Transcervical Versus Laparotomy Embryo Recovery: What Strategy Is Best for Embryo Bank Formation in the Canindé Goat Conservation Program?

By allowing for the creation of embryo banks, reproductive biotechnologies play an essential role in the preservation of endangered goat breeds' genetic diversity. This study focused on comparing both available embryo collection methods [laparotomy (LAP) vs. nonsurgical embryo recovery (NSER)] in Canindé goats to create an embryo bank for later use in a breed conservation program. Twelve females were superovulated and subjected to either the LAP or NSER technique for embryo recovery. The recovery rate was similar (p > 0.05) between NSER (86.8% ± 5.6%) and LAP (92.8% ± 4.0%). Moreover, there were no differences (p > 0.05) in the number of structures recovered, the viable embryos, and the freezable embryos per goat, respectively, for NSER (11.7 ± 1.3, 11.2 ± 1.5, and 10.2 ± 1.1) and LAP (10.3 ± 1.0, 8.7 ± 0.7, and 8.0 ± 0.8). Overall, 132 structures were collected out of 151 ovulations (∼12.6 ± 1.2 corpora lutea per goat). Finally, the procedure duration time was also similar (p > 0.05) for NSER versus LAP, respectively: 32.3 ± 3.3 versus 30.8 ± 3.9 minutes. In conclusion, the NSER method results proved to be similar to the LAP technique in small-sized Canindé goats. It was noticeable, however, that the NSER technique is simpler and provides the possibility for successive procedures with few health risks and sequels for females. This study may hopefully boost in vivo embryo production programs in the Canindé breed, facilitating the formation of embryo banks and so assuring the availability of genetic diversity before any decline becomes irreversible.

Reproductive Seasonality Affects In Vitro Embryo Production Outcomes in Adult Goats.

Reproductive seasonality may have a considerable influence on the efficiency of assisted reproductive technologies in seasonal species. This study evaluated the effect of season on cleavage, blastocyst rates and quality of in vitro produced (IVP) goat embryos. In total, 2348 cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and subjected to the same IVP system throughout 1.5 years (49 replicates). The odds ratio (OR) among seasons was calculated from values of cleavage and blastocyst rates in each season. Cleavage rate was lower (p < 0.05) in spring (anestrus), in comparison with either autumn (peak of breeding season) or summer, while the winter had intermediate values. Furthermore, lower OR of cleavage was observed in spring. Blastocyst formation rate (from initial number of COCs) was higher (p < 0.05) in autumn (52 ± 2.5%) when compared with the other seasons (combined rates: 40 ± 1.9%). Moreover, its OR was higher (p < 0.05) in autumn compared to all other seasons and impaired in the spring compared to winter (OR: 0.54) and summer (OR: 0.48). Embryo hatchability and blastocyst cell number were similar (p > 0.05) among seasons. In conclusion, the breeding season leads to improved oocyte developmental competence, resulting in higher cleavage and blastocyst yield, whereas embryo quality remained similar throughout the years.

Ovarian gene expression in collared peccary (Pecari tajacu Linnaeus, 1758) subjected to gonadotropin stimulation protocols.

Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.

Addition of antifreeze protein type I or III to extenders for ram sperm cryopreservation.

Antifreeze proteins (AFP) play an important role in cellular survival at sub-zero temperatures. This study assessed the effect of AFP type I or III in semen extender (TRIS-egg yolk) for ram sperm cryopreservation. Pooled semen of four rams were allocated into five treatments: Control (CONT, without AFP); AFP Type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) µg/mL]; or III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) µg/mL], and then frozen in six replicates. Treatments affected kinetic parameters, plasma membrane integrity and morphology (P < 0.05). The AFPIII-0.1 presented lesser total motility. Linearity was greater in AFPI-0.1, AFPI-0.5 and AFPIII-0.5 and straightness was greater in all AFP-supplemented extenders. Plasma membrane integrity was greater in AFPI-0.1 and AFPI-0.5. All AFP groups had greater percentage of normal sperm than CONT. No differences (P > 0.05) were observed in hypoosmotic test, sperm acrosome status, mitochondrial activity, chromatin condensation, perivitelline membrane binding rate and lipoperoxidation. In conclusion, the use of AFP, predominantly type I, may increase sperm cell protection during cryopreservation, with no adverse effect on potential fertilization capacity or increase in reactive oxygen species, being a potential cryoprotectant to ram sperm.

In vitro production of small ruminant embryos: latest improvements and further research.

This review presents the latest advances in and main obstacles to the application of invitro embryo production (IVEP) systems in small ruminants. This biotechnology is an extremely important tool for genetic improvement for livestock and is essential for the establishment of other biotechnologies, such as cloning and transgenesis. At present, the IVEP market is almost non-existent for small ruminants, in contrast with the trends observed in cattle. This is probably related to the lower added value of small ruminants, lower commercial demand and fewer qualified professionals interested in this area. Moreover, there are fewer research groups working on small ruminant IVEP than those working with cattle and pigs. The heterogeneity of oocytes collected from growing follicles in live females or from ovaries collected from abattoirs remains a challenge for IVEP dissemination in goats and sheep. Of note, although the logistics of oocyte collection from live small ruminant females are more complex than in the bovine, in general the IVEP outcomes, in terms of blastocyst production, are similar. We anticipate that after appropriate training and repeatable results, the commercial demand for small ruminant invitro -produced embryos may increase.

In Vitro Development and Mitochondrial Gene Expression in Brown Brocket Deer (Mazama gouazoubira) Embryos Obtained by Interspecific Somatic Cell Nuclear Transfer.

The genetic diversity of Neotropical deer is increasingly jeopardized, owing to declining population size. Thus, the formation of cryobanking of somatic cells is important for the preservation of these species using cloning. The transformation of these cells into viable embryos has been hampered by a lack of endangered species oocytes. Accordingly, the aim of this study was to produce brown brocket deer embryos by interspecific somatic cell nuclear transfer (iSCNT), using goat or cattle oocytes as cytoplasts, and to elucidate embryo mitochondrial activity by measuring the expression levels of ATP6, COX3, and ND5. Cattle embryos produced by in vitro fertilization (IVF) were used as a control. There were no differences in the development of embryos produced by traditional SCNT and iSCNT when using either the goat cytoplasts (38.4% vs. 25.0% cleaved and 40.0% vs. 50.0% morula rates, respectively) or cattle cytoplast (72.8% vs. 65.5% cleaved and 11.3% vs. 5.9% blastocyst rates, respectively). Concerning the gene expression, no significant difference was observed when goat oocytes were used as cytoplasts. However, when using cattle oocytes and 16S as a reference gene, the iSCNT upregulated COX3, when compared with SCNT group. In contrast, when GAPDH was used as a reference gene, all the evaluated genes were upregulated in the iSCNT group, when compared with the IVF group. When compared with the SCNT group, only the expression of ATP6 was statistically different. In conclusion, it was demonstrated that interspecific nuclear transfer is a potentially useful tool for conservation programs of endangered similar deer species.

Heterotopic ovarian allotransplantation in goats: Preantral follicle viability and tissue remodeling.

An appropriate implantation site favors angiogenesis and avoids ovarian tissue damage after tissue grafting. The objective of this study was to evaluate the effects of intramuscular (IM) and subcutaneous (SC) sites for ovarian grafts in goats by evaluating follicular morphology and activation, preantral follicle and stromal cell densities, tissue DNA fragmentation, collagen types I and III depositions, and graft revascularizations. Ovarian cortical tissue was transplanted in IM or SC sites and recovered 7 or 15 days post-transplantation. There was a greater percentage of developing follicles and lesser follicular and stromal cell densities in all grafted tissues as compared to ovarian tissues of the control group. The stromal cell density and percentage of normal follicles were positively associated. At 15 days post-transplantation, tissues at the SC and IM sites had similar amounts of DNA fragmentation and type III collagen content. In contrast, tissues at the SC, as compared with IM site, had greater abundances of collagen type I. Furthermore, there was a positive association between collagen type I and percentage of morphologically normal follicles post-transplantation. In addition to a marked decrease in follicular density 15 days post-transplantation in ovarian grafts at the SC and IM sites, low percentages of normal follicles and follicular activation were observed similarly in both transplantation sites. There were also positive associations of stromal cell density and abundance of type I collagen fibers with the percentage of intact follicles in grafted ovarian tissues.

Disulphide-less crotamine is effective for formation of DNA-peptide complex but is unable to improve bovine embryo transfection.

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA-dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA-dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA-dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA-dLCr 1:25 and the controls. The DNA-dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA-dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA-dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

Protein profile of the ovarian follicular fluid of brown brocket deer (Mazama gouazoubira; Fisher, 1814).

The aim of this study was to characterize the protein profile of ovarian follicular fluid (FF) of brown brocket deer (Mazama gouazoubira). Five adult females received an ovarian stimulation treatment and the FF was collected by laparoscopy from small/medium (≤3.5 mm) and large (>3.5 mm) follicles. Concentrations of soluble proteins in FF samples were measured and proteins were analyzed by 1-D SDS-PAGE followed by tryptic digestion and tandem mass spectrometry. Data from protein list defined after a Mascot database search were analyzed using the STRAP software tool. For the protein concentration, no significant difference (P > 0.05) was observed between small/medium and large follicles: 49.2 ± 22.8 and 56.7 ± 27.4 µg/µl, respectively. Mass spectrometry analysis identified 13 major proteins, but with no significant difference (P > 0.05) between follicle size class. This study provides insight into elucidating folliculogenesis in brown brocket deer.

Etiology and severity of diarrheal diseases in infants at the semiarid region of Brazil: A case-control study.

BACKGROUND: Diarrheal diseases are an important cause of morbidity and mortality among children in developing countries. We aimed to study the etiology and severity of diarrhea in children living in the low-income semiarid region of Brazil. METHODOLOGY: This is a cross-sectional, age-matched case-control study of diarrhea in children aged 2-36 months from six cities in Brazil's semiarid region. Clinical, epidemiological, and anthropometric data were matched with fecal samples collected for the identification of enteropathogens. RESULTS: We enrolled 1,200 children, 596 cases and 604 controls. By univariate analysis, eight enteropathogens were associated with diarrhea: Norovirus GII (OR 5.08, 95% CI 2.10, 12.30), Adenovirus (OR 3.79, 95% CI 1.41, 10.23), typical enteropathogenic Escherichia coli (tEPEC), (OR 3.28, 95% CI 1.39, 7.73), enterotoxigenic E. coli (ETEC LT and ST producing toxins), (OR 2.58, 95% CI 0.99, 6.69), rotavirus (OR 1.91, 95% CI 1.20, 3.02), shiga toxin-producing E. coli (STEC; OR 1.77, 95% CI 1.16, 2.69), enteroaggregative E. coli (EAEC), (OR 1.45, 95% CI 1.16, 1.83) and Giardia spp. (OR 1.39, 95% CI 1.05, 1.84). By logistic regression of all enteropathogens, the best predictors of diarrhea were norovirus, adenovirus, rotavirus, STEC, Giardia spp. and EAEC. A high diarrhea severity score was associated with EAEC. CONCLUSIONS: Six enteropathogens: Norovirus, Adenovirus, Rotavirus, STEC, Giardia spp., and EAEC were associated with diarrhea in children from Brazil's semiarid region. EAEC was associated with increased diarrhea severity.

Efficiency of different incubation systems for the in vitro production of bovine embryos.

SummaryThis study aimed to compare the efficiency of different incubation systems for in vitro embryo production in bovine. Oocytes/embryos were cultured in three incubators: conventional - CONV, mini bench - MINI and portable - PORT. After in vitro maturation (IVM), oocytes were verified for maturation rate. The remaining structures were submitted to in vitro fertilization and culture to verify cleavage (day 2) and blastocyst (day 7) rates. Reactive oxygen species (ROS) were evaluated in post-IVM oocytes and embryos (days 2 and 7) using arbitrary fluorescence units (AFUs). No significant difference (P>0.05) was observed for maturation rate. The CONV system (74.0%) produced the highest cleavage rate (P0.05) to MINI (65.0%). The same pattern and differences were observed for blastocyst rate: CONV (33.3%), MINI (32.3%) and PORT (21.9%). ROS levels were not different (P>0.05) in post-IVM oocytes: CONV (35.6±4.5), MINI (29.4±4.0) and PORT (35.6±4.5). For day-2 embryos, ROS levels were higher (P0.05) was observed in blastocysts. In conclusion, although it produced high ROS levels at day 2 of culture, the MINI system was as efficient as the CONV system for blastocyst production. This option may be an interesting and economical for the in vitro embryo industry.

Laparoscopic ovarian biopsy pick-up method for goats.

Biopsy pick-up (BPU) has been considered a safe method to harvest ovarian fragments from live animals. However, no studies have been reported on the use of BPU to collect in vivo ovarian tissue in goats. The goals of this study were: (i) to test different biopsy needle sizes to collect ovarian tissue in situ using the BPU method (Experiment 1), and (ii) to study ovarian tissue features such as preantral follicle density, morphology, class distribution, and stromal cell density in ovarian fragments obtained in vivo through a laparoscopic BPU method (Experiment 2). In Experiment 1, goat ovaries (n = 20) were collected in a slaughterhouse and subjected to in situ BPU. Three needles (16, 18, and 20G) were tested. In Experiment 2, the most efficient biopsy needle from Experiment 1 was used to perform laparoscopic BPU in goats (n = 8). In Experiment 1, the recovery rate was greater (P < 0.05; range 50-62%) with 16G and 18G needles than the 20G (17%) needle. The mean weight of ovarian fragments collected by the 16G needle was greater (P < 0.05) than the 18G and the 20G needle. In Experiment 2, 62 biopsy attempts were performed and 52 ovarian fragments were collected (90% success rate). Overall, 2054 preantral follicles were recorded in 5882 histological sections analyzed. Mean preantral follicular density was 28.4 ± 1.3 follicles per cm2. The follicular density differed (P < 0.05) among animals and ovarian fragments within the same animal. The mean stromal cell density in the ovarian fragments was 37.1 ± 0.5 cells per 2500 µm2, and differed (P < 0.05) among animals. Moreover, preantral follicle density and stromal cell density were associated (P < 0.001). The percentage of morphologically normal follicles was 70.1 ± 1.2, and differed (P < 0.05) among animals. The majority (79%) of the morphologically normal follicles was classified as primordial follicles, and differed (P < 0.05) among animals and between ovaries. In summary, a laparoscopic BPU method has been developed to harvest ovarian tissue in vivo with a satisfactory success rate in goats. Furthermore, this study described for the first time that goat ovarian biopsy fragments have a high heterogeneity in follicular density, morphology, class distribution, and stromal cell density.

Proteomic analysis of follicular fluid from tropically-adapted goats.

The present study was conducted to characterize the major proteome of ovarian follicular fluid from locally-adapted, "Canindé" goats in the northeast of Brazil. Eight estrous cycling goats received a hormonal treatment consisting of medroxyprogesterone acetate, D-cloprostenol and FSH. Fluid was collected by laparoscopy from small (<3mm), medium (3-4mm) and large (>4mm) follicles and then, proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Thirty-six proteins were identified in the goat follicular fluid, including albumin, immunoglobulins, ceruloplasmin, complement factor B, alpha-1B-glycoprotein precursor, serotransferrin, complement C3 and serpins, among others. Albumin and immunoglobulins were the most abundant proteins. Protein concentrations were similar in the fluid from small (45.3±3.1mg/mL), medium (44.2±3.3mg/mL) and large follicles (45.1±2.3mg/mL). The intensities of spots identified in 2-D gels as serotransferrin, zinc-alpha-2-glycoprotein-like, complement factor B and complement protein C3 differed (P<0.05) among follicle categories. The amount of serotransferrin was greater in the medium than small follicles (P<0.05). Content of zinc-alpha-2-glycoprotein-like, complement factor B and complement C3 was greater (P<0.05) in the fluid of large follicles than in medium follicles. Based on gene ontology, the major molecular functions associated with goat follicular fluid proteins were binding and catalytic activity, while the main biological processes were related to regulation, cellular processing, location and the immune system. In conclusion, the major proteome of the follicular fluid from goats subjected to hormonal stimulation was elucidated in the present study. Also, molecules associated with follicle development are potential biomarkers of oocyte competence were prevalent.

The Effects of Cryopreservation on Different Passages of Fibroblast Cell Culture in Brown Brocket Deer (Mazama gouazoubira).

The brown brocket deer Mazama gouazoubira is 1 of the 10 recognized brocket deer of the Neotropical region. Recently, this species has suffered a population decline due to current threats, mainly poaching and habitat loss. Several studies have shown that some endangered species can benefit from interspecies somatic cell nuclear transfer technology through the use of their somatic cells, such as the fibroblasts. Thus, the aim of this study was to verify the viability and the effect of cryopreservation on fibroblasts after several passages. For this purpose, fibroblast cells were cultured until passages 4, 7, and 10 (cultured control groups) and cryopreserved in cryotubes (frozen/warmed groups). The cellular viability, functionality, and percentage of cells undergoing necrosis and apoptosis were evaluated. The survival rates were always higher than 80% irrespective of the tested group, except for passage 10 in the frozen/warmed group. Population doubling time of cultured cells from passage 10 was significantly higher than that of passages 4 and 7, exhibiting low metabolic activity and a higher percentage of cells in initial apoptosis. In conclusion, the M. gouazoubira fibroblast-derived cell line provides an essential resource for further studies regarding reproductive biotechniques and is likely to be useful as an ex situ conservation strategy.

Mitotic index and morphological characteristics of ovarian small follicles from goats submitted to nutritionally unbalanced regimens.

The objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding-refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis.

Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis.

The use of antifreeze protein type III for vitrification of in vitro matured bovine oocytes.

The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation.

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression.

PURPOSE: Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes. METHODS: PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid. RESULTS: Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgene-expressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05). CONCLUSIONS: Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.

Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation.

Hormonal ovarian stimulation may affect transcripts in somatic cells of cumulus-oocyte complexes (COCs) and affect the resulting oocyte quality. Here, in parallel with morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of hyaluronan synthase 2 (HAS2), gonadotropic receptors (FSHR and LHR) and connexin 43 (GJA1) in cumulus cells (CCs) from goat COCs after multi-dose FSH (MD) or one-shot FSH/eCG (OS) treatments, using bovine COCs as control groups. The MD treatment produced more large follicles, and the resulting COCs had a better morphology and IVM rate than were obtained with OS. The OS treatment produced COCs with increased HAS2, FSHR, LHR and GJA1 expression. This gene expression pattern was also observed in the CCs of COCs that showed poor morphological characteristics. On the other hand, the mRNA levels were more similar between groups after IVM; FSHR and LHR were the main genes that showed decreased expression. Some events that occurred in bovine CCs during IVM, such as cell expansion, increased HAS2 expression and decreased GJA1 expression, were less evident or did not occur in goat COCs. In conclusion, increasing HAS2, FSHR, LHR and GJA1 expression in goat COCs does not confer greater meiotic competence to oocytes. Instead, it may result from poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion, together with HAS2 upregulation and GJA1 downregulation, seems to be more important for bovine COCs than for goat COCs. Additional studies are needed to investigate the importance of other HAS isoforms and connexins in goat COCs.

Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 µM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 µM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 µM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 µM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 µM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 µM for 6-24 h.