Physical activity has been recognized as an important lifestyle factor, which contributes to good health and delays the onset of many diseases later in life . By doing exercise in a proper way can give good effects to prevent many diseases such as coronary heart disease, hypertension, obesity, heart failure, depression, and diabetes mellitus . The regular exercise can prevent many severe diseases for human and animal but it must in a proper way. Most data indicate that excessive and chronic oxidative stress may cause cellular damage . The side effects on this excessive exercise can cause increasing oxidant in the body. If this oxidant could not be neutralized by the body, it can be defined as oxidative stress .
When free radicals overwhelm antioxidant defenses, the cells are damaged. This damage is called oxidative stress . But, if the formations of free radicals are increased induced by excessive physical exercise, the amount of free radicals will exceed the ability of the defense system body and cannot be neutralized by antioxidants in the body. This could damage in to the cell membrane, muscle, and tissues .
Free radical is a group of an atom that has unpaired electrons, unstable, and highly reactive those tend to attack cells, leading damage cell itself . In the excessive physical exercise, the free radicals formed primarily are Reactive Oxygen Species (ROS), through electron transport chain . In the report of , high concentrations of ROS could damage organs, tissue and cells included male reproduction system. Reactive oxygen species were dangerous for polyunsaturated fatty acid leading lipid peroxidation.
According to , the testicular interstitial tissue is the place of Leydig cells as a producer of testosterone that has a role in the differentiation process of spermatozoa. Excessive physical exercise may cause interference on the endocrine systems, such as increased cortisol levels and decreased testosterone levels . CRH induce increased of ACTH leading to activation of cortisol. Activation of cortisol may cause disturbance of testosterone .
Spirulina platensis is a blue-green alga belongs to the group cyanobacterium . It is rich in proteins, lipids, carbohydrates, β-carotene, riboflavin, α-tocopherol and α-linoleic acid . In the research of , the addition of Spirulina plantensis in diet can have beneficial health effect due to antioxidant properties. It is rich in proteins, lipids, carbohydrates, β-carotene, riboflavin, α-tocopherol and α-linoleic acid.Protective effect of Spirulina plantesis pre-treatment could reduce the lipid peroxidation, histopathological changes of testes and spermatozoa abnormalities .
In the report of , antioxidants present in Spirulina platensis (C-phycocyanin and β-carotene) acts as a free radical scavenger and improve the testicular condition. Vitamin C (ascorbic acid) and Vitamin E (α-tocopherol) also contribute to the support as an antioxidant in the testis with protecting role from the oxidative damage .
2. Materials and methods
The experimental animal used in this study are 20 healthy Wistar strained male rats (Rattus norvegicus) at age of 2-3 month, and the weight about 150-200 grams.
Ethanolic extract of Spirulina platensis obtained by means of maceration method. Materials used in this research included Spirulina platensis powder obtained from Yogyakarta, ethanol 96%for maceration, and rotavapor.
Chemicals used in histopathological preparation are 70%, 80%, 90% and 96% alcohol, xylol, paraffin, entellan, and Hematoxylin Eosin.
The equipment used for the treatment includes a scale to measure the weight of rat, five units of rat cage in the form of rectangular plastic tubs covered with wire, a container for feed and drink, 3 ml tuberculin syringes, feeding tube, a bucket of water measuring with a diameter of 35 cm.
The equipment used for the Spirulina platensis extract solution is beaker glass, digital scales, spoon, a bottle of extract, sterile aquadest, and 0.5% Na-CMC as suspension.
20 white male rats reared in Animal Laboratory of Experimental Animal at Medicine Faculty of Universitas Airlangga, randomized by lottery and were divided into five groups, and then adapted to the environment for one week. In the second week of experimental, T(1), T(2), and T(3) adapted to feeding orally of Spirulina platensis extract. In the third week of experimental, rats have been treated with swimming as excessive physical exercise respectively for 35 days, feeding and drinking given twice a day ad libitum.
2.1. Dose and Treatment
Negative control (C -): Neither received Spirulina platensis extract not swimming
Positive control (C+): Rats carry out swimming for 60 minutes
Treatment 1 (T1): Received 300 mg/kg bw Spirulina platensis extract and swimming for 60 minutes
Treatment 2 (T2): Received 600 mg/kg bw Spirulina platensis extract and swimming for 60 minutes
Treatment 3 (T3): Received 1200 mg/kg bw Spirulina platensis extract and swimming for 60 minutes
The treatments administered using 3 ml disposable tuberculin syringe with Feeding tube by intragastric gavage. The experimental treatment has been done every day for 35 days. The next day from the last treatment, treatment groups of C(-), C(+), T(1), T(2) and T(3) sacrificed by doing anesthesia (ether) and the testes have been collected.
2.2. Leydig cells observation and seminiferous tubules diameter measurement
The data observed from the amount of Leydig cell, the amount of Leydig cell undergoing necrotic, and diameter of seminiferous tubules. Each histopathological slide has been observed with five different views randomly. The evaluation of Leydig cells amount has been observed per field of view in each histopathological slide. Necrotic evaluation has seen from the changes of Leydig cells' nucleus, which may change into pycnosis, karyorrhexis, or karyolysis. The slide was observed under a microscope at 400x magnification.
The diameter measured on the shortest distance between two opposite points on the shortest of the central line and measure the furthest distance between two opposite points that perpendicular, and then divided by two. The measurement result is in micrometer at 100x magnification. Every slide measured by ten seminiferous tubules and then divided by two .
2.3. Data Analysis
The design of this research is using Completely Randomized Design with 5 treatments and 4 replicates. Data of research were analyzed with Analysis of Variance (ANOVA) followed by Duncan's test. Statistical analyzer used Statistical Program Service and Solution (SPSS) 22.0 for windows
3.1. Amount of Leydig cells
Based on the result, there are significant differences between treatments after counting the number of Leydig cells (p<0.05). Between C+ and the other groups such as C-, T1, T2, and T3 (Table 1). C+ with mean value 5.95 ± 0.10. Each group has a mean value of Leydig cells there are C- with mean value 12.5 ± 1.88, T1 with mean value 9.05 ± 1.45, T2 with mean value 10.50 ± 3.38, and T3 with mean value 12.20 ± 1.72.
3.2. Amount of Necrotic Leydig cells
Based on the result, showed a significant difference (p<0.05). C+ has a significant difference with the others group such as T1, T2, T3, and C- (Table 2). T1 and T2 group showed no significantly different, each group has mean value 38.80 ± 2.09 and 36.5 ± 1.56. T3 and C- group showed no significantly different, each group has mean value 14.27 ± 4.14 and 13.09 ± 4.87.
3.3. Seminiferous tubules diameter
C- has mean value 439.0 ± 18.9 significant difference with the others group such as T1, T2, T3, and C+ (Table 3). Based on the mean value, between T2 and T3 group, there is an increase of diameter number 297.0 ± 11.6 and 354.9 ± 26.1. T3 group has higher mean value than C+, T1, and T2.
The amount of Leydig cells in each testis are related to the total volume of Leydig cells and with the content of LH, if LH content is decreased then the amount of Leydig cells are decreased too . Excessive physical exercise could affect the endocrine system, which were FSH and LH. FSH and LH were gonadotropin hormone stimulating spermatogenesis in the male reproductive system . FSH and LH were produced and secreted by the gonadotropic cells of the anterior pituitary, which were derivate by GnRH. When excessive physical exercise, CRH induce increased of ACTH leading to activation of cortisol. CRH induce β-endorphin and suppressing GnRH leading inhibition of reproductive axis, which is a potent negative regulator of LH in Leydig cells causing decreased the level of LH . So the decreasing of the amount of Leydig cells because of LH content is decreasing too. C+ group which did not receive Spirulina platensis extract showed decreasing of Leydig cells amount (Figure 1).
Leydig cells are large, polygonal, eosinophilic cells that typically contain lipid droplets. The cytoplasm of Leydig cells also contains many inclusions like lipid . This was related to the statement of , high concentrations of ROS could damage organs, tissue and cells included male reproduction system. Reactive oxygen species were dangerous for polyunsaturated fatty acid leading lipid peroxidation. T3 is group received a dose of 1200 mg/kg bw of Spirulina platensis extract and it could protect Leydig cells against necrotic in testes (Figure 2).
The result of diameter measurement of seminiferous tubules and data analysing showed a significant difference (p<0.05). T3 which received dose 1200 mg/kg bw of Spirulina platensis has the optimal result among each group. It could be indicate that administration of Spirulina platensis extract at a dose of 1200 mg/kg bw has increased the diameter of seminiferous tubules (Figure 3).
This was related to the research of Spirulina platensis extract could maintain on the number of spermatogenic cells in seminiferous tubules. In addition to  Anindita and Sutyarso (2012), Diameter of seminiferous tubules is affected by spermatozoa production.