ISSN: 2637-4749
Muna Kholghi2, Jalal Rostamzadeh2, Mohammad Razmkabir2 and Farid Heidari1*
Received: August 03, 2020; Published: August 18, 2020
Corresponding author: FaridHeidari, Department of Animal Biotechnology, Institute of Agricultural Biotechnology, National Institute of Genetic Engineering and BiotechnologyShahrak-e Pajoohesh, km 15 Tehran - Karaj Highway, Tehran, Iran
DOI: 10.32474/CDVS.2020.04.000177
Sex ratio has a direct impact on livestock economy and controling sex-linked genetically diseases. Offspring sex ratio is affected by such various factors. One of these factors is the Y/X-hromosome bearing sperm ratio in fertile specimen. This study was conducted to explore the effect of testosterone concentrations of blood and semen on the relative frequency of Y/X -chromosome bearing sperm in Holstein bovine semen. Blood and semen testosterone level were measured by ELISA technique. Quantitative real-time PCR was performed to estimate the ratio between sperm with Proteolipid Protein (PLP) and the Sex-Related Y (SRY) genes, locating on non-homologous regions of X and Y chromosomes. Blood and semen samples of 26 Holstein bovines were taken simultaneously. DNA was extracted from semen sampls and real-time PCR was performed to amplify the fragments of 90, 89, and 79 base pairs (bp) for PLP, SRY and PAR (as reference) genes, respectively. Wide variation was shown in Y- and X - chromosome bearing sperm, ranging between 18-82%. The least mean square of Y-bearing sperm (1.23±0.15) was significantly higher than that of X-bearing sperm (0.71±0.02). The correlation coefficients of SRY and PLP with blood and semen concentration of testosterone were 0.38, 0.47, -0.67 and -0.60, respectively. The results demonstrated that higher testosterone levels are probably associated with a higher proportion of Y- bearing sperm. A significant positive correlation (P<0.05) was detected between the age of cattle and the ratio of Y-bearing sperm. The testosterone concentration of blood and semen was positively correlated to the cattle age (P<0.05). The results may provide insights into the effects of paternal testosterone on sex ratio of sperm transferred to females.
Keywords: Real-time PCR, Semen, Sex ratio, Testosterone
Offspring sex ratio is an important statistic index defined as the proportion of males to females newborn[1], which expected to be 1:1 in populations. But recent researches have shown this proportion can vary significantly from the expected rate [2,3]. In the livestock industry, sex preselection of offspring has a special economic importance, therefore causes and mechanisms of sex selection are hot subjects for investigators[4-6]. From 1970 onwards, factors affecting sex ratio have been studied and role of breed and genetic, season, nutrition, age and weight of parent, gestation periods, male ejaculation frequency, time of insemination, different movement speed of the X- and Y-chromosome bearing sperm were studied[7-10]. These factors may be affect cervical mucus, metabolites and female reproductive tract secretion and vaginal pH [11]. Ability of father to bias offspring sex ratio has been dismissed given the expectation of an equal proportion of Y/Xchromosome bearing sperm during ejaculation. This expectation has been recently refuted[12]. Gomendio[13] reported a strongly sexually dimorphic species and a classic example for large variance across males in reproductive success-to show that fathers can bias sex ratio at birth. More fertile fathers produce more sons and less fertile produce more daughters. Saragusty [14] shown that variation in the ratio of Y/ X-chromosome bearing sperm in the ejaculation associates with variation in the sex of the offspring produced. Analysis of large dataset was shown males with higher reproductive success have a higher proportion of male offspring, and also such sex ratio bias is adaptive[15].
It has been reported that the concentration of testosterone
plays a key role in offspring sex ratios in different mammals, as
there was a positive correlation between high concentration of
testosterone and bias in sex ratio of offspring toward males[16,17].
Recent advances in molecular genetics has been resulted in the
development of a variety of techniques (such as real-time PCR,
fluorescent in-situ hybridization (FISH), and flow cytometry)
for accurate estimation of X- and Y-chromosome bearing sperm.
Among these techniques, real-time PCR provides an easy-to-use
context for estimation of the copy number of genes [18]. The
sex-related Y gene (SRY), which is located on the short arm of Y
chromosome close to the centromere, has been frequently used as a
marker for detection of Y-chromosome bearing sperm. SRY involves
in initiation of transcription, processing of mRNA, participation in
spermatogenesis, motility of sperms, interaction between sperm
and ovum, and testosterone production[19-21]. SRY can affect
the viability of Y chromosome and its role in population ratio of
X and Y chromosome-bearing sperm suggested to examined[22].
The proteolipid protein (PLP) gene, on the other hand, is routinely
used for detection of X-chromosome bearing sperm. PLP locates
on non-homologous region of X chromosome and is expressed in
all nervous and non-nervous tissues. The increasing expression of
PLP under different physiological conditions has been shown to
stimulate apoptosis process [23].
Despite the high frequency of studies on the role of testosterone
on offspring sex ratio in different mammals, the potential effect
of testosterone concentration of bovine in the ratio of his Y/Xchromosome-
bearing sperm has been poorly studied. In this
research, The effect of blood and semen testosterone concentration
on the viability and proportion of Y/X -chromosome bearing sperm
in male Holstein bovine was Studied.
Soils Bio Dyne kit (Cat. No: 08-24-0000S) was used to amplify DNA fragments in qPCR.
Twenty-six healthy male Holstein bovine were used in this study. Semen samples were taken using artificial vagina during early morning. Simultaneously, blood samples were collected from the caudal vein using gel-clot activator tubes. Rectal temperature of all bovines were taken immediately after blood sampling.
Serum and semen testosterone level were measured by AccuBind ELISA commercial kit (Monobind Inc. Lake Forest, USA, Cat. No: 3725-300). Immediately after blood collection, all tubes were puted in incubator 37˚C for 10 minutes to promotion clot formation. All tubes were centrifuged at3000 × g for 10 minute. Serums were collected and transfer to 2ml microtube and freezed at -70 untile hormone misurment. 0.5cc of semen samples were transfer to 1.5 ml microtube and freezed at -70 until hormone misurment.
Collected semen was analyzed macroscopic and microscopic. Volume, color, density, contamination, concentration, viability ratio and motility were evaluated, high quality semen was used in this study.
Total DNAs were extracted from sperm using salting-out protocol [24]. All chemical materials used for DNA extraction were obtained from Merck (Darmstadt, Germany). Concentration and purity of extracted DNAs were estimated by Nanodrop spectrophotometry absorption ratios at 260 nm and 260/280 nm respectively. The quality of extracted DNA was assessed by electrophoresis at 1% agarose-gel containing Ethidium Bromide. PAR gene was used as reference gene for normalization of expression data obtained from qPCR. The nucleotide sequences of genes, SRY (NCBI number: EU581861.1) and PLP (NCBI number: AJ009913.1) and PAR (NCBI number: AC234910.2) were obtained from NCBI (GenBank, National Center for Biotechnology Information). Primer pairs were designed using primer3Plus software and were shown in Table 1. The specificity of designed primers were evaluated using PrimerBLAST software of NCBI database.
Quantitative PCR was performed using SYBR Green super mix. The reactions consisted of 4μl SYBR Green PCR Master Mix (SYBR biopars, GUASNR, Iran), 0.5μl of each specific forward and reverse primers, 1μl of DNA, and 14 μl nuclease free water to a final volume of 20μl.
The data obtained from qPCR were analyzed according to the method of Livak&Schmittgen [25]. The mean Ct value was calculated for PAR and each of the two studied genes (PLP and SRY) and ΔCt value was determined for each gene in each sample using following formula:
ΔCt = Ct (target gene) - Ct (reference gene)
After calculation of ΔCt for all samples, the expression status of PLP and SRY genes relative to PAR was estimated using the following formula:Copy number of chromosome (X- or Y-chromosome) = 2-ΔΔCt = 2-(ΔCt (target gene) - ΔCt (PAR)
Finally, the ratio of PLP and SRY was considered as the ratio of X and Y chromosomes, respectively.All data were statistically analyzed using SAS computer software version 9.1 (SAS Institute Inc., Cary, NC, USA). The normality of data was tested by univariate procedure, and then the mean values were exposed to t-tests. Additionally, Pearson’s correlation coefficients between the ratio of PLP/SRY with some biological traits including blood and semen testosterone, semen concentration, age, rectum temperature, and viability of sperm were calculated using Corr in SAS software.
Electrophoresis of the extracted DNA had acceptable quality. Additionally, the least mean square values for the quantity of the extracted DNA were determined as 813.27±114.88 ng/μl, respectively. The amplification plots of PLP, SRY, and PAR genes plotted by Step One software (v.2.1) have been shown in Figure 1.The three studied genes provided a single peak in the melting curve. This implies on absence of primer-dimer formation during the reaction (Figure 2). The blood and semen testosterone concentrations are shown in Table 2. Wide variation was found in testosterone concentration of both blood and semen samples. The blood testosterone concentrations were reported ranging between 6- 12.5 ng⁄ml (Mean±SD: 9.45± 2.39 ng⁄ml) and also semen testosterone concentrations were reported ranging between 0.32- 4ng⁄ml (Mean±SD: 2.07± 1.59 ng⁄ml). There was a positive correlation between blood and semen testosterone concentration.A significant difference was found in the frequency of SRY- and PLPcarrying sperm of the sperm samples among the studied. The Y/X-chromosome bearing sperm was 1.75±0.44 (Mean±SD), ranging between 0.44-4.65 (Table 3). Y- Chromosome bearing sperm percentage was 53.7±12.5% (Mean±SD), Ranging between 18-82%. These data show a large variety in X and Y- chromosome bearing sperm population in different bovine semen.
Table 3: Compare mean 2−ΔcT SRY and PLP-carrying Sperm.
a,bValues with different superscripts within the same row differ significantly (p<0.05).
A strong correlation (0.98) was found between testosterone
concentration of blood and semen, meaning that cattle with
higher blood testosterone content have also higher testosterone
concentrations in their semen (Table 4). Furthermore, the correlation
coefficients of testosterone concentration of blood and semen and
the ratio of SRY-carrying sperm were 0.38 and 0.47, respectively,
meaning that bovine with higher testosterone concentrations
have significantly higher proportion of Y-chromosome bearing
sperm than those with lower blood and semen testosterone
contents. Additionally, the correlation coefficients of testosterone
concentration of blood and semen with the ratio of PLP-carrying
sperm were -0.67 and -0.60, respectively (Table 4).
A significant positive correlation (0.63, P<0.05) was detected
between the age of bovine and the ratio of SRY-carrying sperm
(Table 4). However, the correlation between age and the ratio of
PLP-carrying sperm was not statistically significant (-0.26, P>0.05)
(Table 5). Testosterone concentration of blood and semen was
positively correlated with age of bovine(0.72 and 0.79 respectively,
P<0.05). Rectum temperature was positively correlated to blood
testosterone concentration (0.41, P<0.05), but not correlated with
the ratio of SRY- and PLP-carrying sperm (-0.20, P>0.05) (Table 5).
The results of correlation analyses between different biological
characteristics of Holstein bovine have been summarized in Table
5. The correlation of rectum temperature with population of sperm
and sperm stimulation was 0.05 and -0.1, respectively, which were
not statistically significant (p>0.05).
Table 4: The correlation coefficient between PLP gene, SRY gene, blood testosterone, semen testosterone, age and rectum temperature among Holstein bovine.
*P < 0.05; **P< 0.01; and ns: non-significant (P > 0.05).
Table 5: The correlation coefficient between blood testosterone, semen testosterone, age, volume of semen, population and viability of sperm among Holstein bovine
*P< 0.05; **P< 0.01; and ns: non-significant (P > 0.05).
There is a strong evidence of effect of environment and social
factors on sex ratio [26]. Several factors have been reported in bovine
that affect secondary sex ratio and also male calf is significantly
higher when Artificial Insemination (AI) used compared to natural
service [27,28]. Checa [29] reported that 50.02 ± 2.79% of sperm
bearing X-chromosome, but another study found that 44% of the
sperm carrier X-chromosome[30]. Lobel[31]examined 98 human
semen. They reported that 41.9-56.7% of the sperms bearing Y
chromosome. Another research reported similar data, 46.9-52.7%
Y-sperms in each ejaculation [32]. Madrid-Bury [33] reported
neither bovines nor semen didn’t have effect on Y-chromosome
bearing sperm percent or sex ratio of embryos produced in vitro
but the method of sperm preparation affected the primary sex ratio.
However, double swim-up sperm preparation method produced
differences in %Y- chromosome bearing sperm in some of sperm
fractions. They suggested that there are intrinsic differences in
capacitating of X and Y-bearing sperm that might be used to produce embryos of the desired sex in laboratory production of embryo.
However in two different studies on bovine semen, it became clear
that Y-chromosome bearing sperm population was between 24-
84% in each ejaculation[34,35]. The difference between previous
studies may be due to the breeds of the employed bulls (Holstein
and Galicia) or PCR techniques.
Wide variation was found in testosterone concentration in
both blood and semen samples. This variation may be related to
some factors such as individual difference, breed, and age. Although
the main source of testosterone in male is testicular tissue, the
concentration of semen testosterone in all studied animals was
much lower than blood. Lower levels of testosterone in semen
is due to transfer testosterone from blood in semen. Laydig cells
produce and release testosterone in blood, and semen testosterone
comes from blood[36].
Recently, studies have shown that offspring sex ratio is
significantly influenced by maternal dominance, a characteristic
which has been shown to be linked to testosterone in mothers[37,38].
Testosterone has been suggested to play an important role in the
viability of germ cells, probably by regulating a specific pathway for
apoptosis [39,40]. A lot of studies have been carried out to verify the
role of testosterone in alteration of sex ratio in different mammals
and our results were in agreement with these investigations. Helle
reported that a 1pg/ml increase in serum testosterone content of
rats could lead to 19% biased in sex ratio of offspring toward males.
They also showed that mothers with higher testosterone
produced more male offspring than those with low testosterone
content. James demonstrated that increase in blood testosterone
level in males can bias the sex ratio of offspring toward males.
Similarly, Shargal found that female ibexes (Capra Nubiana) with
higher fecal testosterone produced more male offspring. Grant
and Irwin and Grant focused on follicular testosterone, instead of
conventional serum and fecal testosterone, and demonstrated that
ova, developing in follicular fluid with high levels of testosterone,
were subsequently more likely to be fertilized by Y-chromosomebearing
spermatozoa, probably due to the differential stimulation
or viability. These authors proposed that there might be a critical
time, in which the follicular testosterone level affects the molecular
composition of zonapellucida and alters the susceptibility of
oocyte to be fertilized by a Y-bearing spermatozoon. According
to García-Herreros when the average testosterone level of bovine
follicular liquid exceeded 32.12 ng/ml, the probability of male birth
increased, while higher proportion of female birth was observed
when the follicular testosterone was around 23.98 ng/ml.
The important role of SRY in the viability of Y chromosome,
stimulation of apoptosis signaling pathway by PLP (due to
lipoprotein synthesis) and increasing of testosterone level, seems
to trigger the apoptosis of the X-chromosome bearing sperm
during the early stages of spermatogenesis. Additionally, some
other biological parameters of male Holstein cattle were found to
be influenced by testosterone concentration. Although, this is the
first study on the effects of paternal testosterone on offspring sex
ratio, a relatively large volume of studies on different mammalians
have demonstrated that mothers with higher blood, follicle or
fecal testosterone produce significantly more male offspring than
females with lower testosterone levels.
It has been reported that in terms of association between
the weather change and secondary sex ratio, by increasing of
1°Centigrades above the average value in the temperature of
weather during the week before fertilization, the probability of
male calf birth will increase about 1 % [41]. Evaporation rate
has the same effect on the birth of male calf. In addition, it has
been expressed that the birth of male and female in the hot and
cold weather is increased, respectively [42]. Perez-crespo[43,44]
reported that the birth of male can be increased by increasing the
temperature of environment and scrotum.
The results of the current study revealed a difference in percentages of Y and X -chromosome bearing sperm. A positive correlation between the frequency of SRY and testosterone was shown. Therefore, investigating the molecular mechanism involved in the effects of paternal testosterone on the ratio of its X/Y-chromosome bearing sperm may be an interesting subject for future studies.
The authors wish to acknowledge of Abbasabad Rearing and Breeding Station especially M. Jafari who has assisted in samples collection. We appreciate the staff of Laboratory of genetics, National Institute of Genetic Engineering and Biotechnology for technical assistance during this research.
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