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

Crioprotectors are the main component of any vitrification media. The penetrant crioprotectors areessential for cell dehydration and for the decrease of the freezing point of the solution, allowing a longertime for dehydration to set in. The aim of our paper was to make a series of experiments in order todetermine the concentration at which four cryoprotectants (ethylene glycol, sucrose, Ficoll 70 andgalactose) singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous whenthawed in water bath. A total of 156 solutions were tested. During freezing, vitrification was evidenced bythe formation of transparent glass when the unsealed straws were plunged into liquid nitrogen, at -196°C. Crystallization (ice formation) resulted in a milky appearance. Solutions that vitrify on freezingwere tested if they remain vitreous on thawing. For thawing we tested three temperatures 20°C, 25°C and37°C. During thawing, solutions that did not devitrified were transformed from solid clear state to theliquid state without evidence of a milky appearance. From the combinations of two cryoprotectors weretested a number of 51 solutions vitrify on freezing (19 solutions with ethylene glycol and galactose; 19solutions with ethylene glycol and sucrose; 13 solutions with ethylene glycol and Ficoll). The ethyleneglycol and galacose pair give the best results on thawing (3 combinations remained vitreous on thawing)at 37°C

THE USE OF HORMONAL METHODS ON GILTS REPRODUCTIVE CYCLES

The main purpose of the current research was to conduct the reproductive cycle ongilts, using hormonal methods to induce estrus in non-cycling and late pubertal giltsand to group in a short period of time the breedings and, in the same time, to inductfarrowings. The gilts that have made the object of this experiment were distribute intwo equal lots and they were treated with PG 600 (400 I.U. PMSG and 200 I.U.hCG) to induce estrus in two consecutive weeks. The main reproductive objectivesthat we have observed were the percentage of gilts that came into heat, the timerange when the gilts showed signs of estrus and the gestation rate after pregnancycheck at 28 and 56 days from breeding. The percentage of the gilts that were in heatafter PG 600 was 67 %. The majority (44.8 %) of gilts were in heat after 72-96hours from PG 600. The gestation rate at 28 days after insemination was 64.6 % andat 56 days after insemination was 53.0%

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 K-CASEIN ALLELES ON MILK PRODUCTION AND QUALITY IN A HOLSTEIN-FRISIAN COW POPULATION

Milk production and its composition are determined by quantitative loci, whichunder the influence of some environmental factors are producing an allelicvariability, meaning a genetic polymorphism of the gene. K-casein is a milk proteinwhose genetic polymorphism can serve as molecular marker for milk production,composition and industrial processing suitability. The allelic variants for k-casein Aand B are the most common and the most important of them. The experiments wereconducted on 24 Holstein-Friesian milking cows from a private farm in Giroc. Themilk production on a normal lactation is 8444 milk kg/305 days, with a fat percentof 3.9 and a protein percent of 3.3. The cows were divided in three groups AA, ABand BB in function of the genotyped obtained after the allelic variantsdetermination. The DNA isolation was made from hair roots and blood, the cowpopulation studied is not in genetic equilibrium fore k-casein gene, the frequency ofallele A is 0.43 and the frequency of B allele is 0.58. The highest genotype frequencywas 0.5 for CSN3-AB genotype, the BB genotype had 0.33 frequency, and the lowestfrequency was 0.17 for AA genotype. The mean daily milk production from cowswith BB genotype for k-casein is significant (p<5%) higher compared to the allelicvariant AA. The fat percent is significant higher at the allelic variant AA comparedto the other allelic variants (AB and BB) of the k-casein gene. Between the fatpercent of the three genotypes variants of K-casein (AA, AB and BB) there are nosignificant differences

PROSTAGLANDIN F2α SUPPLEMENTED SEMEN IMPROVES LANDRACE BOARS SPERM MOTILITY

This study investigated whether the sperm motility from Landrace boars improveswhen PGF2α (Dinolytic®; 5 mg PGF2α /ml) was added to diluted semen. Boars fromone large production unit, were manually collected; semen was either enriched withPGF2α (group 1, n=38), either untreated (group 2, n=32). Total volume of semencollected, percent of motility and number of obtained doses were recorded. Thehighest sperm volume collected from the two groups is corresponding to ejaculatesfrom Landrace boars with PGF2α supplemented semen (267.6 m)l. Regardingmotility, the sperm collected from Landrace boars with PGF2α supplemented semenwas higher from the one collected from Landrace boars with untreated semen(81.37%) and very significant differences were statistically determined. Theejaculates with highest number of obtained doses is corresponding to the onescollected from boars with PGF2α supplemented semen (25.21). Only boars from thefirst group (with PGF2α supplemented semen) showed motility over 70% and even100%. The untreated semen showed motility values around 65-70%

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 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