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.
In vitro-produced Bos taurus indicus (zebu) and Bos taurus indicus × Bos taurus taurus (cross-bred) embryos behave differently when vitrified. The present experiment aimed to examine the effect of vitrification on embryos produced in the presence or absence of FCS. Cumulus-oocyte complexes (COC) were matured in TCM-199 and fertilized in human tubal fluid medium with frozen Nelore bull semen. On Day 1 (Day 0 = IVF), presumptive zygotes were cultured with SOFaa + BSA in the presence of FCS (Group 2.5%) or in the absence of FCS (Group 0%) until Day 7. The cleavage was analysed on Day 3 and the blastocyst rate on Day 7. Blastocysts were vitrified and, after warming (Campos-Chillòn et al. 2006) the viability was evaluated. Data were analysed with ANOVA, using the general linear model (GLM) of SAS (SAS Inst Inc., Cary, NC, USA). Sources of variation in the model included FCS concentration and first-order interactions; all factors were considered fixed effects. The arcsine transformation (√y/100) was applied to percentage data. If the ANOVA was significant, means were separated using the Tukey test. There was no difference in cleavage (for zebu embryos: Group 0%: 87.2 ± 6.8; Group 2.5%: 87.4 ± 9.5; for cross-bred embryos: Group 0%: 79.6 ± 11.9; Group 2.5%: 73.1 ± 13.7; P > 0.05). On the other hand, zebu embryos cultured in the presence of FCS reached blastocysts at a higher rate than cross-bred embryos in the absence of FCS (for zebu embryos: Group 0%: 33.3 ± 12.4ab; Group 2.5%: 46.8 ± 13.2a; for cross-bred embryos: Group 0%: 21.8 ± 8.3b; Group 2.5%: 33.6 ± 10.1ab; P < 0.05). After vitrification and warming, no significant differences in re-expansion rate (zebu embryos: Group 0%: 82.7 ± 13.1; Group 2.5%: 75.0 ± 9.8; cross-bred embryos: Group 0%: 93.7 ± 8.8; Group 2.5%: 84.1 ± 11.3; P > 0.05) and cell number per embryo (zebu embryos: Group 0%: 65.1 ± 34.7; Group 2.5%: 42.6 ± 17.2; cross-bred embryos: Group 0%: 64.3 ± 44.2; Group 2.5%: 52.0 ± 31.5; P > 0.05) between species groups and within species were seen. However for zebu embryos, Group 0% showed a lower damaged cell rate than Group 2.5%. The same effect was not observed in the cross-bred embryos (zebu embryos: Group 0%: 20.3 ± 22.7c; Group 2.5%: 63.3 ± 27.0d; cross-bred embryos: Group 0%: 25.4 ± 24.3cd; Group 2.5%: 45.8 ± 34.6cd; P < 0.05). The addition of 2.5% FCS had a higher deleterious effect on zebu embryos than cross-bred (zebu × taurine) embryos after vitrification. These results also reinforce the species differences observed between zebu and cross-bred, as they behaved differently in relation to the addition of FCS in the culture medium and in relation to their cryopreservation sensitivity. Supported by FAPESP 10/50410-2.
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 + 0.3 μM PES (started on Day 4); SOFaa + 10% FCS; SOFaa + 10% FCS + 0.3 μM PES (started on Day 4). Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2 and 90% N2). 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. FAPESP 09/54513-3, 10/09922-0.
Over the past decades, there have been great advances in in vitro production (IVP) systems with improved culture methods and new knowledge regarding embryo genetics, physiology, ultrastructure, and morphology. Nevertheless, a major obstacle for dissemination of this technology is the great sensitivity of IVP embryos to cryopreservation. The objective was to study the global gene-expression patterns of fresh and vitrified IVP bovine embryos. Oocytes (N = 1290) were matured and fertilized in vitro (Day 0). Presumptive zygotes were cultured in SOFaa with 0.5% BSA and 2.5% of FCS. Cleavage and blastocyst production was evaluated after 3 and 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2, and 90% N2). On Day 7, half of the blastocysts were vitrified (n = 94), warmed (Sudano et al. 2011 Theriogenology 75, 1211–1220), and returned for 24 h of additional culture (re-expansion and hatching; hatched was evaluated 12 and 24 h after warming, respectively) when their RNA was extracted (vitrified group). The remaining embryos returned to culture until Day 8 when their RNA was extracted (fresh group). Total RNA extraction of a single blastocyst was performed using the PicoPure Kit (Applied Biosystems®, Foster City, CA, USA). The RNA samples were DNAse treated (Qiagen®, Valencia, CA, USA), and mRNA was amplified (RiboAmp Kit®). The aRNA output was evaluated with a NanoDrop (Thermo®, Wilmington, DE, USA) and Bioanalyzer (Agilent®, Santa Clara, CA, USA). Biotin-labelled and fragmented cRNA were obtained with the 3′IVT Kit (Affymetrix®, Santa Clara, CA, USA) to perform hybridization (N = 6–7, respectively, for vitrified and fresh groups) using the GeneChip Bovine Array (Affymetrix®). Microarray data analysis was performed with the FlexArray 1.6.1.1. Genes with a fold change of at least 2 and a probability of P ≤ 0.05 were considered differentially expressed. Real-time PCR was used to validate microarray results (N = 11–15, respectively, for vitrified and fresh groups). As a control, a pool of 200 blastocysts was submitted or not to mRNA amplification followed by the reverse transcription and qPCR of 17 genes. For statistical analyses, PROC GLIMMIX, PROC LOGISTIC, and PROC CORR were used. Cleavage and blastocyst production rates were 86.8 ± 1.0 and 32.5 ± 1.9%, respectively. Re-expansion and hatching/hatched rates were 69.3 and 19.3%, respectively. Messenger RNA abundance of amplified and nonamplified RNA had a high correlation (r = 0.89, P < 0.01). The microarray analysis indicated 383 differentially expressed genes (P ≤ 0.05) between fresh and vitrified blastocysts. Genes involved in apoptosis (PRDX2), heat shock (HSPA5), maternal recognition of pregnancy (IFNT2 and PAG2), and cell differentiation and placenta formation (KRT18) were downregulated in vitrified embryos. According to qPCR analysis, mRNA abundance of IFNT2, PRDX2, and KRT18 was downregulated, whereas HSPA5 mRNA levels were upregulated in vitrified blastocysts. Messenger RNA abundance of PAG2 was not different (P = 0.46) between fresh and vitrified embryos. In conclusion, vitrification alters the expression profile of the genes IFNT2, PRDX2, KRT18, and HSPA5 that can be related with embryo postcryopreservation survival capacity. FAPESP and LNBio-CNPEM are acknowledged.
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