).
The C24:1 percentage had the highest level in FO group after feeding on FA supplemented diet and 35 days after removal of FA sources.
Interestingly, C22:6 percentage was highest in FO treatment (8.5, 8.9 vs. 13.6 % of total sperm FA in RP-10, SO and FO, respectively; p < 0.01) 35 days after excluding supplementary fat source.
Previous studies observed a decrease in C18:1 in
response to feeding of FO, marine algae, or fish
meal in ruminant products such as milk. This decrease
of C18:1 is a typical response to FO supplementation
which appears to be a result of ruminal
biohydrogenation and release of FAs to ruminant
products (
As expected, DHA concentration increased dramatically
with FO at the end of this study which
supports Samadian et al. findings (
Our results also conform the results of Salem et
al. (
It seems that the use of PUFA sources can have positive effects on C18:0 percentages due to the biohydrogenation of PUFA in ruminants which leads to the production of C18:0.
In boars, changes in fatty acid proportions of
sperm phospholipids and improvement in sperm
quality appeared only after 5 weeks of feeding
them marine oil. It was noteworthy that spermatogenesis
and epididymal transport required 34 and
10 days respectively in boars, whereas the corresponding
intervals were approximately 49 and 9
days in rams (
It was therefore surprising to find that diet FAs changed the sperm fatty acids profile after 35 days, because sperm FA patterns in previous studies were not measured after removing FAs from the diet.
Such a modification might have accounted for the
aforementioned changes in the sperm FA profile.
The vast majority of research on the supplementation
of unsaturated fatty acids for improved sperm
production or cryosurvival has resulted in positive
or at least neutral findings, with improvements noted
in roosters (
In 1961, Hartree and Mann (
The composition of LC-PUFAs in sperm was changed by the dietary supplementation of FA sources. Fish oil supplementation significantly increased the concentration of DHA in sperm lipids. Our experiment demonstrated that after the consumption of commercial diets, C16:0 and C18:1 are the most prominent FAs in ram sperm. Consumption of FA in ram diet, changes FAs profile and this change has a persistency up to 35 days after excluding of FO in ration.
Nutritionists and physiologists should pay attention to this crucial point. The use of PUFA in ruminant ration is very useful, in spite of the fact that biohydroganation can cause lots of changes in physico-chemical characteristic and function in FA.
)