Palumbo, Veronica (2020) Valutazione della riduzione del danno testicolare e spermatico indotto da Cannabis Sativa (Marijuana) nel topo dopo somministrazione orale di Lepidium Mejenii (MACA). [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: Italiano
Title: Valutazione della riduzione del danno testicolare e spermatico indotto da Cannabis Sativa (Marijuana) nel topo dopo somministrazione orale di Lepidium Mejenii (MACA)
Date: 31 January 2020
Number of Pages: 137
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Veterinaria e Produzioni Animali
Dottorato: Scienze veterinarie
Ciclo di dottorato: 32
Coordinatore del Corso di dottorato:
Cocchia, NatasciaUNSPECIFIED
Date: 31 January 2020
Number of Pages: 137
Keywords: MACA, SPERM, THC
Settori scientifico-disciplinari del MIUR: Area 07 - Scienze agrarie e veterinarie > VET/10 - Clinica ostetrica e ginecologia veterinaria
Date Deposited: 23 Mar 2020 08:34
Last Modified: 08 Nov 2021 14:45

Collection description

Tetrahidrocannabinol (THC) administration has been associated with testicular damage and reduced semen quality. The oral administration of hypocotyl- roots of MACA improved spermatogenesis, sperm motility and count and reduced spermatogenic damage induced by different causes (high altitude, lead acetate injections, malathion). The aim of this study was to evaluate the effect of administration of THC, MACA and of their association on testicular tissue and semen parameters. Thirty 6-weeks old male c57/BL mice were divided into 4 groups: 1) control mice that not received any treatment (n=6); 2) received 10 mg/kg of Δ9-THC in 0.1 ml of sesam oil subcutaneously for 30 days (n=9); 3) received 50 mg/Kg of MACA powder PO for 30 days (n=10); 4) received the same doses of both Δ9-THC and MACA (n=5). Immediately after euthanasia, epididymis, testes, liver and kidney were collected. Epydidimiswas incised and placed into 500 µl of PBS solution. Spermatozoa were allowed to swim-up into the medium for at least 30 minutes at 35°C. Sperm concentration (SC) was determined with a Bürker chamber; total (TM), rapid (RPM) and slow progressive (SPM) sperm motilities were assessed by placing 10 µl of pre-warmed (37°C) semen suspension onto a pre-warmed slide and examined by light microscope. Testes, liver, kidney and colon were preserved in 10% neutral buffered formalin), dehydrated and embedded in paraffin Paraffin blocks were cut into 4 μm thick sections and stained with hematoxylin and eosin (HE) for morphology. For liver, kidney and colon histologic assessment, several parameters were semiquantitatively evaluated separately by two independent experienced pathologists in a blinded fashion with good concordance (Cohen's κ = 0.913, P < 0.001). For the morphometry of the testis, transverse sections of testes with at least 20 round or nearly round seminiferous tubules were chosen randomly to measure tubular diameters and seminiferous epithelium height for each animal regardless the stage of the seminiferous epithelium cycle using images obtained at × 100 magnifications. The diameter (D) of seminiferous tubules was measured across the minor and major axes of the tubules by taking the average of two diameters, D1 and D2. The same tissue section used for measuring tubular diameters was used to measure the seminiferous epithelium height. For this analysis, two perpendicular lines in each field were drawn from the basement membrane (tunica propria) to the tubule lumen (luminal border); the mean of these two values were considered as the height of the seminiferous tubule. For tubular spermatogenesis index evaluation and quantification, we applied a ten-point scoring system used both in human and experimental pathology for its good reproducibility. Statistical analyses between groups were performed using Student’s t-test (two-tailed) or MANOVA for parameters with normal test based on distribution. Statistical significance was set at p < .05. Mean and standard deviation of semen parameters for the different group were reported in the Table; different small letters (a-d) indicate differences between groups evaluated by MANOVA and Fisher’s LSD (p<0.05). Groups Semen parameters Control 1 2 3 SC(x106spz/ml) 50.5±8.4a 23.1±3.6 b 36.6±6.1c 54±8.3 a TM (%) 76.7±2.6 a 33±2.5 b 55.8±6.2 c 79.4±4.4 a RPM (%) 69.2±3.8 a 15.8±5.5 b 37.8±4.7 c 51.6±4.7 d SPM (%) 5.8±2 a 5.9±1.8 a 15.5±6 b 13.6±2.5 b Histological examination of formalin-fixed and paraffin-embedded sections of liver, kidney and cecum showed no evident histopathological changes for the selected parameters and no statistically significant difference among the groups. Histological assessment of testes from mice of CONTROL group showed no alterations with a normal histoarchitecture. In experimental group MACA, no severe and significant alterations were observed in testicular parenchyma nor in spermatogenesis. In experimental group THC, transverse sections of testis showed mild to moderate alterations accounting 45% of the testicular parenchyma and consisting mostly in multifocal detachment of germinal epithelium, irregular and buckled basement membrane, tubular deformation and degeneration, shrunken seminiferous tubules and increased luminal diameter. In experimental group THC+MACA, transverse sections of testis showed an overall normal histoarchitecture of testicular parenchyma but about the 25% of testicular parenchyma showed mild alterations such as detachment of germinal epithelium and reduced population of mature spermatozoa. In morphometric measurements, tubular diameter had a significant increasing thickening in groups THC and THC+MACA compared to group MACA and control group (p<0.05). Moreover, seminiferous epithelium height decreased significantly in groups THC compared to control group and experimental groups MACA and THC+MACA (p<0.01). Spermatogenic Index had a level of 10 in control group and experimental group MACA but shifted from a level of 10 to 9in experimental group THC and THC+MACA. Therefore, a slight but not statistically significant reduction in the spermatogenic index was observed in group 2 (p<0.001) compared to group MACA and THC+MACA. Epididymal cross sections of CONTROL groups as well as experimental group THC, MACA, THC+MACA showed no significant alterations: epididymal lumen were filled with spermatozoa and epithelium showed intact basement membrane, epididymal tubules, pseudostratified columnar epithelium and interstitial areas. In Vivo MACA administration reduced the deleterious effect on testicular parenchima and semen production caused by THC exposure.


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