Efek Serbuk Kunyit dan Kurkumin pada Spermatogenesis Mencit (Mus musculus) yang Diberi Minuman Beralkohol

Kunyit mengandung senyawa kurkumin yang dapat digunakan sebagai zat antiinflamasi dan membantu memperbaiki sel-sel yang rusak. Tujuan dari penelitian ini menganalisis pengaruh serbuk kunyit dan kurkumin pada jumlah dan ukuran sel spermatogonium; spermatosit primer; dan spermatosit sekunder; bobot testis serta diameter tubulus seminiferus Mus musculus yang diberi minuman beralkohol. Penelitian ini merupakan penelitian eksperimental dengan Rancangan Acak Lengkap (RAL), menggunakan 12 ekor Mus musculus jantan yang dibagi kedalam 4 kelompok perlakuan dan 3 kali ulangan. R0 merupakan kontrol, R1 kontrol alkohol, R2 pemberian serbuk kunyit sebanyak 0,1 mg/hari, R3 pemberian kurkumin sebanyak 0,01 mg/hari. Perlakuan diberikan selama 30 hari. Data penelitian dianalisis menggunakan analysis of variance (ANOVA) pada taraf kepercayaan 95%. Berdasarkan hasil penelitian yang diperoleh dapat disimpulkan bahwa tidak terdapat perbedaan bermakna (p>0.05) pada jumlah spermatogonium dan ukuran sel (spermatogonium, spermatosit primer, dan spermatosit sekunder), namun terdapat perbedaan bermakna pada (P<0,05) pada bobot testis, diameter tubulus seminiferus dan jumlah sel (spermatosit primer, dan spermatosit sekunder). Turmeric contains curcumin compounds that can be used as anti-inflammatory substances and help repair damaged cells. The purpose of this study was to analyze the effect of turmeric powder and curcumin on the number and size of spermatogonia cells; primary spermatocytes; and secondary spermatocytes; testicular weight and diameter of the seminiferous tubules of Mus musculus given alcoholic beverages. This study is an experimental study with a completely randomized design (CRD), using 12 male Mus musculus which were divided into 4 treatment groups and 3 replications. R0 is control, R1 is alcohol control, R2 is 0.1 mg/day of turmeric powder, R3 is 0.01 mg/day of curcumin. The treatment was given for 30 days. The research data were analyzed using analysis of variance (ANOVA) at the 95% confidence level. Based on the results obtained, it can be concluded that there is no significant difference (p>0.05) in the number of spermatogonia and cell size (spermatogonia, primary spermatocytes, and secondary spermatocytes), but there is a significant difference (P<0.05) in testicular weight, diameter of the seminiferous tubules and the number of cells (primary spermatocytes, and secondary spermatocytes).

POTENSI MADU PADA PENINGKATAN JUMLAH SEL SPERMATOGENIK TIKUS (Rattus norvegicus) YANG KEKURANGAN NUTRISI

This study aimed to determine honey's potential in increasing the number of spermatogenic cells (spermatogonium, spermatocyte and spermatid) of rats (Rattus norvegicus) with nutritional deficiencies. Twenty four male rats (Rattus norvegicus) were randomly divided into four groups. Negative control (K-) mice was not fasted nor given honey. Positive control (K+) mice was fasted for 5 days without the administration of honey. The treatment group rats were fasted for 5 days followed by administration of honey 30% (P1) and 50% (P2) in drinking water for 10 days. On the 76th day all rats were sacrificed and their testes were collected for histological slides using Hematoxylin-Eosin staining. The results showed that fasting treatment for five days (group K +) caused a decrease (p <0.05) in the number of spermatogonia cells, spermatocytes and spermatids compared to those of the normal mice (group K–). Administration of 50% honey (group P2) for 10 days caused an increase (p <0.05) in the number of spermatogonia cells, spermatocytes and spermatids compared to the K + group mice. In P2 group, the number of spermatogonia cells was lower (p <0.05), while the number of spermatocytes and spermatids was not significantly different than in the K– group mice. It could be concluded that the administration of honey was able to regenerate the testicular tissue of rats with nutritional deficiency by increasing the number of spermatogenic cells in the seminiferous tubules.

Edible Bird’s Nest Supplementation Improves Male Reproductive Parameters of Sprague Dawley Rat

Edible bird’s nest (EBN) is reported to have a positive in vitro proliferative effect and contain male reproductive hormones. Spermatogonia cells proliferate during spermatogenesis under male reproductive hormones stimulation that include testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Characterization of EBN through liquid chromatography-mass spectrometry (LCMS) has found testosterone as a base peak. Six types of amino acids, estradiol and sialic acid were among the major peaks that have been characterized. Based on the presence of these reproductive components, this study evaluated different doses of EBN on sperm parameters and male reproductive hormones of Sprague Dawley rats. Sixteen Sprague Dawley rats at the age of eight weeks were randomly and equally divided into four groups, which are Control, 10 mg/kg BW/d 50 mg/kg BW/d, and 250 mg/kg BW/d EBN group. The rats were fed with EBN enriched pellet daily and water ad-libitum. Rats were sacrificed and the organ was weighed for organ coefficients after eight weeks of treatment. Sperm concentration, percentage of sperm motility, and sperm viability were evaluated. Meanwhile, ELISA method was used to measure testosterone, FSH, and LH. Findings showed that there were no significant differences in organ coefficient between groups. Supplementation of 250 mg/kg BW/d EBN demonstrated a significant increase in sperm concentration, percentage of sperm motility as well as FSH and LH level compared to 10 mg/kg BW/d group. There was a dose-dependent increase in testosterone level but was not significant between groups. Based on these findings, EBN is concluded to have crucial effects on male reproductive parameters.

Acetyl-L-Carnitine Induces Autophagy to Promote Mouse Spermatogonia Cell Recovery after Heat Stress Damage

Acetyl-L-carnitine (ALC) is an effective substrate for mitochondrial energy metabolism and is known to prevent neurodegeneration and attenuate heavy metal-induced injury. In this study, we investigated the function of ALC in the recovery of mouse spermatogonia cells (GC-1 cells) after heat stress (HS). The cells were randomly divided into three groups: control group, HS group (incubated at 42°C for 90 min), and HS + ALC group (treatment of 150 μM ALC after incubated at 42°C for 90 min). After heat stress, all of the cells were recovered at 37°C for 6 h. In this study, the content of intracellular lactate dehydrogenase (LDH) in the cell supernatant and the malondialdehyde (MDA) levels, catalase (CAT) levels, and total antioxidant capacity (T-AOC) were significantly increased in the HS group compared to the CON group. In addition, the mitochondrial membrane potential (MMP) was markedly decreased, while the apoptosis rate and the expression of apoptosis-related genes (Bcl-2, Bax, and caspase3) were significantly increased in the HS group compared to the CON group. Furthermore, the number of autophagosomes and the expression of autophagy-related genes (Atg5, Beclin1, and LC3II) and protein levels of p62 were increased, but the expression of LAMP1 was decreased in the HS group compared to the CON group. However, treatment with ALC remarkably improved cell survival and decreased cell oxidative stress. It was unexpected that levels of autophagy were markedly increased in the HS + ALC group compared to the HS group. Taken together, our present study evidenced that ALC could alleviate oxidative stress and improve the level of autophagy to accelerate the recovery of GC-1 cells after heat stress.

PSII-36 The application of busulfan to inhibit the spermatogenesis in quail testis

The use of testicular stem cells (spermatogonia) is of most interest for obtaining individuals with predetermined traits and genome genetic modification and for conservation of poultry gene pool. A significant population of mature donor germ cells (sperm) is formed upon successful spermatogonia cells transplantation into the testes of male recipients. Obtained sperm can be used to produce offspring with the desired traits. A key step in this technology is the removal of own spermatogenic cells (inhibition of spermatogenesis) in male recipients. The aim of research was to develop and optimize methodological approaches to inhibit the spermatogenesis in quail using busulfan. This drug was injected directly into the testes parenchyma of mature males by multiple injection at the concentration from 20 to 100 mg per 1kg of body weight (n = 25). Histological preparations of testes from the experimental quails were obtained to study composition of spermatogenic cells in the seminiferous tubules after busulfan administration. The male peers who were not injected with busulfan were used as a control. Experimental quails showed a decrease in the number of spermatogenic cells in the seminiferous tubules 32, 75, 111, 119 and 118 times compared with the control when using busulfan in concentrations 20, 40, 60, 80 and 100 mg/kg of weight, respectively (P &lt; 0.001). The cells composition in the seminiferous tubules from experimental quails was represented mainly by Sertoli cells and spermatogonia. After busulfan introduction at the concentrations 20, 40, 60, 80 and 100 mg/kg, the percentage of spermatogonia was 55±5 %, 24±4 %, 6±2 %, 5±2 % and 4±1 %, respectively. The use of busulfan at the concentration of 80–100 mg/kg led to high mortality of quails. Thus, it was found that the optimal busulfan concentration for elimination of quail spermatogenic cells was 60 mg/kg. Supported by RFBR within Project №18-29-07079.

PSII-35 The age-related characteristics of spermatogenesis in gander

Cryopreservation of testicular stem cells - spermatogonia is of interest along with the creation of semen cryobanks. During transplantation into recipients’ testes, spermatogenic cells can create a significant population of germ cells in the process of differentiation. The knowledge about spermatogenesis course in males is necessary for the effective selection spermatogenic cells. The research aim was to study the age-related characteristics of spermatogenesis in geese. The histostructure of gander testes (n = 35) at the age of 1 to 7 months was studied. The diameter of seminiferous tubules, and the types and number of spermatogenic cells in them were evaluated. From each gander at least 30 seminiferous tubules were examined. At the age of 1 month, the diameter of the seminiferous tubules was 51±1 μm. In subsequent age periods, this indicator increased and amounted to 63±2, 65±3, 66±2, 79±3, 98±6 and 170±5 μm at the age of 2, 3, 4, 5, 6 and 7 months, respectively. Diameter increase with the age was associated with an increase of spermatogenic cells number inside tubules. At the 1 months age, the number of spermatogenic cells in one seminiferous tubule did not exceed 22±1. At the age of 2, 3, 4, 5, 6, and 7 months, this indicator increased by 2.1, 2.7, 3.1, 6.4, 8.5 and 21.2 times. At the age from 1 to 3 months, the main cells types were Sertoli and spermatogonia cells. Primary and secondary spermatocytes from 4 months of age and spermatids from 5 months of age were visualized in the seminiferous tubules. Sperm were detected in the seminiferous tubules at 6 months old, the number of which increased towards the age of 7 months. The study was supported by RSF within Project №16–16–04104.

Effects Of Short And Long-Term Administration Of Alfalfa On Testicular Histomorphometry In Rats

Alfalfa (Medicago sativa) is a plant with phytoestrogenic properties, which has been used as a major part of diets in husbandry. Since there are controversial reports related to the effects of alfalfa consumption on animal fertility, its effects on rat testicular tissue were assessed in the present study. Control (n=15) and alfalfa (n=15) groups were fed with ordinary rat chow and ordinary rat chow plus alfalfa, respectively. Testicles were removed after 30, 45, and 60 days of consumption, and tissue sections were prepared to assess histomorphometric changes related to alfalfa consumption. Based on the results, there was no significant difference in length, width, and volume of testes of treated rats to control in all groups. But the number of testicular spermatogonia cells, primary spermatocyte cells, primary spermatid cells, testicular spermatozoid cells and Leydig cells significantly or insignificantly increased in rats that received alfalfa for 30 days but all of these parameters insignificantly decreased in rats that received alfalfa for 60 days. The cause of these changes may be due to estrogenic or anti-estrogenic, antioxidant and endocrine effects of alfalfa. Conclusion: Consumption of alfalfa for short time had only a transient positive effects on testicular tissues but use of alfalfa for 60 days had little destructive effects on testicular tissue in rats. So longer durations of time could be suggested for further research on the effects of alfalfa on rat’s reproduction index.

In Vitro Cytotoxicity Effects of Zinc Oxide Nanoparticles on Spermatogonia Cells

Zinc Oxide Nanoparticles (ZnO NPs) are a type of metal oxide nanoparticle with an extensive use in biomedicine. Several studies have focused on the biosafety of ZnO NPs, since their size and surface area favor entrance and accumulation in the body, which can induce toxic effects. In previous studies, ZnO NPs have been identified as a dose- and time-dependent cytotoxic inducer in testis and male germ cells. However, the consequences for the first cell stage of spermatogenesis, spermatogonia, have never been evaluated. Therefore, the aim of the present work is to evaluate in vitro the cytotoxic effects of ZnO NPs in spermatogonia cells, focusing on changes in cytoskeleton and nucleoskeleton. For that purpose, GC-1 cell line derived from mouse testes was selected as a model of spermatogenesis. These cells were treated with different doses of ZnO NPs for 6 h and 12 h. The impact of GC-1 cells exposure to ZnO NPs on cell viability, cell damage, and cytoskeleton and nucleoskeleton dynamics was assessed. Our results clearly indicate that higher concentrations of ZnO NPs have a cytotoxic effect in GC-1 cells, leading to an increase of intracellular Reactive Oxygen Species (ROS) levels, DNA damage, cytoskeleton and nucleoskeleton dynamics alterations, and consequently cell death. In conclusion, it is here reported for the first time that ZnO NPs induce cytotoxic effects, including changes in cytoskeleton and nucleoskeleton in mouse spermatogonia cells, which may compromise the progression of spermatogenesis in a time- and dose-dependent manner.

Effect of Bleomycin, Etoposide, and Cisplatin Treatment on Spermatogonia Cell and Malondialdehyde Level in Male Rats

Co-administration of bleomycin, etoposide, and cisplatin (BEP) becomes standart chemotherapy for testicular cancer because it has brought a cure rate of more than 90%. Impact of the treatment to the outcome become a concern, particularly the adverse effect on a long-term reproductive health risk to cancer survivors. There is no evidence, when the damage to the testes began due to the administration of BEP chemotherapy, makes the indication of treatment still controversial. The aim of this study is to determine the effects of BEP on Spermatogonial cell and MDA levels outcome in an animal model. Male wistar rats (Rattus norvegicus) aged 13-15 weekswere treated daily with BEP for three cycles, 33 hours each. It was divided into one control group received 1cc of normal saline, and three groups received three cycles of 0.5 x dose-levels of BEP (Intraperitoneally; 0.75 mg/kg, 7.5 mg/kg, and 1.5 mg/kg). Cell number of Spermatogonia cells were calculated from HE-stained specimens and observed under light microscope (Olympus BX-51) using 400x magnification (high power field) Thiobarbituric acid (TBA) test method used to measure malondialdehyde (MDA) level by spectrophotometry. The result was a significant decrease in the average number of Spermatogonia cells (p = 0.003) between the control group and others. This is caused by excessive exposure to BEP chemotherapy, which cause atrophy of the seminiferous tubules and content of germ cells in the tubules has decreased, accompanied by the appearance of immature germ cells that enter the lumen. A significant increase in MDA levels (p = 0.001) occurred after the administration of the third cycle of BEP chemotherapy. In conclusion, BEP chemotherapy adversely affect the number of Spermatogonia cells and MDA level. The third cycle BEP chemotherapy significantly more destructive compared to the first and second cycle.