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Biomedical subjects

G Lazzari

Publications and source records attributed to G Lazzari.

At least 19 recordsLinked to original sources
PubMed2006

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

Animals↗
PubMed2006

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes.

Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified-warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.

Animals↗
PubMed2005

Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.

Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.

Animals↗
PubMed2005

Susceptibility of bovine mesenchymal stem cells to bovine herpesvirus 4.

Bovine herpesvirus 4 (BoHV-4) is a gamma herpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbour persistent BoHV-4. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of adjacent haematopoietic precursors, such as macrophages, the interaction between BoHV-4 and mesenchymal stem cells was investigated. Primary bovine mesenchymal stem cells were highly permissive to support full replication of BoHV-4. This finding could be considered a new important step in studies on the potential pathogenesis related to BoHV-4.

Animals↗
PubMed2005

Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.

Animals↗
PubMed2004

Expression pattern of the maternal factor zygote arrest 1 (Zar1) in bovine tissues, oocytes, and embryos.

Zygote arrest 1 (Zar1) is an ovary-specific maternal factor that plays an essential role during the oocyte-to-embryo transition in mouse. In this species, Zar1 expression is strictly limited to the oocyte, the zygote and, at a lower level, the 2-cell embryo. Aim of the present study was to analyze the presence and the expression pattern of the Zar1 ortholog in bovine tissues and embryos. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis was performed in a panel of bovine tissues, in oocytes and pre-implantation in vitro produced embryos. The results demonstrated that a Zar1 ortholog is present in cattle. In the adult, the gene is expressed in ovary, testis, muscle, and myocardium. The gene is also expressed in the oocyte, the zygote, and in all the stages of embryonic development until blastocyst formation. A semi-quantitative RT-PCR analysis revealed that Zar1 levels are constant through in vitro development with the exception of the 4-cell stage, when a significant increase is observed. The exposure of fertilized oocytes to the RNA polymerase II inhibitor alpha-amanitin was able to suppress this Zar1 increase indicating that transcription of this gene occurs at the 4-cell stage. Zar1 is conserved in cattle but has an expression pattern different from the mouse. In particular, Zar1 expression in the adult is not limited to the ovary and in the embryo is expressed well beyond the oocyte to embryo transition. Moreover, the identification of Zar1 transcription at the 4-cell stage represents the first characterization of one of the genes expressed in cattle embryos before the major onset of embryonic transcription.

Animals↗
PubMed2004

Production and quality of bovine oocytes and embryos.

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.

Animals↗
PubMed2004

Zootechnical performance of cloned cattle and offspring: preliminary results.

This paper presents information on the evolution of sets of cloned heifers of Holstein breed in comparison to that of control heifers derived from artificial insemination (AI) in the same farm, as well as data on a set of cloned bulls and their semen characteristics. Preliminary observations on a group of calves sired by a cloned bull and offspring of cloned females are reported. Mean birth weight in the clone group (50 females) was statistically higher than that of 68 contemporary female controls obtained by AI (49.27 +/- 10.98 vs. 40.57 +/- 5.55 kg, respectively, p < 0.05). Growth rate was within normal values for Holstein heifers (from 0.7 to 0.8 kg/day) and daily gain was not influenced by the high or low birth weight of clones. Within animals of the same clone, variability of daily gain was reduced compared to their control counterparts. Semen production from three cloned bulls was within the parameters expected for young bull of the same age. A direct comparison of morphological analysis was made between the frozen thawed semen of the donor bull and of his three clones collected at the same age. The overall semen picture appeared within acceptable limits and the clones presented similar percentages of sperm abnormalities (80% of morphologically normal spermatozoa) as the donor. These preliminary results suggest no deleterious effect of cloning on the semen picture of cloned sires. Frozen semen from one clone bull was used for an AI trial, resulting in 65% pregnancies, 25 live calves were naturally delivered. Concerning the offspring of both female and male clones, the phenotypical and clinical observation of the calves in the first week of age did not reveal any clinical abnormality, suggesting that the deviations observed in clones are not transmitted to the progeny.

Animals↗
PubMed2003

Bovine embryo technologies.

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Animals↗
PubMed2003

Interaction of a green recombinant bovine herpesvirus 4 with in vitro-produced bovine embryos.

The objective of the present study was to assess whether bovine herpesvirus 4 (BHV-4) is able to infect in vitro-produced bovine embryos. A green recombinant BHV-4 (BHV-4EGFP deltaTK), obtained by insertion of an EGFP gene into the TK locus of BHV-4, was used. The presence of this marker protein made it possible easily to detect infected cells under physiological conditions, without harmful manipulation of the cells or the addition of exogenous substrates, so that the spread of the virus could be followed in real time. Zona pellucida intact (ZP-I) and zona pellucida open (ZP-O) blastocytes were exposed to 10(6) TCID50 viral particles and infection was monitored by fluorescent microscopy for 48 h. Expression of EGFP and degeneration of embryonic cells was observed in three of the 18 ZP-O embryos, but in none of the ZP-I embryos. It was concluded from this preliminary study that BHV-4 has only a low ability to infect in vitro-produced bovine embryos, depending on the absence of ZP, the amount of virus present and the stage of embryonic development. However, embryonic stem cells could be transduced by BHV-4EGFP deltaTK just after differentiation, as shown by expression of EGFP.

Animals↗
PubMed2001

Embryo production by ovum pick up from live donors.

Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique. It can be performed twice a week for many weeks without side effects on the donor's reproductive career. The donor can be in almost any physiological status and still be suitable for oocyte recovery. A scanner with a sectorial or convex probe and a vacuum pump are required. Collection is performed with minimal stress to the donor. An average of 8 to 10 oocytes are collected per OPU with an average production of 2 transferable embryos. The laboratory production of embryos from such oocytes does not differ from that of oocytes harvested at slaughter as the results after transfer to final recipients. For other species such as buffalo and horses OPU has been attempted similarly to cattle and data will be presented and reviewed. For small ruminants, laparotomy or laparoscopy seems the only reliable route so far to collect oocytes from live donors.

Animals↗
PubMed2001

Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity.

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.

4-Butyrolactone↗
PubMed2000

The insulin-dependent glucose transporter isoform 4 is expressed in bovine blastocysts.

We have investigated the expression of two glucose transporter isoforms, Glut1 and 4, in 14- and 16-day-old bovine blastocysts (d14, d16) using RT-PCR, competitive RT-PCR and in situ hybridization. The blastocysts were grown in vivo or had been produced in vitro. Glut1 mRNA was detected in all blastocysts studied, Glut4 in all d14 blastocysts, but only in a few d16 blastocysts. Glut4 mRNA was localized in trophoblast and endoderm cells. Glut1 mRNA increased from d14 to d16 while Glut4 transcription was down-regulated in d16 blastocysts. The mRNA amounts varied between 0.8 to 23 pg and 3.9 to 65 fg per 100 ng embryonic RNA for Glut1 and Glut4, respectively, displaying a 100- to 1500-fold lower expression of Glut4 compared with Glut1 during blastocyst elongation. This is the first report on the expression of the insulin-sensitive Glut4 isoform in mammalian preimplantation embryos.

Animals↗
PubMed1999

Mammalian leukocytes contain all the genetic information necessary for the development of a new individual.

We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.

Animals↗
PubMed1998

Oral iron chelating therapy. A single center interim report on deferiprone (L1) in thalassemia.

BACKGROUND AND OBJECTIVE: Deferiprone (L1) is a largely studied oral chelator in clinical setting, however, no definite conclusions concerning efficacy and toxicity still could be drawn. In an ongoing prospective trial with L1, we evaluated the efficacy and tolerance-toxicity in patients with thalassemia major previously treated by desferrioxamine (DFO); the specific aim of the study is to demonstrate that L1 could be an alternative to DFO in some patients with an acceptable toxicity. DESIGN AND METHODS: Sixty-nine patients over 13 years of age with poor compliance to DFO were considered for the study. The design included a liver biopsy before starting L1 in all patients in order to define liver siderosis either by histologic grading or by hepatic iron concentration (HIC); only patients with a minimum HIC of 4 mg/g dry weight entered the study. A repetition of the liver biopsy after one year of L1 was planned; further evaluations included serum ferritin, plasma iron, transferrin TIBC and iron urine excretion. L1 was given at 70 mg/kg/day in three divided doses. Toxicity was monitored either clinically or by controlling liver, kidney and marrow function by specific tests. Concerning clinical characteristics 52 patients showed hypogonadism (78%), 39 growth retardation (58%), 6 diabetes (9%), 4 cardiomyopathy (6%), 9 hypothyroidism (12%); 45 patients had chronic liver damage (65%). RESULTS: We focus this report on data collected in a group of 29 patients with a minimum follow-up of one year (14-33 months). The mean ferritin value was 3748 ng/mL (range: 200-10,000) and 2550 ng/mL (range: 80-14,500), before and while on L1 therapy, respectively (p = 0.001); the mean sideruria changed from 17.25 mg/dL (range: 5.4-50) to 20.98 mg/dL (range: 10-40), on DFO and L1, respectively (p = 0.078); the ratio between plasma iron (sideremia) and transferrin TIBC changed from 0.96 with DFO to 0.86 with L1 (0.014). A correlation with grade of liver siderosis and serum ferritin (p = 0.069) and iron urine excretion (p = 0.008) was recorded. The judgement of efficacy showed that L1 was effective (EF) in 9 patients, no assessable (UN) in 11 patients, not effective (NE) in 2 patients and with no advantages with respect to DFO in 7 patients. Liver biopsy was repeated in 20 patients showing a reduction of grade of liver siderosis and iron content in 7 patients. Clinical toxic effects of L1 were gastric intolerance (one patient), joint pain (three patients) and mild and temporary neutropenia (one patient). INTERPRETATION AND CONCLUSIONS: This preliminary experience shows that L1 is effective in several patients with thalassemia with poor compliance to DFO and to improve iron burden and iron excretion with generally minor side effects. L1 could be an alternative to DFO in some patients, however the recognition of neutropenia warrants a careful evaluation of patients and efforts finalized to early recognition of those to be addressed with this new and still experimental therapy.

Adolescent↗