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J A Visintin

Publications and source records attributed to J A Visintin.

9 recordsLinked to original sources
PubMed2007

Morphological changes in mouse embryos cryopreserved by different techniques.

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.

Animals↗
PubMed2003

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).

Animals↗
PubMed2002

Cryopreservation of Bos taurus vs Bos indicus embryos: are they really different?

Cryopreservation with storage at very low temperatures is essential to make full use of this technology for both biological and commercial reasons. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Lowering temperature below 0 degree C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls. Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Comparisons of the toxicity of various cryoprotectants indicated that ethylene glycol (EG) is a nontoxic compound for murine and bovine embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing and thawing require further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. This paper describes the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification.

Animals↗
PubMed2002

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases.

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Alkaline Phosphatase↗
PubMed2000

Effects of dominant follicle aspiration and treatment with recombinant bovine somatotropin (BST) on ovarian follicular development in nelore (Bos indicus) heifers.

Follicle ablation has been recognized as an efficient method of follicular wave synchronization. Treatment with recombinant bovine somatotropin (BST) has been shown to enhance follicular development in Bos taurus. This experiment assessed the effects of these treatments in Nelore (B. indicus) heifers. Eight cycling Nelore heifers were randomly assigned to 3 different treatments. On Day 2 of a synchronized cycle (Day 0 = day of ovulation), heifers assigned to Treatments 1 and 2 received 2 mL of saline, whereas heifers assigned to Treatment 3 received 320 mg of BST. On Day 5, the first-wave dominant follicle was ablated by ultrasound-guided transvaginal aspiration in heifers in Treatments 2 and 3, and all heifers received an injection of prostaglandin on Day 11. Aspiration of the dominant follicle advanced and synchronized (P < 0.05) the day of second-wave emergence (6.9 +/- 0.1 vs. 8.4 +/- 0.4) and the day of the pre-wave FSH peak (6.0 +/- 0.0 vs. 6.9 +/- 0.4), and increased FSH peak concentrations (381 +/- 21 vs. 292 +/- 30; pg/mL; P < 0.01). Recombinant bovine somatotropin treatment caused a two-fold increase in plasma insulin-like growth factor-I (IGF-I) concentrations (P < 0.001) and resulted in a 36% increase in the number of small follicles (<5 mm; P < 0.001) compared with saline-treated heifers. In summary, in agreement with previous reports on B. taurus, dominant follicle aspiration synchronized ovarian follicular development, and BST treatment increased peripheral concentrations of IGF-I in Nelore heifers. Recombinant bovine somatotropin also increased the number of small follicles, but this response appeared to be inferior to that reported for B. taurus.

Animals↗
PubMed1999

Detection of leptospires in bovine semen by polymerase chain reaction.

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

Animals↗
PubMed1997

Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis).

The growth, selection, regression and ovulation of ovarian follicles was ultrasonically monitored in 30 Murrah buffalo throughout a spontaneous estrous cycle during the breeding season (autumn). Examinations revealed that follicular growth during the estrous cycle occurs in waves; the buffalo showed 1-wave (3.3%, n = 1), 2-wave (63.3%, n = 19) or 3-wave (33.3%, n = 10) follicular growth. The first wave began at 1.00, 1.16 +/-0.50 and 1.10 +/- 0.32 d in buffalo with 1, 2 and 3 waves, respectively (ovulation = Day 0). The second wave appeared at 10.83 +/- 1.09 and 9.30 +/- 1.25 d (P < 0.01) for the 2 and 3 wave cycle animals, respectively. The third wave started at 16.80 +/- 1.22 d. Structural persistence of the first dominant follicle was longer in the 2- than 3-wave cycles (20.67 +/- 1.18 vs 17.90 +/- 3.47 d ; P < 0.05). The duration of the growth and static phases of the first dominant follicle differed between the 2 and 3 wave cycles (P < 0.05), whereas there were no differences in linear growth rates (cm/d). Two and three wave cycles differed (P < 0.05) with respect to the maximum diameter of both the first dominant follicle (1.51 +/- 0.24 vs 1.33 +/- 0.18 cm) and the ovulatory follicles (1.55 +/- 0.16 vs 1.34 +/- 0.13 cm). No relationship was found between dominant follicle development and the presence of either a CL or a previous dominant follicle in either ovary. Two and three wave cycles also differed with respect to the mean length of intervals between ovulation (22.27 +/- 0.89 vs 24.50 +/- 1.88 d; P < 0.01) and the mean length of luteal phases (10.40 +/- 2.11 vs 12.66 +/- 2.91 d; P < 0.05). These results demonstrate that buffalo have estrous cycles with 1, 2 or 3 follicular waves; that 2-wave cycles are the most common; and that the number of waves in a cycle is associated with the luteal phase and with estrous cycle length.

Journal Article↗