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Advances in Clinical Toxicology Research Article 14 min read

Effect of Sodium Nitrate (NaNO3) on Sperm Motility and Abnormality: An In vitro Approach

Rajini SV, Chaithra B and Shivabasavaiah*
* Corresponding author
ISSN: 2577-4328  10.23880/act-16000210  Received: February 10, 2021  Published: April 28, 2021
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Keywords
Abnormality In Vitro Motility Sodium nitrate Spermatozoa
Abstract

Nitrates (NO3) are the naturally occurring inorganic ions with beneficial effects in both plants and animals. However, NO3 in excess has adverse effect on male and female reproductive systems. The study was undertaken to investigate the effect of 10mg/ ml and 100mg/ml of sodium nitrate (NaNO3) on sperm motility and abnormality at different time intervals (5,10,15,20,25 and 30 min). Treatment of cauda epididymal sperm suspension with 10mg/ml of NaNO3 significantly reduced the sperm motility only at 25 and 30 min time intervals compared to controls. However, 100mg/ml of NaNO3 resulted in significant reduction of sperm motility from 5 min to 30 min time interval in time dependent manner and complete loss of sperm motility was observed at 15 min of incubation. In addition, the number of abnormal spermatozoa was significantly high in 100mg/ml of NaNO3 treated groups compared to controls and 10 mg/ml of NaNO3. These results clearly reflected the toxic effect of NO3 on spermatozoa and its capability in fertilization.

Introduction

Nitrate (NO3) and nitrite (NO2) are the naturally occurring inorganic ions involved in nitrogen cycle. There are numerous sources of nitrates which can be classified into exogenous sources and endogenous sources. Leafy vegetables are the exogenous sources of nitrates which account for more than 70% of nitrates ingested in human diet. Nitrites are produced endogenously through the oxidation of nitric oxide (NO) and through a reduction of NO3 by commensal bacteria in mouth and gastrointestinal tract [1]. In biological system, NO3 gets converted to NO2 and NO and are interchangeable [2].

Besides having beneficial effect, NO3 has adverse effects on the body. The connection between human health and harmful effect of NO3 was first reported by Comly in 1945 after observing cyanosis in residents of Iowa (USA). The primary effect of NO3 is the formation of methemoglobinemia in infants which reduces capacity to release oxygen to tissues due to oxidation of iron group of haem group [3, 4]. Further, NO3 causes improper thyroid functioning via inhibition of sodium iodide symporter [5]. Increase in NO3 concentration causes myocardial infraction and hypotension [6]. And also affects liver by increasing bilirubin and transaminase levels [7].

Numerous studies have reported the toxic effect of NO3 on reproductive system [8, 9, 10, 11, 12]. It is evident that NO3 is a potential endocrine disruptor of reproductive endocrinology [13]. The adverse effects of NO3 on reproductive system viz, mummified foetuses, lesions on the cervix, uterus and placenta and maternal death were observed in NO3

exposed guinea pigs [14]. Further, higher risk of ovarian cancers in women drinking NO3 contaminated water [15] and incidences of abortions in potassium nitrate (KNO3) administered pregnant cows [16, 17] were reported. In male rat and mice models, treatment of different doses of sodium nitrate (NaNO3), sodium nitrite (NaNO2) and KNO3 caused a significant reduction in weight of testis and epididymis, sperm count and sperm motility and increased sperm abnormality [9, 10, 11, 12]. In addition NO3 causes lesions in spermatocytes and spermatids of germ layers, degenerations of leydig cells and arrest of spermatogenesis [9, 10]. Further, NO3 affects steroidogenesis of male reproductive system by decreasing the activities of 3βHSD (3-beta-hydroxysteroid dehydrogenase) and 17βHSD (17-beta-hydrosteroid dehydrogenase) and serum concentration of testosterone [10, 11, 18].

Besides in vivo studies, few in vitro studies have focused on the toxic effect of NO on human spermatozoa. For instance, treatment of NO declined forward progressive movement of spermatozoa in in vitro [19]. Studies have reported that the reduced sperm DNA integrity with the association and high concentration of NO in seminal plasma [20]. In addition, high concentration of NO was reported in semen samples of patients suffering from asthenozoospermia than that of normozoospermic individuals [21]. Further, higher concentration of NO caused decreased zona binding capacity of sperm and embryonic development [22]. These results clearly indicate that NO has adverse effect on sperm functions. Though there are enough reports on effect of NO on human spermatozoa, the studies related to the action of NO3 on spermatozoa in in vitro is lacking. Hence, there is a need for investigation to assess the toxic effect of NaNO3 (source of NO3) on motility and abnormality of spermatozoa at different time intervals. with this background, the present study was conducted to investigate the dose and time dependent effect of two different concentrations of NaNO3 (10mg/ml and 100mg/ml) on sperm motility and sperm abnormality in in vitro.

Materials and Methods

Animals

The study included adult male wistar rats weighing 180- 200g procured from central animal facility of University of Mysore, Mysore. Polypropylene cages were used to maintain the animals under standard laboratory conditions and were provided with rat chow and water ad libitum and relative 12h light/dark cycle. The ethical acceptance to conduct the experiment was obtained from Institutional Animal Ethics Committee of University of Mysore, India (reference number: UOM/IAEC/04/2018) and the study was carried out as per the guidelines of the committee.

Experimental Design

An in vitro experiment was conducted to assess the effects of two different concentrations of NaNO3 on sperm motility and abnormality at different time intervals viz.,0, 5, 10, 15, 20, 25 and 30 min. The epididymal sperm suspension (60 million/ml) was incubated with different concentrations of NaNO3 (10mg/ml and 100mg/ml) at different time intervals. The group devoid of NaNO3 was treated as control. After incubation, sperm motility and sperm abnormalities were analysed at regular time intervals.

Isolation of spermatozoa

Epididymal spermatozoa were isolated from cauda epididymis of adult male rats. The epididymis was minced in 1 ml of phosphate buffered saline and the suspension was filtered through muslin cloth. The filtered sperm suspension was used for the in vitro experiment.

Estimation of sperm motility

Progressive sperm motility was considered for estimating sperm motility. Motility was estimated by placing the sperm suspension on a slide and number of motile spermatozoa was counted from three different microscopic fields under light microscope. The mean of the three estimations was calculated in percentage [23].

Estimation of sperm abnormality

Sperm abnormality was analysed by staining the sperm suspension with eosin and a uniform smear was made on a glass slide. One thousand spermatozoa were observed under higher magnification (40X) and the number of spermatozoa showing head and tail abnormalities were counted. The aggregate of different types of spermatozoa showing abnormality were considered to compute percentage of spermatozoa with abnormal morphology [24, 25].

Statistical Analysis

The mean ± standard error of each parameter was computed by considering the data and the mean values of each parameter of different groups were compared using one way analysis of variance followed by Duncan’s multiple range test and judged significant if p< 0.05.

Results

Effect of different concentration of NaNO3 on sperm motility at 0, 5, 10, 15, 20, 25 and30min intervals

There was a significant decrease in sperm motility in sperm suspension treated with 100 mg/ml of NaNO3 at 5, 10, 15, 20, 25 and 30 min of time interval compared to control. However, 10mg/ml of NaNO3 significantly decreased the sperm motility only at 25 and 30 min time interval compared to control. No significant changes in sperm motility was observed at 5, 10, 15 and 20 min time interval of 10mg/ml of NaNO3 treated groups compared to controls. Treatment of 100mg/ml of NaNO3 caused complete loss of sperm motility at 15, 20, 25 and 30 min of incubation (Table 1).

GroupsMean percentage of motility ± SE
0 min5 min10 min15 min20 min25 min30 min
Control91.00±3.21a91.33±2.72a94.33±2.33a89.00±3.05a76.66±2.40a67.33±1.76a45.33±3.711a
10mg/ml of NaNO392.33±1.76a95.33±0.88a91.66±0.88a82.00±3.05a69.33±2.96a45.66±2.33b27.66±4.33 b
100mg/ml of NaNO391.00±2.64a37.66±6.48b15.00±5.13b0.00±0.00 b0.00±0.00 b0.00±0.00 c0.00±0.00 c
ANOVA F
value(df=2,6)		0.87 *p>0.91884.31
***p<0.001		187.04
***p<0.001		393.58
***p<0.001		368.88
***p<0.001		414.27
***p<0.001		48.11
***p<0.001

Table 1: Effect of different concentration of NaNO3 on sperm motility. Note: Mean values were compared by one-way ANOVA followed

Table 1: Effect of different concentration of NaNO3 on sperm motility. Note: Mean values were compared by one-way ANOVA followed by Duncan’s multiple range tests. Values with same superscript letters in the given column are not significantly different, whereas those with different superscript letters are significantly (*p< 0.05) different from each other. df - degree of freedom, NaNO3 - sodium nitrate.

Effect of different concentration of NaNO3 on sperm abnormality at 0, 5, 10, 15, 20, 25 and 30 min interval

A significant increase in sperm abnormality was observed in 100mg/ml of NaNO3 treated groups compared to that of controls and 10mg/ml of NaNO3. However, no significant difference in sperm abnormality was observed between control groups and 10mg/ml of NaNO3 with increase in duration of incubation (Table 2).

GroupsMean percentage of abnormality ± SE
0 min5 min10 min15 min20 min25 min30 min
Control0.00±0.00a0.66±0.33a0.66±0.33a0.66±0.33a0.66±0.33a0.66±0.33a0.66±0.33a
10mg/ml of
NaNO
3		0.00±0.00a1.33±0.33a1.66±0.66a2.00±0.57a2.66±1.20a3.00±0.57a3.33±0.33a
100mg/ml
of NaNO
3		1.00±0.57a11.00±1.15b14.66±1.76b23.66±3.48b23.66±3.48b23.66±3.48b23.66±3.48b
ANOVA
F value
(df=2,6)		3 *p<0.12564.5
***p<0.001		49.9
***p<0.001		39.83
***p<0.001		35.63
***p<0.001		38.29
***p<0.001		38.49
***p<0.001

Table 2: Effect of different concentration of NaNO3 on sperm abnormality. Note: Mean values were compared by one-way ANOVA follow

Table 2: Effect of different concentration of NaNO3 on sperm abnormality. Note: Mean values were compared by one-way ANOVA followed by Duncan’s multiple range tests. Values with same superscript letters in the given column are not significantly different, whereas those with different superscript letters are significantly (*p< 0.05) different from each other. df - degree of freedom, NaNO3 - sodium nitrate.

Discussion

Present study was undertaken to investigate the toxic effect of two different concentration of NaNO3 on sperm motility and sperm abnormality at different time intervals. The study revealed that treatment of NaNO3 decreased the sperm motility and increased the number of abnormal spermatozoa compared to controls.

Sperm motility is an important aspect of spermatozoa to reach the fallopian tube during fertilization. The quality of spermatozoa depends on its motility and number of abnormal spermatozoa [26]. Earlier in vivo studies in rat and mice showed a decline in sperm motility under NO3 treatment [9, 10, 11, 12]. Similarly, present study showed NO3 induced reduced sperm motility in in vitro condition. Between the two doses of NaNO3, a higher concentration that is 100mg/ml showed severe toxicity as it resulted in complete cessation of sperm motility at 15 min of incubation with epididymal spermatozoa. However, the lower dose of NaNO3, that is 10mg/ml showed a time dependent decrease in sperm motility from 5 min to 30 min compared to controls.

A significant dose dependent effect was observed between two doses of NaNO3 at different durations of incubations studied. Thus, the present study clearly demonstrates that treatment of NaNO3 affected sperm motility in dose and time dependent manner. Similarly, earlier study have reported the dose dependent inhibition of sperm motility by NO donors viz, nitroprusside and pure NO gas [27]. In contrast, few investigations reported the beneficial aspects of NO3 on spermatozoa wherein treatment with NO3 caused enhanced capacitation [28], motility, viability [29] and vigour [30] of spermatozoa. In addition, it has been reported that NO and nitric oxide synthase (NOS) is essential for capacitation and fertilization [31].

The NO3 induced decrease in sperm motility is due to increased level of NO, a product of NO3 and NO2 [2]. Higher concentration of NO3 enhances the synthesis of NO which in turn affects sperm motility. The peroxy nitrate (ONOO- ) formed due to the interaction between NO and reactive oxygen species (ROS) can cause damage to lipids and thiol proteins of sperm membrane [32] thereby affects sperm motility. In addition, NO inhibit sperm motility by affecting mitochondrial electron transport protein and thereby obstruct cellular respiration and ATP production in spermatozoa [27, 33]. Further, NO3 inhibit sperm motility by inducing oxidative stress [34]. Spermatozoa are very sensitive to ROS and ONOO- which can readily damage the cell membrane [32, 34].These are the possible mechanisms through which NO3 can affect sperm motility.

The morphology of spermatozoa is another important aspect which plays a vital role in fertilization. Earlier in vivo studies have proved enhanced number of abnormal spermatozoa under NO3 treatment [9, 10, 11, 12]. In the present in vitro study, treatment of 100mg/ml of NaNO3 caused a significant increase in the number of abnormal spermatozoa compared to 10mg/ml of NaNO3 and control groups. The NaNO3 induced increase in abnormal spermatozoa increased with increasing duration of exposure. These results clearly indicate that NaNO3 affect the sperm morphology by increasing head and tail abnormalities, thereby affect the fertilization capacity of spermatozoa. The NO3 induced sperm abnormality may be due to obstruction in cellular respiration and production of ATP and formation of ROS which can cause damage to the lipid and thiol proteins present in the sperm membrane [27, 32, 33, 34]. Therefore, NO3 exposure increases the number of abnormal spermatozoa by affecting cell membrane.

The present in vitro study clearly demonstrated that NaNO3 has adverse effect on spermatozoa and the effect is time and dose dependent. The study reports the toxic effects of NaNO3 on spermatozoa in in vitro condition for the first time as earlier in vitro studies were focused on effect NO on human spermatozoa [19, 21, 27]. The study also provides evidence that higher concentration of NaNO3 that is 100mg/ ml has severe effect on sperm motility and abnormality. These results gain importance in male infertility of human beings residing in the areas where there is high level of NO3 in drinking ground water. Therefore, present investigation appears to be an evidence for the effect of NO3 on male reproduction. However, future studies are needed to reflect mechanistic action of NO3 in affecting sperm motility.

Conclusion

The present study demonstrated dose and time dependent effect of NO3 on spermatozoa. The higher dose of NaNO3 that is 100mg/ml adversely affected sperm motility and abnormality compared to lower dose. The results of the present study for the first time showed reproductive toxicity of NaNO3 in in vitro condition.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

The first author is thankful to BCWD (Backward classes welfare department) Government of Karnataka. India.

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@article{rajini2021,
  title   = {Effect of Sodium Nitrate (NaNO3) on Sperm Motility and Abnormality: An In vitro Approach},
  author  = {Rajini SV, Chaithra B and Shivabasavaiah},
  journal = {Advances in Clinical Toxicology},
  year    = {2021},
  volume  = {6},
  number  = {2},
  doi     = {10.23880/act-16000210}
}
Rajini SV, Chaithra B and Shivabasavaiah (2021). Effect of Sodium Nitrate (NaNO3) on Sperm Motility and Abnormality: An In vitro Approach. Advances in Clinical Toxicology, 6(2). https://doi.org/10.23880/act-16000210
TY  - JOUR
TI  - Effect of Sodium Nitrate (NaNO3) on Sperm Motility and Abnormality: An In vitro Approach
AU  - Rajini SV, Chaithra B and Shivabasavaiah
JO  - Advances in Clinical Toxicology
PY  - 2021
VL  - 6
IS  - 2
DO  - 10.23880/act-16000210
ER  -