Moderate to strong PRA immuno-expression were observed in the proliferated theca interna cells of the atretic antral follicles. The atretic large lutein cells of the regressed corpus luteum showed negative immunostaining for PRA. This study showed that the PRA positive small lutein cells of the regressed corpus luteum and the PRA positive proliferated theca interna cells of the atretic antral follicles were transformed into PRA positive interstitial gland cells. In conclusion, the present study had described the distribution of PRA in the ovary of pseudopregnant rabbit, which is not discussed before in the available literature. It also gives more information about follicular dynamic, formation and origin of interstitial glands, mechanism of ovulation, formation and regression of the corpus luteum.The aim was to evaluate pregnancy success after transfer of embryos vitrified in micropipette tips in Merino sheep under extensive conditions. A second objective was to evaluate the influence of embryo stage in such pregnancy rate. One hundred and twenty-seven embryos were rewarmed and transferred into recipient ewes. On rewarming, the embryos were placed into three-step cryoprotectant dilutions. Finally, prior to transfer to recipient females, embryos were maintained in Basic Medium for 5 min at 25ºC and were re-evaluated by morphological criteria; all degenerated embryos were eliminated. Recipient ewes (n = 150) were treated for estrus with sponges placed for 14 days and 300 IU of eCG. At embryo transfer, three experimental groups were defined morulae transferred on Day 7, blastocysts transferred on Day 7 and blastocysts transferred on Day 8 after sponge removal. In all groups, semi-laparoscopic transfer of one rewarmed embryo per recipient was performed. Pregnancy was diagnosed by ultrasonography on day 28 after embryo transfer. The embryo selection rate after rewarming was higher for blastocysts (89.3% – 67/75) compared to morulae (65.9% – 60/91) (P less then 0.05). Pregnancy diagnosis showed a 38.3% (23/60) of success after morula transfer on Day 7 post progestagen removal. The day of transfer showed a significant influence on pregnancy rate after blastocyst transfer (Day 8, 55.9% – 19/34 vs Day 7, 21.2% – 7/33) (P less then 0.05). Blastocysts transfer on Day 8 showed the highest global efficiency (pregnancies/total embryos after rewarming) (47.5% – 19/40) (P less then 0.05). In conclusion, reproductive efficiency obtained by vitrified embryo transfer allows its recommendation for embryo transfer programs under extensive conditions. The importance of considering the synchrony between the embryo age and the recipient uterus stage is emphasized.Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. 1-Azakenpaullone supplier Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification HPRT1 and B2M and for bovine cumulus samples four were indicated HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P less then 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).The objective of this study was to assess the stemness marker expressions (Oct4, Nanog, and Sox2) of granulosa cells (GCs) collected from bovine ovarian follicles and in vitro expansion. The single bovine ovarian follicles were isolated and categorized into 4 groups according to their diameter including group A (4 mm). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunostaining were applied to evaluate the stemness marker expression of bovine GCs from ovarian follicles. We also estimated the stemness marker transcript expressions of GCs during in vitro expression by qRT-PCR. qRT-PCR analysis demonstrated that fresh GCs from bovine ovarian follicles expressed the stemness markers (Oct4, Nanog, Sox2). These markers were down-regulated during antral stage follicular development. We also estimated stemness marker transcript expressions of GCs which were isolated and in vitro expanded from ovarian follicles of group A. The qRT-PCR results showed that Oct4 and Sox2 transcript expressions were reduced during in vitro expansion while Nanog transcript was not expressed.