Document Type : Research Article
Author
Abstract
Keywords
It is well known that the lipid composition of
a mammal’s spermatozoa and seminal plasma
differ in both structure and function. Evidence
exists to suggest that dietary supplementation
with appropriate polyunsaturated fatty acids
(PUFAs) affects spermatogenesis, semen quality
and sperm motility (
Lipoproteins transfer various lipids such as cholesterol
from the tissue of origin to target sites,
where the lipid complex is delivered via lipoprotein
receptor-mediated uptake. Also, the supply
of the Steroids required for cellular activities,
including membrane formation, steroid hormone
secretion, and the post-translational modification
of proteins is regulated by lipoproteins (
The ratios of triglycerides and cholesterol to
a polipoprotein B in the plasma are inversely
related to testosterone levels (
Although goats are economically a very important
domestic mammal, there are very few
data in the literature concerning the testicular
cell populations and endocrine secretion in this
species (
In this analytic, cross-sectional study, a total of ten mature Iranian male goats that comprised a homogenous group (aged 28.7 ± 2.2 months and 43.7 ± 3.1 kg average body weight) were selected from local intensively managed goat flocks (Ilam, Iran) through simple random sampling. The experiments were carried out from late May to July 2009, at the research farm of Ilam University. All animals were in good health and of proven fertility since they were being used as sires in a specialized goat farm. All goats received a daily diet of 702 g alfalfa hay, 69.9 g barley and 141 g of a commercial concentrate. The diet contained 2.4 Mcal ME/ kg, 12.1% crude protein, 1.1% calcium, and 0.31% phosphorus. All goats had free access to mineral blocks and fresh water. This unit was supervised by an experienced veterinarian and the prevention of internal and external parasites were a routine practice.
Blood samples (15 ml) were taken from the
jugular vein between 09:00 and 10:00 am after
overnight fasting and collected in vacutainers
(Becton Dickinson Co., USA). Serum was separated
by centrifugation at 2500 rpm for 20 minutes
and stored at -20˚C until analysis. The sera
were analyzed for total cholesterol and triglyceride
levels using enzymatic kits (Pars Azmoon, Iran).
Lipoproteins were isolated from sera by a combination
of precipitation and ultracentrifugation.
The precipitation method was used for measuring
high density lipoprotein (HDL-c) particles. In this
method, after addition of sodium phosphotungstate-
magnesium to the serum, the non-HDL lipoproteins that sedimented by centrifugation (10000
g for 5 minutes) were aggregated. Then, the residual
cholesterol was enzymatically measured (
Semen was collected in the morning by using
an artificial vagina and transported to the laboratory
(at 37˚C) within 10-15 minutes, then placed
in a water bath at 37˚C. All ejaculates were evaluated
for volume, sperm concentration (photometer,
IMV, L’Aigle, France), percent of live sperm
(eosin-nigrosin method), and percentage of normal
sperm in the stained smear (
Scrotal circumference was measured using a tape-measure. The combined testis width, then testis length in the scrotum was determined by the use of a caliper (Mitutoyo, Kanagawa, Japan).
The right testis from each animal was removed
from the body under local anesthesia. Before
surgery, all animals received intramuscular
(IM) injections of 1 ml of Rompum (Bayer)
per 100 kg of body weight. All surgical procedures
were performed by a licensed veterinarian
and the obtained testes were trimmed out from
the scrotum and epididymis. Then, testes were
weighed and cut along the longitudinal axis to
obtain transverse sections of the seminiferous
tubules (STs). Tissue samples, 1-3 mm thick,
were taken near the tunica albuginea. Sections
of 4 μm thickness were stained with hematoxylin
and eosin (H & E) and viewed by a light microscope
(Nikon Eclipse E800). Fields with sections
of STs in circular transverse section were
selected at 100x magnification; photographs
were taken with a digital camera (Coolpix 950,
Nikon, China) and stored. Histometric and cytometric
studies were conducted using Image
Tool® 3.0 software (UTHSCSA, San Antonio,
USA). For this purpose, the boundary demarcation
of STs was performed using the software and the
area of the STs in each field was deducted from the
total area of the field. Thus, the area of the STs and
the area of the interstitial tissue were deduced. To
measure the tubular diameter and the height of STs
epithelium, at least thirty round or nearly round ST
cross-sections were chosen at random and measured
for each animal. In addition, the number of
Leydig cells per 103 mm of the interstitial area was
counted in five fields from each animal, at 1000x
magnification. Finally, the diameters of the Leydig
cell nuclei were measured according to the Faridha
et al. (
Results were presented as mean ± standard error of mean (SEM) for serum lipid profile, histological, anatomical and seminal parameters and the levels of serum testosterone. The data were statistically analyzed using the SPSS statistical software package, version 10.0.1 (SPSS Inc., Chicago, IL, USA. Prior to analysis, the Kolmogorov-Smirnova test was used to check normal distribution of the data. Then, to evaluate the correlation of the serum lipid profile with testicular cell populations and endocrine secretion, Pearson’s partial correlation coefficient analyses were conducted. A p value <0.05 was considered statistically significant.
Mean ± SEM and correlation coefficients between serum lipids and lipoproteins with histological measurements of testis in the Iranian male goat are presented in tables 1-3. In the present work, the level of serum HDL-c showed a positive, highly significant correlation with interstitial testicular tissue area (r=0.73; p<0.001) and STs area (r=0.61; p<0.01). There was a weak positive correlation of these attributes with the number of Leydig cells (r=0.53; p<0.05) and the nuclei diameters of the Leydig cells (r=0.54; p<0.05). In addition, significant but negative correlations were found between the level of triglycerides in the serum and STs area (r=-0.53; p<0.05) or diameter of the Leydig cell nuclei (r=-0.55; p<0.05).
Mean ± standard error (SEM) of serum lipids and lipoproteins in the Iranian male goat (n=10)
| Total cholesterol (mg/dl) | Total triglycerides (mg/dl) | Very low density lipoprotein (mg/dl) | Low density lipoprotein (LDL-c) (mg/dl) | High density lipoprotein (HDL-c) (mg/dl) |
|---|---|---|---|---|
| 47.25 ± 2.9 | 46.6 ± 1.5 | 10.3 ± 2.8 | 17.8 ± 1.1 | 18.1 ± 0.8 |
Mean ± standard error (SEM) of the histological measurements of testis in the Iranian male goat (n=10)
| Interstitial testicular tissue area (%) | Area of seminiferous tubules (STs) (%) | Height of STs epithelium (μm) | STs diameter (μm) | Number of Leydig cells (×103 mm area of the testis) | Diameter of Leydig cell nuclei (μm) |
|---|---|---|---|---|---|
| 9.3 ± 1.3 | 88.8 ± 0.88 | 72.90 ± 1.9 | 215.7 ± 3.7 | 19. 5 ± 1.6 | 3.2 ± 0.1 |
Correlation coefficients between serum lipids and lipopr oteins with histological measurements of testis in the Iranian male goat (n=10)
| Serum lipid profile/ histological measurements of testes | Total cholesterol (mg/dl) | Total triglycerides (mg/dl) | Very low density lipoprotein (VLDL-c) (mg/dl) | Low density lipoprotein (LDL-c) (mg/dl) | High density lipoprotein (HDL-c) (mg/dl) |
|---|---|---|---|---|---|
| 0.16 | 0.19 | 0.21 | 0.11 | 0.73*** | |
| 0.23 | -0.53* | 0.18 | 0.18 | 0.61** | |
| 0.12 | 0.29 | -0.21 | 0.15 | 0.18 | |
| 0.11 | 0.13 | 0.25 | 0.12 | 0.15 | |
| 0.15 | 0.24 | 0.31 | -0.23 | 0.53* | |
| 0.15 | -0.55* | 0.13 | 0.27 | 0.54* | |
*; Significant at p<0.05, **; Significant at p<0.01 and *** ; Significant at p<0.001.
The mean ± SEM of anatomical measurements of the testes, live body weight of the animals and their correlation coefficients with the serum lipid profile are presented in tables 4 and 5. Correlation coefficients of HDL-c with the scrotal circumference (r=0.83; p<0.001) and testis weight (r=0.72; p<0.001) were highly significant. There were significant negative correlations observed between total serum triglyceride level and testis weight (r=- 0.64; p<0.01).
Mean ± standard error (SEM) of anatomical measurements of the testes and live body weight of the Iranian male goat (n=10)
| Scrotal circumference (cm) | Testis width (cm) | Testis length (cm) | Testis weight (g) | Live body weight (kg) |
|---|---|---|---|---|
| 42.23 ± 2.5 | 265.17 ± 5.3 | 7.38 ± 1.6 | 3.59 ± 1.2 | 29.61 ± 1.6 |
Correlation coefficients between serum lipid profile with anatomical measurements of the testis and live body weight of Iranian male goat (n=10)
| Serum lipid profile/ histological measurements of testes | Total cholesterol (mg/dl) | Total triglycerides (mg/dl) | Very low density lipoprotein (VLDL-c) (mg/dl) | Low density lipoprotein (LDL-c) (mg/dl) | High density lipoprotein (HDL-c) (mg/dl) |
|---|---|---|---|---|---|
| 0.09 | 0.04 | 0.02 | 0.21 | 0.83*** | |
| 0.06 | 0.01 | 0.03 | 0.02 | 0.11 | |
| 0.21 | 0.11 | 0.31 | 0.08 | 0.06 | |
| 0.07 | -0.64** | 0.38 | 0.12 | 0.72*** | |
| 0.05 | 0.19 | 0.01 | 0.17 | 0.03 | |
*; Significant at p<0.05, **; Significant at p<0.01 and ***; Significant at p<0.001.
Mean ± SEM of the semen characteristics and serum testosterone levels and their correlation coefficients with the serum lipid profiles are shown in tables 6 and 7. The correlation coefficients of HDLc with the number of live, normal sperm (r=0.94; p<0.001) or serum testosterone levels (r=0.88; p<0.001) was highly significant. However there were negative significant correlation coefficients between serum VLDL-c concentration and the percent of live sperm (r=-0.67; p<0.01) or serum testosterone levels (r=-0.65; p<0.01). Negative correlations were found between serum triglyceride concentration and total sperm number (r=-0.82; p<0.001), number of live, normal sperm (r=-0.55; p<0.05) or serum testosterone levels (r=-0.79; p<0.001).
Mean ± standard error (SEM) of semen characteristics an d serum testosterone concentration in the Iranian male goat (n=10)
| Semen volume (mL) | Sperm concentration (109/mL) | Percent of live sperm (%) | Percent of abnormal sperm (%) | Total sperm number (109) | Number of live sperm (109) | Number of live and normal sperm (109) | Serum testosterone concentration (ng/ml) |
|---|---|---|---|---|---|---|---|
| 1.20 ± 0.32 | 2.85 ± 0.53 | 76.5 ± 0.34 | 7.5 ± 2.6 | 2.89 ± 0.4 | 1.34 ± 0.2 | 1.06 ± 0.8 | 7.3 ± 0.7 |
Correlation coefficients between serum lipid profile with semen characteristics and serum testosterone concentration of the Iranian male goat (n=10)
| Serum lipid profile/ histological measurements of testes | Total cholesterol (mg/dl) | Total triglycerides (mg/dl) | Very low density lipoprotein (VLDL-c) (mg/dl) | Low density lipoprotein (LDL-c) (mg/dl) | High density lipoprotein (HDL-c) (mg/dl) |
|---|---|---|---|---|---|
| 0.13 | 0.27 | 0.17 | 0.42 | 0.21 | |
| 0.19 | 0.07 | 0.16 | 0.11 | 0.26 | |
| 0.04 | 0.07 | -0.67** | 0.03 | 0.29 | |
| 0.37 | 0.15 | 0.22 | 0.05 | 0.39 | |
| 0.24 | -0.82*** | 0.08 | 0.09 | 0.33 | |
| 0.06 | 0.09 | 0.34 | 0.07 | 0.19 | |
| 0.01 | -0.55* | 0.14 | 0.26 | 0.94*** | |
| 0.13 | -0.79*** | -0.65** | 0.42 | 0.88*** | |
*; Significant at p<0.05, **; Significant at p<0.01 and ***; Significant at p<0.001.
The findings demonstrated that among serum
lipids, only the HDL-c levels had a positive correlation
with testicular histomorphology, serum
testosterone values, and seminal parameters. The
steroidogenic capacity of Leydig cells in the goat’s
testis and its changes during development of cellular
organelles including the mitochondria and
smooth endoplasmic reticulum responsible for
steroid biosynthesis has been mentioned previously
(
The mechanism(s) of beneficial effects of HDLc
on testis are unknown but it seems that its cholesterol
content is a major source of substrate for steroidogenic
process in the Leydig cells of the testis.
The main arguments for this case are as follows. In
human (
Receptor-mediated endocytosis of HDL-c and
the amount of its affinity for binding to the receptor
sites have been reported by other researchers
as a probable mechanism for its functions in cattle
fertility (
It has been shown that normal Sertoli and Leydig
cell secretory functions play a key role in the
initiation and maintenance of spermatogenesis
(
The present data demonstrated a significant negative
correlation in serum levels of triglycerides
and VLDL-c with total sperm number, percent of
live sperm, number of live and normal sperm, and
serum testosterone concentration. In this regard,
Ergün et al. reported that increased serum total
triglyceride and VLDL-c values had deleterious
effects on spermatogenesis and were significantly
correlated with decreased sperm motion characteristics
(
Additional studies are necessary to clarify whether there is a high concentration of VLDL-c in the interstitial testicular tissue, since harmful consequences within the STs may be secondary to the secretory deficiency of Leydig cells. Alternatively other compounds induced by high serum triglyceride levels may exert a direct detrimental action outside of the seminiferous tubule (i.e. on Leydig cells).
The present results seem to indicate that among serum lipids only the levels of HDL-c positively correlate with testicular parameters. The high serum triglyceride levels exert direct adverse effects at the testicular level, which are observed mainly in STs, Leydig cell characterization and semen quality. Obviously, further studies are required to elucidate the underlying mechanism(s) and to identify the sites of lipids action on the goat testis.
)