Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B®) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V® 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin®, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean ± S.E.M.) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8 ± 1.8, 6.1 ± 1.3, 51.5), P48 (12.6 ± 1.9, 7.1 ± 1.0, 52.3), P60 (10.5 ± 1.6, 5.7 ± 1.3, 40.0) and D60 (10.3 ± 1.7, 5.0 ± 1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol. © 2002 Elsevier B.V. All rights reserved.

Molecular characterization and chromosomal localization of two families of satellite DNA in Prochilodus lineatus (Pisces, Prochilodontidae), a species with B chromosomes

Prochilodus lineatus, an abundant species in the Mogi-Guaçu river basin, represents a large part of the region's fishing potential. Karyotypic analyses based on classic cytogenetic techniques have revealed the presence of 54 metasubmetacentric type chromosomes, together with the occurrence of small supernumerary chromosomes with intra and interindividual variations. This paper describes the genomic organization of two families of satellite DNA in the P. lineatus genome. The chromosomal localization these two repetitive DNA families through fluorescence in situ hybridization (FISH) demonstrated that the SATH1 satellite DNA family, composed of approximately 900 bp, was located in the pericentromeric region of a group of chromosomes of the standard complement, as well as on all the B chromosomes. The SATH2 satellite family has a monomeric unit of 441 bp and was located in the pericentromeric regions of some chromosomes of the standard complement, but was absent in the B chromosomes. Double FISH analyses showed that these two families participate jointly in the pericentromeric organization of several chromosomes of this species. The data obtained in this study support the hypothesis that the B chromosomes derive from chromosomes of the standard complement, which are carriers of the SATH1 satellite DNA.

Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings

Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Superovulation in Cycling Mares Using Equine Follicle Stimulating Hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation. In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation. Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares. In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3). In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Implantation failures: Success of assisted hatching with quarter-laser zona thinning

Implantation failure after IVF is one of the factors associated with a reduced chance of pregnancy for some patients. Assisted hatching methodologies are designed to facilitate the embryo's escape from the zona pellucida, and this strategy has been suggested as a means of improving pregnancy rates in patients with previous implantation failure. The aim of this prospective and randomized study was to evaluate the efficacy of quarter-laser zona thinning assisted hatching (qLZT-AH) in improving the implantation of embryos in patients with previous implantation failure. A total of 150 patients with a history of previous implantation failure were treated with intracytoplasmic sperm injection, and allocated into two groups: group 1, only one previous implantation failure, and group 2, repeated implantation failures. The patients in each group were randomized at the time of embryo transfer into a control group (no qLZT-AH) or experimental group where qLZT-AH was performed. For patients with repeated implantation failures, the implantation rate in those who received laser-thinned embryos was significantly higher (P=0.02) than in those whose embryos were not laser-thinned (10.9 and 2.6% respectively). However, this difference was not observed in patients who presented with only one previous implantation failure. The data demonstrate that qLZT-AH is an effective strategy for improving the implantation of embryos in patients with repeated implantation failures.

Desenvolvimento morfolôgico dos ovários em embriões e fetos bovinos da raça Nelore

The morphologic events related to the prenatal development of the ovaries in 81 Nelore breed embryos and fetuses gathered in a local slaughterhouse, with age range from 26 to 240 days following fecundation were studied. The age of fetuses was estimated from measures taken in the cranium-caudal direction. The sex was identified from macroscopic observations and using Polymerase Chain Reaction (PCR) technique. For histology the gonads were fixed in Bourn's fluid for 24 hours and 5 μm thick section's were stained with hematoxylin-eosin. Formation of gonadal ridge and presence of germinal cells were found within it at 29 and 34 days, respectively. Oogonia and primordial follicles, unlike the growing follicles, exhibited significant differences in diameter in the various periods studied. Positive correlation (P<0.05) was found between the diameter of oogonia and their nucleus as well as between primordial and growing follicles with their oocytes and respective nuclei. The gonad was fully formed at 40 days. Primordial follicles, in the growing stage, and antral follicles first appeared, approximately at 95, 140, and 180 days, respectively. Despite the onset and duration of oogenesis being similar to that of taurine breeds, folliculogenesis initiates at an early stages in the Nelore breed.

Evaluation of serum- and animal protein-free media for the production of infectious bronchitis virus (M41) strain in a continuous cell line

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mixture of vitamin C, hesperidin and piperidol exposure in pregnancy: Maternal-fetal repercussions

To evaluate the reproductive performance and the development of their offspring on rat pregnancy. Wistar pregnant rats were gavaged with 0 mg/kg wb/day (control group, n = 20) and 166.5 mg/kg/day of a mixture of vitamin C, hesperidin and piperidol (experimental group, n = 20) during the organogenic period (from day 5 to 14 of pregnancy; positive vaginal smear = day 0). The female rats were killed on day 21 of pregnancy. The number of implantations, resorptions (dead embryos), and live/dead fetuses were counted for the analysis of the postimplantation loss rates. There was neither alteration in maternal reproductive performance, but it was verified an increase of the number of fetuses presenting dilated urether, hydronephrosis, and reduced ossification of skull due to the treatment of female rats with a mixture of vitamin C, hesperidin and piperidol, these abnormalities were considered transitory and may not interfere on offspring development. It was not verified other type of major malformation neither the appearance of fetuses presenting atrophy of upper limbs that it could be associated to use of this drug.

Cleidocranial dysplasia: importance of radiographic images in diagnosis of the condition.

Cleidocranial dysplasia (CCD) is a rare syndrome usually caused by an autosomal dominant gene, although 40% of cases of CCD appear spontaneously with no apparent genetic cause. This condition is characterized by several cranial malformations and underdevelopment, absence of the clavicles, and multiple supernumerary and impacted permanent teeth. The diagnosis of this condition is usually based on the presence of the main features (supernumerary teeth, partial or total absence of one or both the clavicles, and bony malformations) and on clinical and familial evidence. The bony and dental features of CCD may be visualized on radiographic images of the face and skull. Here, we present a familial case of CCD and discuss the importance of dental radiographs in diagnosis of the condition.

Implantation sites after embryo transfer into the central area of the uterine cavity

A total of 63 pregnancies (47 singleton, 15 twin, 1 triplet) from intracytoplasmic sperm injection cycles were analysed. In all embryo transfers, the catheter was introduced into the endometrial cavity guided by abdominal ultrasound, with the catheter tip placed at the middle point of the endometrial cavity. Gestational sacs (GS) were located 21-24 days after transfer (gestational age = 5 weeks) by two-dimensional and three-dimensional transvaginal ultrasound. The uterine cavity was divided into three parts: upper, middle and lower. Furthermore, the upper region was subdivided into right, middle and left areas, and the middle region was subdivided into right and left areas. The frequency of gestational sacs in each area was evaluated. In singleton pregnancies 66.0% (31/47) of the GS were detected in the upper region, 29.8% (14/47) in the middle region and 4.2% (2/47) in the lower region. In multiple pregnancies (twins and triplet) 45.5% (15/33) of the GS were detected in the upper region, 51.5% (17/33) in the middle region and 3.0% (1/33) in the lower region. In conclusion, the results demonstrate that when embryos are transferred to the central area of the uterine cavity there is an increase in implantation rate in the middle region compared with the rate expected in naturally conceived pregnancies (9-15%).

Utilização do primeiro ciclo ovulatório da estação reprodutiva para produção de embriões em éguas sob condições tropicais

The efficacy of the first ovulation of the breeding season was determined through the response of first pre-ovulatory follicle of the breeding season to hCG, embryo recovery rate, viability of recovered embryos, serum concentrations of progesterone, and response of first CL to PGF 2α. Thirteenth mares that were in vernal transition were accompanied until the first pre-ovulatory follicle was detected. At this moment, the ovulation was induced with 2,500 IU hCG (IV) and the mares were inseminated every other day until ovulation. Seven days after the ovulation, embryo recovery was performed and the progesterone concentration was determined. After detection of the first pre-ovulatory follicle of breeding season with ≥ 25mm, it took 14.92 ± 10.80 days for the follicle to reach the preovulatory size and 18.00 ± 11.08 days to ovulation. After administration of hCG, 11/13 mares ovulated in 48 hours. These follicles growth 2.19 ± 0.86 mm/day on average. Nine of 13 mares (69.2%) produced embryos and all were considered viable after morfological evaluation and fluorescence exams. The CL appeared competent producing 7.39 ± 2.11 ng/ml P 4 on average, and responding to PGF 2α. According to these results the first ovulatory cycle of the year can be utilized to produce viable embryos.

The effect of type of vaginal insert and dose of pLH on embryo production, following fixed-time AI in a progestin-based superstimulatory protocol in Nelore cattle

The objective was to analyze and report field data focusing on the effect of type of progesterone-releasing vaginal insert and dose of pLH on embryo production, following a superstimulatory protocol involving fixed-time artificial insemination (FTAI) in Nelore cattle (Bos taurus indicus). Donor heifers and cows (n = 68; 136 superstimulations over 2 years) received an intravaginal, progesterone-releasing insert (CIDR® or DIB®, with 1.9 or 1.0 g progesterone, respectively) and 3-4 mg of estradiol benzoate (EB) i.m. at random stages of the estrous cycle. Five days later (designated Day 0), cattle were superstimulated with a total of 120-200 mg of pFSH (Folltropin-V®), given twice daily in decreasing doses from Days 0 to 3. All cattle received two luteolytic doses of PGF2α at 08:00 and 20:00 h on Day 2 and progesterone inserts were removed at 20:00 h on Day 3 (36 h after the first PGF2α injection). Ovulation was induced with pLH (Lutropin-V®, 12.5 or 25 mg, i.m.) at 08:00 h on Day 4 with FTAI 12, 24 and in several cases, 36 h later. Embryos were recovered on Days 11 or 12, graded and transferred to synchronous recipients. Overall, the mean (±S.E.M.) number of total ova/embryos (13.3 ± 0.8) and viable embryos (9.4 ± 0.6) and pregnancy rate (43.5%; 528/1213) did not differ among groups, but embryo viability rate (overall, 70.8%) was higher in donors with a DIB (72.3%) than a CIDR (68.3%, P = 0.007). In conclusion, the administration of pLH 12 h after progesterone removal in a progestin-based superstimulatory protocol facilitated fixed-time AI in Nelore donors, with embryo production, embryo viability and pregnancy rates after embryo transfer, comparable to published results where estrus detection and AI was done. Results suggested a possible alternative, which would eliminate the need for estrus detection in donors. © 2006 Elsevier Inc. All rights reserved.

Chromosomes of Crossopriza lyoni (Blackwall 1867), intraindividual numerical chromosome variation in Physocyclus globosus (Taczanowski 1874), and the distribution pattern of NORs (Araneomorphae, Haplogynae, Pholcidae)

Pholcidae (Haplogynae) encompasses 967 described species, of which only 14 have been cytogenetic analyzed. Several chromosomal features have already been described including presence of meta- and sub-metacentric chromosomes and sex determination chromosome system (SDCS) of the X, X1X2Y, and X1X2 types, which contrast with the telo- and acrocentric chromosomes and SDCS of the X1X2 type typical of entelegyne spiders. To obtain further cytogenetic information for the family, we examined two pholcid species, Crossopriza lyoni (Blackwall 1867) and Physocyclus globosus (Taczanowski 1874) using both conventional staining and silver staining techniques. Crossopriza lyoni exhibited 2n = 23 = 22 + X in males and 2n = 24 = 22 + XX in females, while P. globosus showed 2n = 15 = 14 + X and 4n = 30 = 28 + 2X, both in male adults, 2n = 16 = 14 + XX in female adults and embryos, and 2n = 15 = 14 + X in male embryos. Both species revealed predominately metacentric and submetacentric chromosomes and a SDCS of the X/XX type. The cytogenetic data obtained in this work and those already recorded for C. lyoni indicate interpopulational and intraspecific numerical chromosome variation, suggesting the presence of chromosomal races or cytotypes in this species. The intraindividual numerical chromosome variation observed in male adult specimens of P. globosus may be explained by the presence of cytoplasmatic bridges between germ cells. The use of the silver staining technique to reveal the nucleolar organizer region (NOR) showed that chromosome pairs 4 and 6 and the X chromosome in C. lyoni are telomeric NOR-bearers, and that the chromosome pair 2 in P. globosus possesses a proximal NOR in the long arm.

Morfogênese do testículo de embriões e fetos de vacas da raça nelore (Bos taurus indicus)

The aim of this study was to accompany the process of testicular development from the non-differentiable phase to its complete formation. Embryos and fetuses of Nelore breed cows (Bos Taurus indicus) were obtained in slaughterhouses near the Uberlandia city, Minas Gerais. The gonads and the embryos were fixed in Bouin's fixative and afterwards processed for conventional optical microscopy. The gonadal was observed firstly in a 1.0 cm long embryo. In 2.5 cm long embryos the presence of the albuginea allows the sex identification. The mean thickness of the albuginea ranged from 29.08 to 558.45 mm. Gradually increase of vascularization of the albuginea and parenchyma is observed. The mediastinum is located centrally. There was a decrease in the space occupied by the testicular cords, from 63.71 to 41.99% of the total testes volume. Its diameter ranged from 31.68 to 48.80 mm. The diameter of germinal cells (and their nuclei) was from 12.27 (6.65) to 16.95 914.21) mm. The quantity of germinal cells by cross section of cord decreased from a maximum of 2.80 to 0.76. The total number of germinal cells was from 16 at the beginning of colonization of the gonad to 18.32 x 106 at the end of the study. The number of Sertoli's cells by cross section of cord ranged from 10.00 to 16.25. The results obtained show that the origin and formation of testes in embryos and fetuses from Nelore breed cows (Bos taurus indicus) does occur in a very similar way to what is described for Bos taurus taurus.

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.