Simona Ciupe

Canine Amniotic Membrane Derived Mesenchymal Stem Cells- Potential Sources for Regenerative Medicine

Abstract Canine mesenchymal stem cells (MSCs) can be defined with self renew potential and specific differentiation capacity. Amiotic membrane represent an important source of MSCs, which can be harvested by minimally invasive methods. The aim of our study was to evaluate the growth characteristics of canine amniotic membrane derived mesenchymal stem cells. The placenta samples were collected after cesarean section from healthy mixed breed dogs. MSCs isolation was performed using enzymatic method. Isolated cells were cultured in propagation medium: Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (Sigma-Aldrich). The medium was changed after 4 days. The cell doubling number, cell proliferation capacity, cell doubling time, daily duplication rate and clonogenic efficacy were evaluated. Our study demonstrate the self renew potential of canine amniotic membrane derived mesenchymal stem cells, and can represent a potential source of stem cells for canine regenerative medicine.

Evaluation of the gametotoxic effect of nandrolone decanoate on the gametogenic function of male rabbit

Abstract The research was conducted on a number of 24 sexually mature male rabbits of common breed, divided into two numerically equal groups. The subjects from the experimental group were given 20 mg/kg nandrolone decanoate, intramuscularly, twice a week for four consecutive weeks. The control group did not receive any treatment. At the end of the experiment the testes were harvested through bilateral orchiectomy and processed for carrying out histopathological investigations. Testicular fragments were fixated in Stieve’s mixture for 24 hours, dehydrated with alcohol, clarified with butyl alcohol and included in paraffin. Serial sections of 5 μ thickness were stained by Massons trichrome method modified by Goldner. The testes taken from the control animals showed no detectable changes in optical microscopy. In the case of the animals belonging to experimental lots, there were alterations revealed evidenced by vacuolar dystrophy in the spermatocytes and spermatids, apoptosis, the edema of sperm head, spermatids syncytialization and cell necrosis followed by the disintegration of the seminal cell line with the formation of cellular debris in the seminiferous tubules lumen. These changes have affected part of the seminal cell line from the adluminal compartment (primary and secondary spermatocytes, spermatids and spermatozoids), but not those from the basal compartment (type A1 and B spermatogonia), which proved to be resistant in the conditions of 30 days exposure, at a dose of 20 mg/kg. In this context spermatogenesis is affected only for the period of drug action, but the risk of nandrolone decanoate transmission to humans through animal origin food consumption remains, with negative consequences on the spermatogenesis. Rezumat Având în vedere faptul că hormonii steroidieni au capacitatea de a modifica atât exprimarea informaţiei genetice codată la nivel genic cât şi nivelul fertilităţii la masculi, lucrarea de faţă şi-a propus testarea efectului nortestosteronului decanoat asupra celulelor spermatogene la iepuroi. Materialul biologic utilizat a fost reprezentat de către 24 iepuroi maturi sexual, împărţiţi în două loturi a câte 12 subiecţi fiecare, un lot experimental la care s-a administrat intramuscular 20 mg/kgc nortestosteron decanoat, de două ori pe săptămână, timp de 4 săptămâni şi un lot martor. La sfârşitul experimentului animalele aparţinând celor două loturi au fost supuse operaţiei de orhidectomie bilaterală iar testiculele au fost fixate în amestec Stieve, incluse în parafină şi secţiuni în grosime de 5 μm au fost colorate prin metoda Tricrom Goldner. Examinarea secţiunilor histologice provenite de la lotul martor a evidenţiat faptul că epiteliul tubilor seminiferi prezenta aspecte normale, fără a fi semnalate leziuni, nici măcar discrete. În cazul preparatelor histologice provenite de la lotul experimental au fost consemnate o serie de modificări cu preponderenţă la nivelul celulelor (spermatocite primare şi secundare, spermatide şi spermatozoizi) situate în compartimentul „adluminal” al tubilor seminiferi. Severitatea şi gradul de extindere al leziunilor a variat semnificativ de la un tub seminifer la altul şi chiar de la o porţiune la alta a aceluiaşi tub seminifer. Aceste modificări au constat în apariţia de celule în apoptoză precum şi numeroşi corpi apoptotici, degenerescenţă balonizantă a spermatocitelor şi spermatidelor, sinciţializarea spermatidelor, necroze însoţite de dezorganizare zonală moderată a epiteliului seminifer. În câteva zone au fost evidenţiate un număr mic de celule afectate (spermatogoniile) şi în compartimentul „bazal”. Dacă leziunile din compartimentul „adluminal” sunt reversibile, cele din compartimentul „bazal” sunt ireversibile deoarece afectează celulele „de rezervă” ale liniei spermatogenetice, compromiţând într-o oarecare măsură fertilitatea mascului. Aspectele evidenţiate ridică semne de întrebare asupra oprtunităţii utilizării nortestosteronului decanoat în terapie sau stimularea producţiei animale deoarece afectează funcţia spermatogenetică la masculi existând totodată riscul transferului lor la om prin consumul de alimente de origine animală.

Researches Regarding Recovery and Identification of Mare Oocytes for In Vitro Maturation

During March to May 2009 morphological studies on the mare oocytes were carried out harvested by two methods (aspiration and scrape), from different classes of ovarian follicles. Ovaries were obtained from 16 slaughtered mares in an approved abattoir and transported to the laboratory at a temperature of 5 degrees C. A total number of 181 antral follicles were identified in the macroscopic evaluation of ovaries. They were engaged in three classes based on size, and were collected using the aspiration method (n = 85) and scrape (n = 96). Recovery rate was 64.7% when harvesting by aspiration and 63.5% for scrape follicles. Oocytes were evaluated in terms of morphological, after standard criteria. Of the total 116 oocytes recovered 50 (43.1%) were considered good for in vitro maturation and a number of 66 (56.9%) were considered fallow. The largest percentage recovery was obtained in the case of follicles first class, 66%.. Oocytes Class II and III were recovered in the number of 20 (43%) and 10 (25%) were considered good for in vitro maturation and 26 (57%) and 30 (75%) were considered fallow. Most oocytes obtained from ovarian follicles of Classes II and III were in various stages of degeneration.