The effects of crocetin supplementation on the blastocyst outcome, transcriptomic and metabolic profile of in vitro produced bovine embryos.

The earliest stages of embryo development are deeply influenced by reactive oxygen species (ROS), byproducts of the mitochondrial oxygen metabolism that play a key role as messengers in normal cell signal transduction and cell cycling. Despite its positive roles, the imbalance caused by the excess of ROS and an inefficient antioxidant system leads to oxidative stress, with negative consequences to the cell such as DNA damage, metabolic changes, mitochondrial stress and cell death. In the present work, crocetin - a natural antioxidant - was added to the culture media of bovine embryos to evaluate the efficiency of its antioxidant capability during embryo culture. Oocytes were in vitro matured (IVM) and fertilized according to standard protocols. Embryos were cultured at 38.5 °C under humidified air with 5% CO2, 7% O2, and 90% N2 in Synthetic Oviduct Fluid (SOF) medium supplemented with amino acids and either 5% of FBS (SOFaa) (control group) or SOFaa supplemented with 1  µM crocetin (crocetin group). After 5 days from the beginning of in vitro culture (IVC) (day 5 - D5), embryos were transferred to individual drops of culture media. At day 7 (D7), embryos were assessed by means of blastocyst rates, morphophysiological analyzes (total cell number, ROS and mitochondrial activity levels), transcript quantitation of 47 genes and metabolomic evaluation of the culture media by Raman spectroscopy. In the crocetin group blastocyst rates were higher and embryos had increased total cell number and decreased intracellular levels of ROS. These embryos also had upregulation of genes related with response to stress and lipid metabolism (ATF4, BAX, FOXO3, GADD45A, GPX1, GPX4, HSF1, SOD2, ACACA, SREBF1 and SREBF2). Raman spectroscopy corroborated these results indicating more active lipid and amino acid production in this group. The absence of crocetin in the culture media resulted in higher ROS level, as well as up regulation of genes related to DNA damage, stress response and energy metabolism (MORF4L2, SOD1, TXN, PFKP, PGK1 and PPARGC1A). In conclusion, crocetin supplementation during culture protects embryos from oxidative stress and influences the adaptive response to stress conditions, leading to an increase in both blastocyst yield and quality, as well as changes in transcriptomic and metabolic profile of in vitro produced bovine embryos.

Morphokinetic-related response to stress in individually cultured bovine embryos.

The kinetics of in vitro-produced (IVP) bovine embryos is related to embryo viability, metabolism, and epigenetic patterns. Therefore, we believe that embryos with different speeds of development also respond differently to stress. In the present study, we performed global metabolic analysis (matrix-assisted laser desorption ionization time of flight mass spectrometry [MALDI-TOF]) of culture media, characterized apoptotic events (Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] and caspase quantitation), and quantified transcript abundance of stress-related gene (real-time quantitative polymerase chain reaction [qRT-PCR]) in IVP bovine embryos with different developmental kinetics to investigate possible markers of stress response. For this purpose, embryos were considered "fast" if they presented four or more cells at 40 hours post insemination (hpi). Embryos presenting two cells at this time were classified as "slow". Evaluations were performed at 40 hpi, 112 hpi, and 186 hpi. Metabolome analysis revealed several metabolites differentially represented between groups at all time points related with energy, lipid and amino acids metabolism, and stress response. There was no difference in TUNEL positive cells between groups in any of the time points analyzed. Nevertheless, at 112 hpi, classified as a critical phase because of the genome activation, the amount of caspase 3 and 7 and total caspase were higher in slow when compared to fast group. Transcript abundance analysis of candidate genes (GRP78, HSP60, SOD1, and MORF4L2) was also different among groups. In conclusion, IVP bovine embryos of different development speeds respond differentially to the environmental stress leading to different metabolome patterns and apoptosis activation throughout the culture.

Rapid and noninvasive technique to assess the metabolomics profile of bovine embryos produced in vitro by Raman spectroscopy.

Morphological assessments are used to select embryos with the highest implantation potential, however it is still very limited. The development of new technologies, such as Raman spectroscopy have improved quantitative and qualitative analysis, and consequently led to a better characterization of embryos and improvements on the prediction of their potential. Therefore, we propose a method based on the conventional in vitro culture system of bovine embryos, and the subsequent analysis of the culture media drops by Raman spectroscopy. Our results obtained by PCA analysis clearly showed a separation of the spectral profiles from culture media drops with and without embryos.

Gene expression of estrogen and oxytocin receptors in the uterus of pregnant and parturient bitches

In the canine species, the precise mechanisms of pregnancy maintenance and the initiation of parturition are not completely understood. The expression of genes encoding the receptors for estrogen (ERα mRNA) and oxytocin (OTR mRNA) was studied in the endometrium and myometrium during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERα mRNA and OTR mRNA in the uterus of bitches during early (up to 20 days of gestation), mid (20 to 40 days) and late pregnancy (41 to 60 days), and parturition (first stage of labor). All tissues expressed ERα and OTR mRNA, and are thus possibly able to respond to eventual estrogen and oxytocin hormonal stimuli. No statistically significant differences in the expression of ERα mRNA were verified in the endometrium and myometrium throughout pregnancy and parturition, but expression of OTR mRNA increased at both parturition and late pregnancy. We concluded that the increase of endometrial and myometrial OTR mRNA expression in dogs is not an event dependent on estrogenic stimulation. Moreover, the contractility response of the canine uterus to oxytocin begins during pregnancy and maintains myometrial activity. The expression of OTR mRNA in canine uterine tissues varied over time, which supports an interpretation that the sensitivity and response to hormone therapy varies during the course of pregnancy and labor. Further studies are needed to elucidate the factors underlying the synthesis of uterine oxytocin receptors and the possible role of ERβ rather than ERα in the uterine tissues during pregnancy and parturition in dogs.

Gene expression of estrogen and oxytocin receptors in the uterus of pregnant and parturient bitches.

In the canine species, the precise mechanisms of pregnancy maintenance and the initiation of parturition are not completely understood. The expression of genes encoding the receptors for estrogen (ERα mRNA) and oxytocin (OTR mRNA) was studied in the endometrium and myometrium during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERα mRNA and OTR mRNA in the uterus of bitches during early (up to 20 days of gestation), mid (20 to 40 days) and late pregnancy (41 to 60 days), and parturition (first stage of labor). All tissues expressed ERα and OTR mRNA, and are thus possibly able to respond to eventual estrogen and oxytocin hormonal stimuli. No statistically significant differences in the expression of ERα mRNA were verified in the endometrium and myometrium throughout pregnancy and parturition, but expression of OTR mRNA increased at both parturition and late pregnancy. We concluded that the increase of endometrial and myometrial OTR mRNA expression in dogs is not an event dependent on estrogenic stimulation. Moreover, the contractility response of the canine uterus to oxytocin begins during pregnancy and maintains myometrial activity. The expression of OTR mRNA in canine uterine tissues varied over time, which supports an interpretation that the sensitivity and response to hormone therapy varies during the course of pregnancy and labor. Further studies are needed to elucidate the factors underlying the synthesis of uterine oxytocin receptors and the possible role of ERß rather than ERα in the uterine tissues during pregnancy and parturition in dogs.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation.

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.

Oestrogen and progesterone receptor gene expression in canine oocytes and cumulus cells throughout the oestrous cycle.

The aim of this research was to analyze oestrogen receptor-alpha (ERalpha), ERbeta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells' total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ERbeta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ERbeta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ERalpha and ERbeta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ERalpha was expressed throughout the oestrous cycle.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows.

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha -lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position - 1689 (g. A > G) and + 15 (g. A > G) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide - 46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A - 1689, A - 46, and A + 15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5'-flanking and the 5'-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Collection and evaluation of semen from the three-toed sloth (Bradypus tridactylus).

Sloths (Bradypus sp.) are extremely sensitive animals that suffer with the destruction and fragmentation of forests. They present a low population growth rate and need to be further studied for the preservation of the specie. Thus, the aim of this study was to establish an efficient semen collection protocol as well as characterize sperm concentration, motility and morphology in order to contribute with information about the reproductive traits of this specie, which has never been described in the literature before. For that, nine Bradypus tridactylus males were captured during the wet season and six during the dry season, in Manaus (AM), located in the north region of Brazil, semen was collected by electroejaculation with shocks given in sequences of progressive intensities (minimum 20mA and maximum 60mA). All animals ejaculated small volumes of semen and in some of them, the volume ejaculated was not enough for a complete spermiogram. Physical characteristics observed on the collections of the wet season were different from those seen in the specimen collected in the dry season. Motility an vigor was very low and did not show forward progression, only oscillatory movement. After Spermac stain, spermatozoa presented a wide variety of defects; however, the differences in morphology were not significant between seasons. The morphology assessed by scanning electron microscopy shows that the head in both groups could be elongated, short or could have a base narrower than the apex and the midpiece narrowed abruptly, forming a nip in its transition to the tail. Although further studies are necessary to verify our preliminary findings concerning seasonal variation in sperm quality, these results demonstrate that semen can be safely collected from sloths by electroejaculation and provide the first reports of semen characteristics in this species.

Effect of chemical or electrical activation of bovine oocytes on blastocyst development and quality.

Activation of in vitro-matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 microM CA, 5 min; n = 88), CA + BSA (5 microM CA, 5 min; BSA, 5 min; n = 90), IO (5 microM IO, 5 min; n = 91), IO + BSA (5 microM IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 micros; n = 120). Blastocyst rates were higher (p < 0.05) for CA (54.4%), IO (51.4%) and EL (54.5%) than for IO + BSA (18.3%). Treatment CA + BSA (39.8%) did not differ from the others. There was no difference (p > 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.

Effects of serum deprivation and cycloheximide on cell cycle of low and high passage porcine fetal fibroblasts.

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 +/- 0.65) when compared with non-starved cells (71.44 +/- 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus.

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5 percent) were successfully fused and 24 (28.9 percent) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8 percent) of which were pregnant on day 35 and 3 (16.7 percent) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Production of a cloned calf from a fetal fibroblast cell line.

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).