ASSESSMENT OF MOUSE EMBRYO VIABILITY BY ESTERASIC ACTIVITY DETECTION

In order to evaluate the esterasic activity within the viable embryos we used the Fluorescein diacetate(FDA) staining test. For staining was used a 0.5 mg/ml FDA stock solution. The embryos were recoveredat 48 hours post coitus from superovulated Swiss mouse females. Before staining the embryos weremicroscopically evaluated by morphological criteria and classified in 4 quality codes. The two methodsused for quality and viability assessment were correlated applying Pearson coefficient. The calculatedvalue of the Pearson coefficient (r=1) showed a strong correlation between the two methods used andindicate FDA staining test and esterasic activity as a fast, easy and reliable method for embryo viabilityassessment

EXPERIMENTAL TRIES TO ESTABLISH THE PREIMPLANTATIONAL MAMMALIAN EMBRYOS VIABILITY THROUGHOUT STAINING

Presently there are more methods to assess embryo quality but, still the wieldy usedremains the morphological criteria method. In this experiment were tested twostaining methods for embryos and oocytes. The embryos were recovered from mousefemale at 72 hours after mating. The recovered embryos were first evaluated aftermorphological criteria and than by Trypan blue exclusion and Neutral red staining.Using Trypan blue exclusion were evaluated 30 embryos from which 19 (63.3) wereclassified as viable and 11 (36.7) were classified as nonviable. By Neutral redstaining were evaluated 37 embryos from which 24 (64.8) were considered viableand 13 (35.2) were considered nonviable. The oocytes recovered were alsoevaluated using the two methods: using Trypan blue exclusion were stained 10oocytes from which 9 remained uncolored and were considered viable and 1 wasstained in blue and was considered nonviable and using Neutral red 13 oocytes werestained from which 9 were evaluated as viable and 4 as nonviable

THE INFLUENCE OF THE PUERPERAL AFFECTIONS ON INSEMINATION INDEX AND UTERINE REPOSE IN COWS

The observations were made, through a year, at SD Timisoara on cows fromHolstein-Friesian and Fleckvieh breed. The puerperal period was observed, theincidence of the endometrites was recorded and there were calculated tworeproduction parameters: the Insemination Index (Ig) and the Uterine Reposeduration (UR) (Open days). The Insemination Index (service/conception) (Ig)represents the mean number of artificial inseminations performed in order to obtaina pregnancy. Uterine Repose represents the time interval, in days, from calving untilthe fecund insemination. The Uterine Repose has two components: VoluntaryWaiting Period (VWP) (time interval from calving until the introduction of thefemale to reproduction) and Service Period (SP) (time interval from the end of theVWP until the fecund insemination). There were noticed that the incidence of theuterine infections were significant higher (p<0.05) at cows from Holstein-Friesianbreed (63.3%), compared to the cows from Fleckvieh breed (41.3%). TheInsemination Index was significant lower (p<0.05) at cows without uterine infections(1.9), compared to the cows with uterine infections (2.5). The mean duration of theUterine Repose was significant lower (p<0.05) at healthy cows (114.7 days),compared with cows with uterine infections after calving (182.2 days). It seams thatthe cows from Fleckvieh breed are more resistant to the exploitation conditions formilk production than compared with cows from Holstein-Friesian breed

Observations Regardin Oocyte in Vitro Maturation after Recovery from Slaughter House Females

The oocytes viability must be taken as an important selection parameter for successful in vitro cultivation. The ovaries were collected from the slaughterhouse and maintained at 4°C for 7 days. Fallowing cumulus -oocytes complexes recovery the viability was tested using two staining methods. For the first experiment we used 27 cumulus - oocytes complexes, stained with Neutral red and for the second experiment we used 11 cumulus - oocytes complexes stained with Trypan blue. Fallowing staining with Neutral red 23 cumulus - oocytes complexes were assessed as viable (were stained in red – enzymatic activity within the cells) and for the Trypan blue staining 11 cumulus - oocytes complexes were assessed as viable (remained unstained – integers cellular membranes)

OBSERVATIONS REGARDING OOCYTES STORAGE POST MENDING FROM SLAUGHTER FEMALES

The oocytes viability must be taken as an important selection parameter for successful in vitro cultivation. The ovaries were collected from the slaughterhouse and maintained at 4°C for 7 days. Fallowing cumulus - oocytes complexes recovery the viability was tested by two staining methods. For the first experiment we used 27 cumulus - oocytes complexes, stained with Neutral red and for the second experiment we used 11 cumulus - oocytes complexes stained with Trypan blue. Fallowing staining with Neutral red 23 cumulus - oocytes complexes were assessed as viable (were stained in red – enzymatic activity within the cells) and for the Trypan blue staining 11 cumulus - oocytes complexes were assessed as viable (remained unstained – integers cellular membranes)

STUDIES REGARDING THE VIABILITY OF MOUSE EMBRYOS RECOVERED IN EARLY DEVELOPMENTAL STAGES

In order to evaluate the embryo viability we tested 2 cultivation media in three combinations (M16, Nutrient mixture F- 12 HAM supplemented with BSA and Nutrient mixture F- 12 HAM without BSA). In the third day of cultivation the embryos viability was assessed by morphological criteria and randomly by staining with fluorescents dyes (Fluorescein diacetate and Propidium iodide). It was observed that both media used for in vitro cultivation assured embryo development up to the expanded blastocyst stage (21.33% for M 16 and 4.34% respectively), exception the Nutrient mixture F- 12 HAM without BSA in which after 3 days cultivation all embryos were degenerated. The M16 medium assured the embryos development up to the hatched blastocyst stage (5.33%). None of the media supported the development of embryos under 8 cell stages and early blockage in stage of 2 cells could not be prevented

EFFECT OF CZB CULTURE MEDIA ON IN VITRO DEVELOPMENT OF CLONED MOUSE EMBRYOS

The effect of simple and sequential embryo culture media on the preimplantationdevelopment of mouse nuclear transfer (NT) embryos reconstructed with cumuluscell nuclei using a mechanical NT technique was studied. Blastocyst formation ratewas evaluated using CZB medium. Nonmanipulated and sham-manipulatedparthenogenetic embryos served as controls for, respectively, the medium and thehandling technique. Rates of blastocyst formation for medium and handling controlembryos were similar in CZB (50% and 53%). Development of NT embryos wassignificantly impaired from the two-cell stage onwards, reaching the blastocyst stage ata rate of 4% in CZB. These data demonstrate not only that NT embryos are moresensitive to in vitro culture conditions than parthenogenetic control embryos but alsothat selection of culture media can influence the preimplantation development of NTembryos

Nutraceuticals: the Link Between Lifestyle and Medicine A review

Numerous studies have reported positive associations between certain biologically active compounds, with pharmacological properties, such as nutraceuticals, contained in some foods and various pathologies. The term "nutraceutical" currently varies from country to country, referring to a number of valuable molecules, derived from organic sources (plants) or foods such as polyphenols, essential amino acids, antioxidants, soluble fiber, polyunsaturated fatty acids ( PUFA), prebiotics, prebiotics, which act at the cellular level, in combating oxidative stress and inflammatory processes and / or in altering the expression of some genes. The discovery of the many benefits attributed to these products and the ever-changing lifestyle have contributed to increasing consumer confidence in nutraceutical and functional foods around the world, and there is a growing interest in improving the quality of life and adopting a healthy lifestyle. to prevent or reduce the risk of disease. Based on these considerations, this paper aims to review some scientific evidence obtained from in vitro / in vivo studies, which supports the beneficial effects of some nutraceuticals and their medical implications in various pathologies

Practical Methods to Assess Mammalian Embryo Quality – Staining Tests Comparative Study

Embryo assessment methods changed during the last 20 years. Morphological criteria used for embryo quality assessment improved in order to increase the selection probability of the most suitable embryos for transfer. This paper is presenting, based on a rich practical experience cumulated in our research group, several practical methods used for embryo assessment. For this study we have comparative evaluate the embryo quality using four staining tests: Trypan blue staining, Neutral red staining, Fluorescein diacetate staining, Propidium iodide staining in order to establish which test is the most reliable and efficient in embryo quality evaluation. The results obtained indicated that the Fluorescein diacetate and Propidium iodide viability test can be successfully used for mouse embryo viability evaluation. This method allows detection of both viable and nonviable cells

STUDIES REGARDING THE CRIOPROTECTIVE PROPRIETIES OF THE VITRIFICATION MEDIA, WITH DMSO, SUCROSE, FICOLL 70 AND GALACTOSE USED IN EMBRYO CRYOPRESERVATION

The aim of our paper was to make a series of experiments in order to determine the concentration at which four cryoprotectants (DMSO, sucrose, Ficoll 70 and galactose) singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous when thawed in water bath. As penetrating cryoprotector we used DMSO (MW=78.13 Da, Sigma D5879) and as nonpenetrating cryoprotectors we used sucrose (MW=342.3 Da, Sigma S7903), Ficoll 70 (MW= 60,000-80,000 Da, Sigma F4375) and Galactose (MW = 180,16 Da; Sigma G 6152). For DMSO there were tested concentrations from 1M to 6.5M, with concentrations step of 0.5M. For the nonpenetrating cryoprotectors there were tested concentrations of 5%, 10%, 15% and 20%. There were a total number of 168 solutions tested. The solutions vitrification ability on freezing was tested by direct plunging into liquid nitrogen at - 196°C. Three thawing temperatures were tested 20°C, 25°C and 37°C. The concentration at which DMSO solutions passed into vitreous state was 5M, but at thawing none of them remained vitreous at thawing. When pairs of cryoprotectors were tested 67 solutions vitrified at freezing (23 for DMSO-sucrose, 23 for DMSOFicoll 70 and 21 for DMSO-galactose) and 26 of them remained vitreous at thawing. The DMSO and galacose pair give the best results on thawing (11 solution remained vitreous on warming) at 37°C