Biomedical subjects

M A Silvestre

Publications and source records attributed to M A Silvestre.

15 recordsLinked to original sources

PubMed2006

Effect of solid storage on caprine semen conservation at 5 degrees C.

In this work, we investigated the effect of storage in solid-phase extender on buck semen conserved at 5 degrees C. Furthermore, we studied the effect of addition of cysteine to the extender and the washing of seminal plasma on sperm survival. In Experiment 1, milk-based extender (M) was used as a control to study the effect of solid media storage (G) and cysteine supplementation (C), and the combination of both (GC), on in vitro sperm survival of washed and non-washed semen, conserved up to 72 h at 5 degrees C. Motility, acrosome integrity (NAR) and hypo-osmotic swelling tests (HOST) were evaluated to assess in vitro sperm survival. In Experiment 2, an artificial insemination (AI) field trial was performed to compare G versus M. Solid media (G) maintained motility of spermatozoa during storage higher than any other extender (67% G versus 62% GC; 61% M and 59% C; P<0.05), but there was no difference in NAR or HOST between extenders (P>0.05). No improvement in sperm viability was obtained by addition of cysteine to the media. Washing of semen improved motility (65% versus 60%; P<0.05), NAR (70% versus 64%; P<0.05) and HOST (37% versus 28%; P<0.05). No significant differences in fertility were obtained between G and M extenders (47% versus 41%; P>0.05). In conclusion, washing of semen and dilution in gelatin-supplemented milk extender (solid storage) appears to be a successful method for goat semen storage at 5 degrees C.

Acrosome Reaction↗

PubMed2006

Morphometric changes in goat sperm heads induced by cryopreservation.

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.

Animals↗

PubMed2005

Vitrification of pre-pubertal ovine cumulus-oocyte complexes: effect of cytochalasin B pre-treatment.

The aim of this study was to evaluate the effect of cytochalasin B (CCB) pre-treatment before vitrification on ability of immature oocytes from lamb ovaries to progress until metaphase II (MII) stage after vitrification/warming procedure. Cumulus-oocyte complexes (COCs) were obtained from ovaries of lambs, from 80 to 90 days old, collected from a local slaughterhouse. Before vitrification, COCs were randomly distributed in two experimental groups corresponding to the incubation with or without 7.5 microg/ml CCB for 30 min. In order to study cryoprotectant and CCB pre-treatment toxicity (toxicity test), oocytes were exposed to cryoprotectants, with or without CCB pre-treatment, but without plunging into N2 liquid. Vitrification solution was composed by 4.48 M EG plus 3.50 M DMSO supplemented with 0.25 M sucrose. Two-step addition was performed. After vitrification or toxicity test, COCs were matured in bicarbonate-buffered TCM 199 containing 10% foetal calf serum and 10 ng/ml epidermal growth factor. A sample of COCs was directly in vitro matured (control group). Rates of MII oocytes of toxicity groups both, with or without CCB pre-treatment were lower than control group (41.1-50.0 versus 79.9, respectively; P<0.05). After vitrification, a lower number of oocytes progressed to MII stage in comparison with non-vitrification groups (P<0.05). In vitrified groups both with or without CCB pre-treatment 8.0 and 12.7%, respectively, of immature oocytes reached MII stage by the end of in vitro maturation culture. No effect of CCB was observed, either in the toxicity or vitrified groups. In conclusion, no effect of CCB pre-treatment before vitrification was detected in this study with immature oocytes of pre-pubertal sheep. More studies are needed in order to increase ovine oocyte survival after vitrification.

Animals↗

PubMed2005

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity.

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.

Animals↗

PubMed2005

Factors affecting pregnancy rate in artificial insemination with frozen semen during non-breeding season in Murciano-Granadina goats: a field assay.

An artificial insemination programme was carried out to study the effect of factors such as depth of semen deposition, inseminator skill, farm, sire and expression of oestrus on pregnancy rate in Murciano-Granadina (MG) goats during non-breeding season and using frozen semen. Frozen-thawed semen from six males was applied by three technicians to inseminate a total of 551 goats in 17 farms distributed throughout the Mediterranean area of Spain. Pregnancy rate was determined at 6 weeks after insemination by transabdominal ecography. Overall pregnancy rate was 57%. Farm and depth of semen deposition affected pregnancy rate, whereas the sire and the technician had no effect. The deeper the semen was deposited in the genital tract, the higher was the rate of pregnancy obtained, being greater when the catheter reached the uterus. In spite of the relevant difference observed (48.2% vs 59.0%), pregnancy rate of females not coming into oestrus until 30 h after sponge removal was not significantly different, compared with those showing oestrus during the OD procedure. In conclusion, our field assay data on AI in MG goat with frozen-thawed semen showed that post-cervical insemination presented significantly greater pregnancy rate in comparison to when semen is deposited in the vagina or in the caudal part of the cervix.

Animals↗

PubMed2004

Effect of changing female stimulus on intensive semen collection in young Murciano-granadina male goats.

The aim of this research was to study the effect of changing female stimulus on libido and semen characteristics from young Murciano-granadina male goats submitted to intensive semen collection using females not in estrus as teasers. Males were submitted to two different sexual stimulation procedures. In the first procedure, the same doe was used as the female stimulus for three consecutive presentations. In the second, the doe was replaced after the second presentation by a new female. Semen volume, concentration, forward progressive motility, and live spermatozoa were scored. To analyze reaction time (RT), three types of analysis were performed. In the first one, RT was analyzed by multifactor ANOVA, taking as a missing value 300 s when a buck did not ejaculate. In the second, RT also was analyzed by multifactor ANOVA, but data from males that did not ejaculate were removed. In the third, a Cox Survival analysis was carried out by censoring data when a buck did not ejaculate within 5 min of entering the test arena. A decrease in semen volume and sperm concentration in the successive ejaculations was observed, being highly marked in the third ejaculation independent of the stimulation procedure (0.62 vs. 0.38 and 0.43 mL, and 2,828 vs. 2,183 and 2,223 million spermatozoa/mL to the first and third ejaculation respectively; P < 0.05). No significant differences were observed either in forward progressive motility or live sperm rate. Changing the female stimulus in the third presentation had no significant effect on any seminal characteristic. Regarding libido and mounting behavior variables, there was a substantial decrease in RT in the third service when the female was changed (with both types of ANOVA). When censored data were taken into account, the relative risk showed that the probability of a male ejaculating in the third presentation increased almost fourfold when the female was replaced than when the female was the same in all services (P < 0.05). In conclusion, young Murciano-granadina bucks can be used as semen donors because none of the most important semen variables used to reject or accept an ejaculate before freezing process decreased after intensive semen collection. We also recommend changing the female stimulus to make the semen collection procedure more efficient and using survival analysis methodology to analyze time data, mainly when a high rate of censored data are scored.

Analysis of Variance↗

PubMed2004

[General anesthesia by infusion of remifentanil for debridement and grafting of large burns in 3 pediatric patients].

Three 13-year-olds with burns on 30%, 70%, and 75% of their body surface areas and with related respiratory tract lesions underwent multiple surgical interventions for debridement, graft placement, and topical treatments. Balanced general anesthesia was provided with sevoflurane and an infusion of remifentanil and cisatracurium. Each patient underwent 41 procedures over a period of 6 months. All interventions were carried out without complications other than the difficulty of establishing an access to veins and monitoring because of the scarcity of intact surfaces. Remifentanil is an effective analgesic for meeting the needs of patients with severe burns. The combination of remifentanil, sevoflurane, and cisatracurium provides good hemodynamic stability and is very appropriate for patients who must undergo a large number of surgical interventions.

Adolescent↗

PubMed2003

Vitrification of in vitro cultured rabbit morulae.

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO-S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG-20DMSO+S) and two-step (20EG-20DMSO-S) groups (0-6% versus 36-50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham's F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG-20DMSO+S) group in s-PBS (35-45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO-S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG-20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG-20DMSO-S), regardless of percentage of FCS in the PBS-based media (81% versus 41-45%; P<0.05). In conclusion, we propose either the one step (20EG-20DMSO+S) or two steps (20EG-20DMSO-S) prepared in PBS plus 20% serum for use in future works.

Animals↗

PubMed2003

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death.

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.

Animals↗

PubMed2002

Vitrification and rapid freezing of rabbit fetal tissues and skin samples from rabbits and pigs.

Vitrification (3.58 M EG and 2.82 M DMSO in PBS with 20% FCS) and rapid-freezing (0.25 M sucrose, 2.25 M EG, and 2.25 M DMSO in PBS with 20% FCS) procedures were assayed to cryopreserve rabbit tissue samples from 12-day fetuses, and skin samples from live born pups and adult rabbits. These methods were also assayed to cryopreserve pig skin samples obtained from abattoir animals. The ability of rabbit tissue samples to attach and colonize the substratum by cell proliferation was not affected by the assayed cryopreservation procedures, regardless of specimen age. In porcines, sample attachment and cell proliferation capability of primary cultures were not affected by applied cryopreservation procedures. Almost all primary cultures from cryopreserved skin samples reached confluency (from 92 to 100%). Results reported here allow us to establish in both species, rabbit and pig, a cryobank of skin samples from adult specimens classified as outliers for longevity (in rabbits) and prolificacy (in pigs).

Animals↗

PubMed2000

Vitrification and rapid-freezing of cumulus cells from rabbits and pigs.

To use adult somatic cloning technology in animal breeding, this technology should be complemented with nuclear donor cell cryopreservation. Two different conventional nonequilibrium methods (vitrification, V: 3.58M EG and 2.82M DMSO in PBS plus 20% FCS and rapid-freezing, RF: 0.25M sucrose, 2.25M EG and 2.25M DMSO in PBS plus 20% FCS) were assayed here on different cumuli types from rabbits and pigs. In rabbits, the cell proliferation capability of fully disaggregated cumuli was not affected by cryopreservation procedures (V: 100% and RF: 82%). Vitrified samples from partially or non-disaggregated cumuli showed the lowest proliferation frequencies (4% and 0%, respectively). In pigs, differences in cell proliferation capability were only observed between vitrified non-disagreggated cumuli and vitrified or rapid-frozen, fully disaggregated cumuli (72% vs 100% or 100%, respectively; P < 0.05). In both species, in vitro cultured sub-confluent samples were able to survive to a second cryopreservation treatment, maintaining the cell proliferation capability in nearly 50% of thawed samples. In conclusion, before cryopreservation, disaggregation of cumulus cells from both species into small clusters of cells improved their viability after thawing. These results allow us to efficiently, easily and rapidly store rabbit and pig cumulus cells, from selected high-merit females.

Animals↗

PubMed1997

Effects of timing, dosage, and duration of morphine intake during pregnancy on the amount of morphine in meconium in a rat model.

Meconium drug analysis is a new and sensitive test for detecting the intrauterine exposure of infants to drugs of abuse. To explore other potential, diagnostic use of the test, we studied, in timed pregnant rats, the relationship between timing, dosage, and duration of morphine administration to the dam and the concentration of morphine, analyzed by radioimmunoassay, in their pups' meconium. The term 'meconium' refers to both the intestine and its contents. Due to the minute size of the pups' intestines, the intestinal contents were not separated from the intestines. The mean morphine concentration in meconium was found to be significantly (p < 0.05) higher in the groups treated with a higher morphine dose (10 vs. 5 mg/ kg/day), longer duration of treatment (7-20 vs. 18-20 days), or treated during the last week of gestation (14-20 vs. 1-6 or 7-13 days). We conclude that the concentration of a drug in the meconium is related to amount, timing, or duration of the drug exposure of the fetus, in utero. The quantitative analysis of drugs in the meconium, therefore, provides added information which enhances the diagnostic use of the test.

Animals↗

PubMed1996

A prospective randomized trial comparing continuous versus intermittent feeding methods in very low birth weight neonates.

OBJECTIVE: To compare the effects of continuous versus intermittent feedings on physical growth, gastrointestinal tolerance, and macronutrient retention in very low birth weight infants ( < 1500 gm). STUDY DESIGN: Very low birth weight neonates stratified by birth weight were randomly assigned to either continuous (24-hour) or intermittent (every 3 hours) nasogastric feedings. Feedings with half-strength Similac Special Care formula were initiated between day 2 and 3 and were advanced isoenergetically to goal. Daily weights, volume/caloric intakes, weekly anthropometric and dynamic skin-fold thickness measurements, and data on feeding milestones and clinical complications were collected. Nitrogen, carbohydrate, and fat balance studies were performed on a subset of male subjects. RESULTS: Eighty-two neonates with birth weights between 750 and 1500 gm who were born between 27 and 34 weeks of gestation were randomly assigned to continuous (n = 42) and intermittent (n = 40) feeding groups. There were no significant differences in baseline demographics and severity of respiratory distress between groups. There were no significant differences in days to regain birth weight, days to full enteral feedings, days to discharge, and discharge anthropometric measurements between continuously fed and intermittently fed infants, both when evaluated together and according to 250 gm weight intervals. Retention rates of nitrogen, fat, total carbohydrate, and lactose were comparable in the continuously fed (n = 17) and intermittently fed (n = 13) male neonates. Very low birth weight neonates who were fed continuously did not have feeding-related complications. CONCLUSION: Very low birth weight infants achieve similar growth and macronutrient retention rates and have comparable lengths of hospital stay whether they are fed with continuous or intermittent feedings.

Body Weight↗

PubMed1996

Prenatal cocaine and neonatal outcome: evaluation of dose-response relationship.

OBJECTIVE: The aim of this study was to evaluate the hypothesis that prenatal cocaine exposure would negatively affect newborn behavior. METHODS: A prospective observational study of term infants recruited from the low-risk nursery used a structured, standardized interview to obtain maternal data. Cocaine exposure was determined by radioimmunoassay of the infant's meconium stool. An examiner blinded to the infant's cocaine status administered the Brazelton Neonatal Behavioral Assessment Scales. RESULTS: The sample was composed of 23 exposed and 29 nonexposed infants. On six of the seven Brazelton Neonatal Behavioral Assessment Scale clusters, cocaine-exposed infants performed less well than control infants, with significant differences observed for autonomic stability. In addition, a dose-response relationship was suggested. Significant negative, within-group relationships were evident in the exposed group, indicating poorer performance with increasing meconium cocaine concentration for orientation (r = -.40) and regulation of state (r = -.40). Regression model testing of the influence of meconium cocaine concentration on neurobehavioral outcomes, after controlling for significant confounders, identified a significant independent, negative effect of meconium cocaine concentration on two clusters-motor and regulation of state. CONCLUSION: In otherwise healthy full-term infants, prenatal cocaine exposure identified by quantitative analysis of cocaine concentration in meconium had a significant, independent negative association with motor and regulation of state that remained after controlling for other significant confounders. A dose-response relationship was evident.

Adult↗

PubMedDate not supplied

Comparison of the effect of two different handling media on rabbit zygote developmental ability.

Despite the attention paid to culture media, the relevance of the handling medium at egg recovery/transfer is frequently overlooked. In the present work, we compare the effect of two different handling media (PBS and HEPES-buffered Ham F10, both supplemented with 20% (v/v) FCS), upon in vitro and in vivo developmental ability of in vivo fertilised rabbit zygotes. Zygotes recovered in HEPES-buffered medium (permanence 1 h as maximum) and subsequently cultured in vitro developed more efficiently to the compacted morula (100%) and blastocyst stage (92%) than those recovered in PBS (83% and 76%, respectively, P < 0.05). Zygotes recovered in such media were then further bilaterally transferred to recipient does following a brief in vitro culture period (for 4 hours). At caesarean section (day 28 of pregnancy), significant differences were observed in both the percentage of pregnant uterine horns (PBS: 60% vs. HEPES-buffered Ham F10: 100%) and live birth rates (PBS: 14% vs. HEPES-buffered Ham F10: 34%). Thus when early rabbit zygotes must be handled, even for short incubation periods, the medium is not innocuous.

Animals↗