C R F Guaitolini

São Paulo State University, São Paulo, Estado de Sao Paulo, Brazil

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Publications (8)17.75 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study was to evaluate re-expansion and ultrastructure of in vivo-produced canine embryos immediately after collection (Co; n=6) and 24h after in vitro culture (Co24; n=6). For embryo collection, two bitches in pro-oestrous were monitored every 48h by vaginal cytology up to the detection of 80 to 90% of superficial cells. Then, they were inseminated 3 times on alternate days with fresh semen from 1 fertile male. Embryo collections were performed after ovariohysterectomies and uterine flushing 12 days after the first artificial insemination. Each uterine horn was flushed 3 times with 60mL of DPBS at 36 to 37°C and collecting embyos in Petri dishes. Embryos culture were performed at 38.5°C under an atmosphere with 5% CO2 and maximum humidity, using SOFaa media added with 20% of fetal bovine serum (FBS). Embryo reexpansion was evaluated after 24h of culture and the embryos were classified as re-expanded or not re-expanded, according the appearance of the blastocoele by stereomicroscopy (Leica MZ 12.5, Leica Microsystems, Wetzlar, Germany). Twelve embryos were collected, 5 from bitch A and 7 from bitch B; 100% of embryos (6/6) showed re-expansion after 24h of culture. Blastocoele reexpansion was used as an embryo viability marker. The group Co showed perivitelline space and apical surface of blastomeres covered with microvilli, elongated mitochondria, rough endoplasmic reticulum (RER), tight junctions, and a large amount of lipid droplets that was similar to the results previously described in others mammals species. The Co24 group showed the same characteristcs of the group Co at the time of collection, however with a reduction of lipid droplets and the presence of myelinic structures. In conclusion, the lipid droplet reduction and presence of myelinic structures after 24h of in vitro culture may indicate lipid consumption associated with embryo expansion.
    Reproduction Fertility and Development 12/2014; 27(1):163. · 2.58 Impact Factor
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    ABSTRACT: Studies have suggested that the prematuration with meiotic blockers can improve oocyte quality promoting embryonic development; however, its exact effects on cytoplasmic characteristics remain unclear. Thus, this study aimed to evaluate the effects of meiosis block of bovine oocytes with the CDK inhibitors roscovitine (ROS) and butyrolactone (BL-I) on nuclear maturation, expression, and localization of ERK 1 and 2, cyclin B1, and p34cdc2 proteins and the ultrastructure of oocytes. Immature oocytes from the slaughterhouse were divided into the following groups: (1) control, in vitro maturated (IVM) in TCM 199 for 24 h; (2) ROS 12.5µM; (3) BL-I 50µM; and (4) ROS (6.25µM)+BL-I (25µM) treatment for 6h followed by IVM in CDK inhibitor-free medium for 18h. Immature oocytes and IVM oocytes in each group were then fixed stained by immunofluorescence for nuclear visualisation (n=600), localization, and expression of ERK1 and 2 proteins, cyclin B1 and p34cdc2 protein (n=350), and prepared for evaluation of the ultrastructure by electron microscopy (n=100). Data were analysed using the ANOVA test (nuclear visualisation), Student-Newman-Keuls test (expression of ERK1 and 2 proteins, cyclin B1 and p43cdc2) by the PROC GLM procedure of SAS (SAS Inst. Inc., Cary, NC, USA). At 6h of IVM, a lower (P<0.05) percentage of oocytes were at the metaphase I (MI) stage in the control group (C=18.2±5.4%) compared with other groups and a higher percentage of oocytes were degenerated in the ROS group (16.3±5.6%) compared with other groups (C=13.6±4.6%, BL-I=10.0±4.5%, BL-I+ROS=8.0±5.6%). At 24h of IVM, higher (P<0.05) percentages of oocytes were at the MI stage in the control and ROS group (8.3±5.9% and 6.8±6.4%, respectively). There was no difference (P>0.05) in percentage of metaphase II (MII) oocytes among the groups. Only the ROS group showed lower fluorescence intensity of ERK1 and 2 proteins in the ooplasma (P<0.05). Immature oocytes showed higher expression of cyclin B1 and p34cdc2 (P<0.05). There was no difference in the localization of these proteins in the ooplasm and there was no difference in the oocyte ultrastructure (mitochondria, cortical granules, endoplasmic reticulum, microvilli, zona pellucida, lipid granules) among treatments (P>0.05). The results suggest that a temporary blocking of oocyte maturation by CDK inhibitors affect neither the expression and distribution of MPF components (cyclin B1/cdc2) nor the distribution of cytoplasmic organelles in IVM oocytes. However, the expression of ERK 1 and 2 in mature oocytes may be reduced by pre-IVM with ROS.
    Reproduction Fertility and Development 12/2014; 27(1):233. · 2.58 Impact Factor
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    ABSTRACT: The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4ngmL(-1)), and the second was performed 48h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n=5), morulae were collected 8 days after the LH surge and Group B (n=5), blastocysts were collected 12 days after the LH surge. The pools (n=10) of embryos (5 embryos/pool) were stored in RNAlater(®) (Ambion, Life Technologies, USA) at -80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express(®) (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl's equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.
    Reproduction Fertility and Development 12/2012; 25(1):248. · 2.58 Impact Factor
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    ABSTRACT: The objective was to evaluate ovarian activity reversibility in domestic queens after short-term contraceptive treatment with deslorelin acetate. Ten mature queens were used. In all queens, the estrous cycle was evaluated every 72 h by vaginal cytology (VC) and behavior assessments. When queens had VC characteristic of interestrus or diestrus, one deslorelin acetate implant (4.7 mg) was placed in the subcutaneous tissue of the interscapular region (day of insertion = Day 0). Thereafter, VC was performed every 48 h and on Day 90, implants were removed. At Day 100, estrus and ovulation were induced with 100 IU eCG (im), followed by 100 IU hCG (im), 84 h later (Day 103.5). Queens were ovariohysterectomized on Day 106. Corpora lutea (CL) were counted, oviducts were flushed, and oocytes were identified, isolated and stained to assess viability. In all queens, blood samples for plasma progesterone concentrations were collected once a week, from Days -21 to 106. After deslorelin acetate application, four queens had VC and behavior typical of estrus, and one ovulated. Furthermore, ovulation occurred in three queens that did not have VC or behavior consistent with estrus. After the initial ovarian stimulation, all females had anestrous VC during the deslorelin treatment period. Implants were readily removed. Following implant removal, all females responded to treatments to induce estrus and ovulation. There were (mean ± SEM) 13.1 ± 5.5 CL and 8.1 ± 5.5 oocytes per queen; the oocyte recovery rate was 56.8 ± 25.4% and all recovered oocytes were viable. We concluded that deslorelin acetate can be used as a reversible short-term contraceptive in domestic cats, because estrus and ovulation were successfully induced following implant removal.
    Theriogenology 05/2012; 78(4):817-22. · 1.85 Impact Factor
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    ABSTRACT: The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from M0 were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5°C under an atmosphere of 5% CO(2) with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 ± 4.8 and 57.3 ± 4.8, respectively, mean ± SEM), or among M0 (62.3 ± 5.7%), M3 (56.9 ± 6.0%), and M6 (66.5 ± 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture.
    Theriogenology 05/2012; 78(3):576-82. · 1.85 Impact Factor
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    ABSTRACT: Over the past decades, there have been great advances in in vitro production (IVP) systems, with improved culture methods and new knowledge regarding embryo physiology, ultrastructure and morphology. Currently, the major obstacle associated with the extensive use of this technology is the great sensitivity of IVP embryos to cryopreservation. According to the literature, the reduced cryotolerance of IVP embryos is frequently associated with their high lipid content. Although is not clear until now how the lipid accumulation occurs, it may be influenced by the use of undefined culture media, supplemented with fetal calf serum (FCS); or as a result of embryo energy metabolism abnormalities that affect mitochondrial function, leading to the decrease in both the embryo quality and survival after cryopreservation. In this context, phenazine ethosulfate (PES), a reducer of NADPH electrons, which favours pentose-phosphate pathways and also inhibits the fatty acids synthesis, has been used to increase IVP embryo cryotolerance (Sudano et al. 2011 Theriogenology 75, 1211-1220). The aim of the present study was to evaluate the phenazine ethosulfate and FCS effect in the ultrastructure of IVP bovine embryos. A 2×2 factorial experiment design was used to test 2 FCS concentrations (0 or 10%) and the addition of PES (without or with PES) in the culture media. Slaughterhouse ovaries were used to obtain oocytes which were matured and fertilized in vitro (Day 0). Presumptive zygotes (n=1440) were divided in 4 culture media: SOFaa without FCS; SOFaa without FCS+0.3μM PES (started on Day 4); SOFaa+10% FCS; SOFaa+10% FCS+0.3μM PES (started on Day 4). Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5%O(2), 5%CO(2) and 90%N(2)). Transmission electron microscopy (TEM) was performed on Day-7 blastocysts from each group (n=5) through standard protocol. For the statistical analysis, the arcsine transformation was applied to blastocyst percentage data and submitted to the ANOVA, followed by Tukeys' test through PROC GLM (SAS Institute Inc., Cary, NC, USA). In the absence of significant interactions, only main effect means are presented. The blastocyst production was not affected (P=0.47) by the use of PES (42.7±3.2 vs 39.3±3.2, respectively for control and PES Day 4). The addition of 10% of FCS increased (P<0.0001) the percentage of blastocysts (48.9±3.2 vs 33.0±3.2, respectively, for 10% and 0% of FCS). The ultrastructure analysis showed similar features in embryos from all studied groups. However, embryos cultured in the absence of FCS presented fewer and smaller lipid droplets. Moreover, embryos cultured without FCS presented more cellular debris in the perivitelinic space and in the blastocoele, indicating loss of blastomeres. The use of PES was able to reduce lipid droplets and increase the mitochondrial number in serum-produced embryos. Therefore, the PES decreased lipid content and increased mitochondrial number without affecting the development and ultrastructure of IVP bovine embryos.
    Reproduction Fertility and Development 12/2011; 24(1):157. · 2.58 Impact Factor
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    ABSTRACT: The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans-surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts.
    Reproduction in Domestic Animals 08/2011; 46(4):724-7. · 1.18 Impact Factor
  • Reproduction Fertility and Development 01/2010; 22(1). · 2.58 Impact Factor

Publication Stats

7 Citations
17.75 Total Impact Points

Institutions

  • 2012
    • São Paulo State University
      • Departamento de Reprodução Animal e Radiologia Veterinária
      São Paulo, Estado de Sao Paulo, Brazil
    • Palo Alto University
      Federal Way, Washington, United States
  • 2011–2012
    • Universidade Federal do Espírito Santo
      Victoria, Espírito Santo, Brazil