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Biomedical subjects

M A Gil

Publications and source records attributed to M A Gil.

At least 19 recordsLinked to original sources
PubMed2006

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium.

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.

Animals↗
PubMed2006

Bloodstream cells phenotypically identical to human mesenchymal bone marrow stem cells circulate in large amounts under the influence of acute large skin damage: new evidence for their use in regenerative medicine.

OBJECTIVES: Recent work has shown that human bone marrow contains mesenchymal stem cells (MSCs). However, little is known about their presence in peripheral blood. Since these cells are potentially responsible for tissue repair after injury, their number should be increased during these situations. To demonstrate their number during these situations, we measured MSCs in the peripheral blood of healthy donors and burn patients. MATERIALS AND METHODS: Blood samples were obtained from 15 acute burn patients and 15 healthy donors. We performed flow cytometric analysis, using a large monoclonal antibody panel: CD44, CD45, CD14, DR, CD34, CD19, CD13, CD29, CD105, CD1a, CD90, CD38, CD25. MSC phenotype was considered positive for CD44, CD13, CD29, CD90, and CD105, and negative for the other monoclonals. The testing was performed on day 3 after injury. We correlated the results with the age, sex, and size and type of burns. RESULTS: Cells expressing the MSC phenotype were detected in the peripheral blood of both groups. Noteworthy, compared with samples from healthy donors (0.0078 +/- 0.0044), blood obtained from burn patients showed a higher MSC percentage (0.1643 +/- 0.115; P < .001). The percentage of MSCs correlated with the size and severity of the burn. Increased values were also observed among younger patients. CONCLUSIONS: MSCs have an important role in regenerative processes of human tissues. We found cells phenotypically identical to MSCs circulating in physiological number in normal subjects, but in significantly higher amounts during acute large burns. Therefore, they may represent a previously unrecognized circulatory component to the process of skin regeneration.

Adult↗
PubMed2006

Incidence of unilateral fertilizations after low dose deep intrauterine insemination in spontaneously ovulating sows under field conditions.

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.

Animals↗
PubMed2006

Challenges in pig artificial insemination.

Semen extended in a liquid state, together with conventional artificial insemination (AI), is the sole sperm technology used by the pig industry. Sperm technologies known for many years, such as cryopreservation, and others developed during recent years, such as sperm sexing, have not yet been integrated into commercial use. Moreover, there has recently been an explosion of new technologies, such as sperm mediated gene transfer or encapsulated spermatozoa which need additional supportive techniques before they can be economically applied to pig breeding. The speed with which the aforementioned sperm technologies are accepted and utilized by the pig industry depends on the availability of efficient insemination procedures. Therefore, AI is entering a new dimension where it will be converted into a tool for the efficient application of current and new sperm technologies. Some new insemination procedures have been recently developed. This review examines the suitability of available insemination procedures for the efficient application of current, emergent and future sperm technology to the pig industry.

Animals↗
PubMed2005

Does multivariate analysis of post-thaw sperm characteristics accurately estimate in vitro fertility of boar individual ejaculates?

The objective of this study was to determine if a multivariate pattern analysis of frozen-thawed sperm characteristics of boar semen of unknown fertility, thus identifying groups of ejaculates as "good" or "bad" freezers, would estimate their fertilizing potential in an in vitro embryo production (IVP) system. Frozen-thawed spermatozoa from a single ejaculate collected from 46 boars were evaluated for sperm motility and kinematic patterns, for sperm viability and for early changes in sperm membrane stability. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all ejaculates within a data set in to one of two groups, categorised as "good" (n = 25) or "bad" (n = 21) according with their freezability. In vitro matured oocytes were exposed to 2000 or 4000 frozen-thawed spermatozoa per oocyte for 6h and then cultured in embryo culture medium for either 6h (assurance of fertilization) or 7 days (to collect data on embryo development). Rates of sperm oocyte penetration and of embryo development significantly (p < 0.05) increased in a sperm:oocyte ratio-dependent manner. A similar pattern was observed when sperm characteristics were grouped. Indeed, ejaculates classified as "good" showed significantly (p < 0.05) higher rates of oocyte penetration, cleavage and of blastocyst formation than those classified as "bad". However, variation was still present among individuals (ejaculates, boars) in their ability to produce blastocysts in vitro. It is therefore concluded that despite the presence of a relationship for ejaculates with good semen quality post-thaw (thus grouped as "good") to higher IVP-results, the presence of individual variation does not allow for an accurate estimation of in vitro fertility based solely on the frozen-thawed semen quality parameters of a single ejaculate from a given boar.

Analysis of Variance↗
PubMed2005

Adjustments in IVF system for individual boars: value of additives and time of sperm-oocyte co-incubation.

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.

Animals↗
PubMed2005

An update on reproductive technologies with potential short-term application in pig production.

Over the past decade, there has been an increase in the development and/or in the improvement of emerging reproductive technologies in pigs. Among emerging reproductive technologies with potential short-term application in pig production are: artificial insemination with low number of spermatozoa, cryopreservation of spermatozoa and embryos, sperm sexing, and non-surgical embryo transfer. The following review will give emphasis to recent advancements in these reproductive technologies that are starting to show possibilities of serious applications under field conditions.

Animals↗
PubMed2004

Hoechst 33342 stain and u.v. laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa.

Sex selection by flow cytometry/cell sorting involves the staining of spermatozoa with Hoechst 33342 in combination with the impact of a u.v. laser beam, two potentially mutagenic agents. A phenotypic and cytogenetic study of lymphocytes of piglets born after insemination with spermatozoa stained with Hoechst 33342 and from piglets obtained from stain-sorted spermatozoa was performed to evaluate the genotoxic effect of Hoechst 33342 staining and u.v. laser irradiation on the offspring. Lymphocytes from piglets born after insemination with unstained spermatozoa, but from the same ejaculate, were used as a control group. Peripheral blood lymphocytes from these piglets were cultured following a standard cell culture protocol. Cells were then collected by centrifugation, subjected to hypotonic solution and fixed and dropped onto slides. Sister chromatid exchanges (SCEs) and chromosome aberrations (CAs: including chromosome and chromatid breaks) per cell were scored in 50-s division metaphase spreads from each donor. Reproductive parameters and litter performance of all inseminations performed were also recorded in all groups. Data were analyzed by ANOVA. No significant increase (P > 0.05) of SCE and CA frequencies were observed in piglets born from stained spermatozoa or from stain-sorted spermatozoa with respect to controls (untreated sperm). The results indicated that no mutagenic effect on spermatozoa, expressed as increases in the incidence of abnormalities in the resulting offspring and also as increases in SCE and CA frequencies on lymphocytes from these individuals, was induced by the staining of boar spermatozoa with Hoechst 33342, nor by combination of staining with laser impact during flow cytometry.

Animals↗
PubMed2003

Influence of follicle size on the penetrability of immature pig oocytes for homologous in vitro penetration assay.

In order to improve the performance of homologous in vitro penetration (hIVP) assays using immature oocytes to assess the penetrating ability of boar sperm, the present study was designed to evaluate the influence of oocyte and follicle size on the penetrability of immature pig oocytes obtained from slaughterhouse ovaries. Nonatretic antral follicles were isolated, measured with a computerized image analysis system and grouped according to their diameter: Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), and Group 4 (2.80-6.50 mm). After sperm coincubation and before penetrability evaluation, the immature oocytes were classified into four size categories according to their diameter excluding zona pellucida: <105, 105-109, 110-114, and > or =115 microm. As regards follicle size, the highest viability and penetrability were obtained with oocytes from follicles >2.20 mm (P>0.05). Regarding oocyte size, significant differences (P<0.05) were observed for all parameters evaluated between oocytes with a diameter above or below 110 microm. However, our results revealed that such differences were due to follicle size rather than oocyte diameter, since oocytes with the same diameter but from different follicle size groups showed different penetration rates. With increasing follicle size, the percentage of penetrated oocytes increased (P<0.05). Finally, our results showed that the greater penetrability of immature oocytes from larger follicles is not due to variations in the thickness of the zona pellucida. There were no significant differences in zona pellucida thickness between oocytes from the four follicular size groups. In summary, these results indicate that follicle size directly affects the penetrability of immature pig oocytes used in hIVP.

Animals↗
PubMed2002

Relationship between antral follicle size, oocyte diameters and nuclear maturation of immature oocytes in pigs.

We designed the present study to examine the possible relationship between oocyte, antral follicle size and the nuclear heterogeneity of immature pig oocytes, in order to study the heterogeneity of oocyte populations in ovaries obtained from slaughterhouses. Previously, we carried out an initial experiment to determine, by histological analysis, the effectiveness of the macroscopic criteria (MC) used to screen atretic and nonatretic antral follicles. We recovered 239 follicles by mechanical dissection, measured them with a computerized image analysis system, and classified them into five size categories according to their diameter (FD): Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), Group 4 (2.80-3.59 mm) and Group 5 (3.60-6.50 mm). In relation to histological analysis, the results showed that MC is an effective method to select atretic and nonatretic antral follicles from 0.40 to 6.50 mm in diameter (overall accuracy was 80.75%, with sensitivity and specificity rates of 79.33 and 82.20%, respectively). In a second experiment, we recovered 454 nonatretic follicles, then measured and classified them as mentioned above. We removed oocytes individually from follicles and measured their size (oocyte diameter without and with zona pellucida, OD and TOD, respectively). Finally, we evaluated the relationship between OD, FD and nuclear maturation of immature oocytes (germinal vesicles (GV) Stages 0, I, II, III and IV; diakinesis, prophase I, and metaphase I). Overall OD was 101.77 +/- 0.65, 109.19 +/- 0.45, 113.55 +/- 0.50, 116.92 +/- 0.46 and 117.13 +/- 0.47 microm (Groups 1, 2, 3, 4, and 5, respectively). Differences in OD between groups were significant (P < 0.01), although from 2.80 to 6.50 mm follicles, the oocytes were not different in size. There was a certain heterogeneity in OD within each follicular group. Although we observed a certain degree of nuclear variability, regardless of FD or OD, the present study showed a clear progression in GV when FD increased from 0.40 to 6.50 mm. A positive correlation (r2 = 0.4248; P > 0.05) was established mainly between the nuclear stage and oocyte diameter.

Animals↗
PubMed2002

Motility characteristics and fertilizing capacity of boar spermatozoa stained with Hoechst 33342.

Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer-aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35 degrees C with 9, 18, 27, 60 and 90 microM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 microM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 microM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 microM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 microM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 +/- 0.32 vs 10.47 +/- 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X- and Y-bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.

Animals↗
PubMed2002

Minimum number of spermatozoa required for normal fertility after deep intrauterine insemination in non-sedated sows.

A fibreoptic endoscope procedure for non-surgical deep intrauterine insemination in non-sedated sows has been reported. However, the endoscope is an expensive and fragile instrument, and is unsuitable for use under field conditions. The aim of this study was to determine the minimum number of spermatozoa required to maintain optimal fertility using a flexible catheter (1.8 m in length, 4 mm in diameter) for deep intrauterine insemination in 2-6 parity non-sedated sows. Crossbred sows were treated with eCG 24 h after weaning and with hCG 72 h later to induce oestrus. Deep intrauterine insemination was performed 36 h after hCG treatment in 117, 126, 60 and 69 sows with 15.0, 5.0, 2.5 or 1.0 x 10(7) spermatozoa in 10 ml, respectively. Weaned sows (n = 147) not treated with hormones and used for standard artificial insemination (AI) (two inseminations per oestrus with 3 x 10(9) spermatozoa in 100 ml) served as controls. The flexible catheter was passed successfully through the cervix into one uterine horn in 95.4% of the sows in an average of 3.7 +/- 0.09 min. Farrowing rates after deep intrauterine insemination with 15 or 5 x 10(7) spermatozoa did not differ from those of the control group (82.9, 76.2 and 83.0%, respectively), but a significant decrease (P < 0.001) was observed in sows inseminated with 2.5 or 1.0 x 10(7) spermatozoa (46.7 and 39.1%, respectively). In contrast, the number of spermatozoa inseminated did not affect prolificacy. Laparotomy revealed that the tip of the flexible catheter reached approximately the anterior third of the uterine horn. Although deep intrauterine insemination was performed in only one uterine horn, the percentages of embryos collected from the tip of both uterine horns 2 days after deep insemination were not significantly different. The results show that in comparison with standard AI, a 20-60-fold reduction in the number of spermatozoa inseminated and an 8-10-fold reduction in the dose volume can be achieved without decreasing fertility when semen is deposited non-surgically into the upper first third of one uterine horn.

Animals↗
PubMed2001

Successful non-surgical deep intrauterine insemination with small numbers of spermatozoa in sows.

A 100-fold reduction of the standard dose for artificial insemination in pigs (3 x 10(9) spermatozoa in 80-100 ml fluid) can be used when spermatozoa are deposited surgically next to the uterotubal junction. The present study was performed to develop a technique for non-surgical deep intrauterine insemination in pigs without sedation of the animal. In Expt 1, sows were weaned, treated to induce oestrus and used to evaluate the difficulties involved in the insertion of a flexible fibre optic endoscope through the cervix and along the uterine horn. Deep uterine catheterizations were performed on each sow at 30-40 h after hCG treatment in the crate in which the animal was housed. The endoscope was inserted through an artificial insemination spirette, moved through the cervical canal and propelled forward along one uterine horn until the entire endoscope was inserted. In 30 sows (90.9%) no or minor difficulties were observed during insertion and in these animals the procedure was completed in 4.1 +/- 0.26 min. Insertion of the endoscope through the cervical canal was not possible in only one sow (3.03%). In Expt 2, endoscopic deep intrauterine insemination at 36 h after hCG treatment was performed in 15, 18 and 13 sows with 100, 20 or 5 x 10(7) spermatozoa, respectively, resulting in farrowing rates of 86.6%, 88.9% and 92.3%, respectively; there were no significant differences among groups. Farrowing rates after deep intrauterine inseminations were also not different from those achieved after standard intracervical insemination with 3 x 10(9) spermatozoa (control group: n = 48; 87.5%). Mean litter size (9.41 +/- 0.38 to 10.02 +/- 0.25) was also similar among the different experimental and control groups. In conclusion, endoscopic non-surgical deep intrauterine inseminations can be performed quickly in sows, and normal farrowing rates and litter sizes can be obtained after insemination with a small number of spermatozoa.

Animals↗
PubMed2001

Deep intrauterine insemination and embryo transfer in pigs.

A new method for non-surgical deep intrauterine catheterization of pigs, without sedation of the sow, is described. Insemination results obtained with this method using fresh spermatozoa demonstrate that, in comparison to conventional artificial insemination (AI) (3 x 10(9) spermatozoa in 80-100 ml), a 20-60-fold reduction in the number of spermatozoa inseminated and at least a 8-10-fold reduction in the dose volume can be used without affecting fertility if spermatozoa are deposited deep (middle or upper) into one of the uterine horns. Results from deep intrauterine insemination with frozen-thawed spermatozoa and flow-sorted spermatozoa are also presented and the effect of deep intrauterine insemination on sperm transport is discussed. In addition, a brief description of the advances made in non-surgical embryo transfer technology is reported.

Animals↗
PubMed1997

An epidural abscess due to resistant Staphylococcus aureus following epidural catheterisation.

We report a case of abscess formation after epidural analgesia, a rare complication that developed in our patient 13 days after placement of a thoracic epidural catheter for patient controlled analgesia. Culture of the pus grew methicillin-resistant Staphylococcus aureus. Although early diagnosis and rapid management have been reported to yield a satisfactory outcome, the case we describe ended in severe sequelae.

Abscess↗
PubMed1997

[Safety and feasibility of dobutamine-atropine stress echocardiography].

PURPOSE: To study the safety and feasibility of dobutamine-atropine stress echocardiography (DASE) in patients with known or suspected coronary artery disease. METHODS: There were 3,000 DASE that were studied in a prospective fashion. All symptoms and side effects were stored in a data base format. RESULTS: Major test-related complications observed included one case of myocardial infarction, four cases of sustained ventricular tachycardia and five cases of atropine intoxication. There was no death or ventricular fibrillation as a direct or indirect consequence of the test. The infusion protocol allowed to us to examine patients using beta blockers, and led to 95% feasibility. CONCLUSION: DASE is a safe and feasible method for the study of patients with suspected or known coronary artery disease.

Adolescent↗