CHANGES IN RAT TESTIS AND SPERM COUNT AFTER … · Changes in Rat Testis and Sperm Count after Acute Treatment with Sodium Nitrite 187 giogenesis and vasodilatation, thus increasing

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Bulgarian Journal of Agricultural Science, 19 (2) 2013, 186–189Agricultural Academy

CHANGES IN RAT TESTIS AND SPERM COUNT AFTER ACUTE TREATMENT WITH SODIUM NITRITE

E. PAVLOVA*, D. DIMOVA, E. PETROVA, Y. GLUHCHEVA and N. ATANASSOVABAS, Institute of Experimental Morphology, Pathology and Anthropology with Museum, BG – 1113 Sofi a, Bulgaria

Abstract

PAVLOVA, E., D. DIMOVA, E. PETROVA, Y. GLUHCHEVA and N. ATANASSOVA, 2013. Changes in rat testis and sperm count after acute treatment with sodium nitrite. Bulg. J. Agric. Sci., Supplement 2, 19: 186–189

Sodium nitrite (NaNO2) is an inorganic salt with various industrial applications. The adverse health effects of NaNO2 in animals and humans are typically due to the formation of methemoglobin in the blood. This can lead to cyanosis and, at very high levels, death. Humans are constantly exposed to sodium nitrite through food and drinking water. The aim of the present study is to follow up the changes in rat testis and sperm count after acute treatment with NaNO2. Four-month-old male Wistar rats were intraperitoneally injected with NaNO2 at dose of 50 mg.kg–1 body weight (distilled water for controls). Treated animals were sacrificed at different time intervals (1 h, 5 h, 24 h and 48 h) following the administration. Testes and epididymides were sampled, weighed and embedded in paraffin using rou-tine histological practice. Spermatozoa were isolated from both vasa deferentia and counted. Preliminary histological observations of the testis of some experimental animals demonstrated disorganization of seminiferous epithelium and assemblance of undifferentiated germ cells in the luminal area of the tubules. Testis weight/body weight index was increased in the first hours after administration, which is probably due to higher seminal fluid volume. Statistically significant reduction in sperm count ranging between 480 g.kg–1 (fifth hour) and 280 g.kg–1 (48 h) was observed. These results may be associated with impaired hormonal balance and tissue anoxia – an adverse environment for germ cell development. In conclusion, acute treatment with NaNO2 affects testis morphology, some weight indices and sperm count in mature rats.

Key words: rat testis, sodium nitrite, sperm countAbbreviations: MetHb – methemoglobin; ns – not signifi cant; BW – body weight; TW – testis weight; EpiW – epididymal weight; Sz count – spermatozoa count

*E-mail: [email protected]

Introduction

Sodium nitrite (NaNO2) is a water soluble, inorganic salt widely used in various industries including agricultural, chemical industry, textile processing industry, disinfectants, colouring agents, etc (U.S.DHHS, 2001). Humans are ex-posed to nitrate and nitrite through food and drinking water, with a minor contribution from the air. Sodium nitrite has been found to inhibit growth of disease causing microorgan-

isms and it is a common food additive (E250) used as a color fi xative and preservative in meats and fi sh. In blood, nitrite undergoes a coupled oxidation reaction with oxyhemoglo-bin to produce methemoglobin (MetHb). Unlike hemoglo-bin, MetHb cannot exchange oxygen; hence, the presence of excess MetHb in the circulation proportionately reduces the ability of the blood to transfer oxygen (RCHAS, 2000) and can cause hemic hypoxia. The body reacts to hypoxia with adaptive responses, such as relaxation of smooth muscle, an-

187Changes in Rat Testis and Sperm Count after Acute Treatment with Sodium Nitrite

giogenesis and vasodilatation, thus increasing blood supply to tissues, compensating for the lack of oxygen (Farias et al., 2005a).

There are literature data mainly for the effect of hypo-baric hypoxia at high altitudes on testis morphology and se-men quality. The effect of NaNO2 and the subsequent hemic hypoxia are poorly investigated and data about its infl uence on reproductive system are controversial. The widespread use of NaNO2 in the food industry contributes to its poten-tial health risk. This motivated us to investigate the effect of NaNO2 on rat spermatogenesis at different time intervals following administration.

Materials and Methods

The experiments were carried out on four-month-old male Wistar rats. The animals were divided into four sodi-um nitrite-treated groups (n = 15 rats per group) and age-matched control group (n = 16). Rats were maintained in the institute’s animal house in standard hard bottom polypropyl-ene cages at 23ºC ± 2ºC and 12:12 h light-dark cycle with free access to laboratory chow and tap water throughout the study.

Sodium nitrite was injected intraperitoneally at 50 mg.kg-1 body weight (one ml dosing volume). Treated ani-mals were sacrifi ced at different time intervals following the administration (1 h, 5 h, 24 h and 48 h) under light diethyl ether anesthesia. The control rats were injected with the same volume of distilled water.

Testes and epididymides were sampled, weighed and embedded in paraffi n using routine histological practice. Spermatozoa were isolated from both vasa deferentia and counted using Buerker’s chamber. Data were statistically processed using Student’s t-test.

The animal experiments were performed in accordance with the animal protection guidelines approved by the Ethics Committee for Experimental Animal Use at IEMPAM, BAS.

Results and Discussion

Hypoxia is known to compromise the fertility in man (Okumira et al., 2003) and in other mammals. It has an im-pact on male testicular functions such as reduced testoster-one level and disturbance of spermatogenesis (Farias et al., 2008). A strong metabolic stress in spermatogenic cells is observed. Several studies provide some evidence for histo-pathological changes of the testis in male rats, but the ob-served effects could not be confi dently attributed to sodium nitrite exposure (RCHAS, OEHHA, CAL/EPA, 2000).

Our histological evaluation of four-month-old old rat testis showed the presence of mature spermatozoa and their release into the tubular lumen in stage VIII of spermatogenic cycle. The spermatogenesis is organized in 14 stages accord-ing to the classifi cation of Clermont and Perey (1957). The germ cells are arranged in fi ve-six layers. Luminal region in all of the seminiferous tubules is clearly distinguished (Fig-ure1A). Besides the intact structure of seminiferous epithe-lium, our preliminary morphological observation of experi-mental rat testis demonstrated the presence of disorganized seminiferous tubules and assemblance of undifferentiated germ cells in the luminal area in some experimental animals.

Fig. 1A. Morphology of the seminiferous tubules of con-trol rat testis. HE, x 200

Fig. 1B. Morphology of the seminiferous tubules on rat testis cross sections fi ve hours fallowing NaNO2 applica-

tion (50 mg.kg-1.bw) Disorganization of seminiferous epithelium was observed. HE, x 200

AA

BB

188 E. Pavlova, D. Dimova, E. Petrova, Y. Gluhcheva and N. Atanassova

There is lack of lumen in some tubules. These fi ndings are most obvious at the fi fth hour after NaNO2 administration but may also be seen at the 24th and 48th hour (Figure 1B). Interestingly, blood vessels with larger diameter are more frequently seen compared to the control (data not shown). We did not expect alterations in seminiferous epithelium so early after sodium nitrite treatment despite its acute applica-tion. The changes are not seen in all experimental animals in group and our future work would elucidate if disorganized seminiferous tubules are due to the effect of sodium nitrite. Literature data for the effect of sodium nitrite-induced hemic hypoxia on the male reproductive system are controversial. Male rats (Farias et al., 2005b) and rhesus monkeys (Sax-ena, 1995) subjected to hypobaric hypoxia showed atrophy of germinal epithelium with Sertoli cell replacing spermato-gonia as well as spermatogenic arrest at the end of the third week of exposure. No evidence for testicular pathology is identifi ed in animals subjected to a three-day regimen of NaNO2 injections (Bond et al., 1981).

The body reacts to hypoxia with adaptive responses, such as angiogenesis and vasodilation, thus increasing blood supply to tissues as a compensatory oxygen delivery mechanism. Local changes in testicles exposed to hypoxia include neovasculari-sation and an increase in temperature that would be correlated with the alterations in spermatogenesis observed in this tissue. In this respect, the response of testis to hypoxia would resemble other hyperthermia-related pathologies, such as varicocele and cryptorhydia (Farias et al., 2005b). Farias et al. (2005b) found signifi cantly greater number and increased diameter of blood vessels in interstitial space of rats subjected to hypobaric hy-poxia. Our fi ndings about the frequently seen blood vessels with higher diameter are in agreement with these observations.

The magnitude and time dependence of testicular mass changes are very relevant for interpreting the underlying physiological changes taking place in this tissue. Our quan-titative results show that the gonado-somatic index (ratio testicular weight to body weight) was elevated by 130 g.kg-1 in the fi rst hours (fi rst and fi fth hour) and returned within the normal range at later stages (Table 1). Epididymal index (ratio epididymal weight to body weight) remained at normal values although it was slightly elevated 24 hours following NaNO2 administration (Table 1). Elevated gonado-somatic index was unexpected at an early stage after hypoxia. Lit-erature data show changes in organs’ mass during hypoxia only if they are engaged in blood fl ow homeostasis (heart’ and lungs’ mass) (Hammond et al., 2001). Therefore, the elevated indices in our study could be due to increased tes-ticular fl uid volume, which may be associated with impaired hormonal balance (dysfunction in the hypothalamo-hypoph-ysial-gonadal axis), and tissue anoxia (Saxena, 1995).

We found reduction of sperm count after NaNO2 induced hemic hypoxia. The sperm count was reduced in all experi-mental groups (1 h, 5 h, 24 h and 48 h) by 300 g.mg–1, 470 g.mg–1, 160 g.mg–1 and 280 g.mg–1, respectively (Figure 2). Saxena (1995) has also reported a signifi cant decrease of se-men volume in rhesus monkeys in the fi rst week after ex-posure to hypoxia. Hypobaric hypoxia associated with high altitude exposure is reported to induce oligoasthenospermia with reduced motility, reduction of the total number of mo-tile sperm and an increase of abnormal or immature sper-matozoa. In animal models (rat, mouse, guinea pig, rabbit, monkey, sheep), it has been shown that hypobaric hypoxia induces partially reversible quantitative changes such as

Sz count

0

5

10

15

20

25

Control 1 h 5 h 24 h 48 h

mill

ion/

ml

*

**

*

30%

47%

16%

28%

Fig. 2. Changes in rat sperm count at different time in-tervals following NaNO2-induced acute hemic hypoxia.

Data represent mean value ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001). Sz count – spermatozoa count

Table 1Changes in rat gonado-somatic index (ratio of the testis weight to body weight) and epididymal index (ratio of the epididymal weight to body weight) at different time in-tervals following NaNO2 administration. Data represent mean value ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001) ns – not signifi cant

TW/BW EpiW/BWControl 0.0055 ± 0.0006 0.0016 ± 0.00021 h 0.0062 ± 0.0006 13%, ** 0.0018 ± 0.0003 ns5 h 0.0061 ± 0.0006 12%, ** 0.0016 ± 0.0002 24 h 0.0057 ± 0.0006 ns 0.0018 ± 0.0003 10%, *48 h 0.0058 ± 0.0006 ns 0.0017 ± 0.0002 ns

189Changes in Rat Testis and Sperm Count after Acute Treatment with Sodium Nitrite

decrease in semen volume, sperm count and sperm motil-ity (Gasco et al., 2005; Petrova and Ormandzhieva, 2012). Established low thyroid hormone concentrations have also been reported to affect semen quality (Chandrasekher et al., 1985).

The observed changes may be indicative of impaired spermatogenesis and suggest decreased testicular functions. Probably testis morphology, some weight indices and sperm count in mature rats are sensitive and undergo changes in the early stages after NaNO2 acute treatment.

AcknowledgementsThis work is supported by a grant No DMU 03/18 for

Young scientists from the Bulgarian National Science Fund and by the European Social Fund and Republic of Bulgaria, Operational Programme “Human Resources Development” 2007-2013 framework, Grant № BG051PO001-3.3.06 - 0048 from 04.10.2012.

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