Associations of feed efficiency with circulating IGF-1 and leptin, carcass traits and meat quality of lambs.

The aim of this study was to investigate the effects of feed efficiency classifications on live animal measurements, circulating IGF-1 and leptin concentrations, and carcass, non-carcass and meat quality traits of lambs. One-hundred and two lambs approximately 70 days-old with initial live weight of 24.6 ± 3.71 kg (mean ± SD) were individually fed for 56 days to determine residual feed intake (RFI) and residual feed intake and gain (RIG). Lambs were then classified as phenotypically Low-, Medium- or High-RFI and Low-, Medium- or High-RIG phenotypes. Circulating leptin and IGF-1 concentrations were higher in more efficient lambs (Low-RFI or High-RIG). Variation in RFI and RIG did not affect meat redness or tenderness, but High-RIG lambs had darker meat. These findings show that the phenotypically more efficient Low-RFI and High-RIG lambs produced carcasses with similar characteristics and meat quality as the less efficient High-RFI and Low-RIG lambs but have a strategic advantage of lower feed intake to achieve similar production outcomes.

Molecular and cellular effects of insulin-like growth factor-1 and LongR3-IGF-1 on in vitro maturation of bovine oocytes: comparative study.

Addition effects of insulin-like growth factor-1 (IGF-1) and its synthetic analogue insulin-like growth factor-1 recombinant-3 (LongR3-IGF-1) after in vitro maturation (IVM) of cattle cumulus-oocyte complexes (COCs) were compared and evaluated on meiotic progression, apoptosis and profile genes of oocyte competence (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 and IGFBP5), and their respective cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). The 739 COCs (n = 10 pools) of bovine ovaries were collected, selected and matured with IGF-1 (100 ng/mL), LongR3-IGF-1 (100 ng/mL), and in two control groups with 0.1% polyvinyl alcohol (PVA) or 10% fetal bovine serum (FBS), for 22-24 h. The statistical analysis was performed by a linear mixed effects model, ANOVA and Tukey tests. There was no statistical difference between experimental groups taken into account the meiotic progression and apoptosis (P > 0.05). Nevertheless, there were statistical differences (P ≤ 0.05) among FBS, IGF-1 and LongR3-IGF-1 groups for IGFBP4 gene expression, and among PVA, IGF-1 and LongR3-IGF-1 for COX2 gene expression in cumulus cells. Moreover, statistical difference was found for BCL2 gene expression between IGF-1, FBS and PVA groups and for IGFBP4 gene expression between LongR3-IGF-1, PVA and FBS in oocytes. There was no statistical difference between experimental groups for other genes evaluated. These results showed a good performance of IVM of bovine oocytes in the presence of LongR3-IGF-1 and the possibility of replacement of IGF-1 and FBS.

Influence of temperature-humidity index on conception rate of Nelore embryos produced in vitro in northern Brazil.

Considering the high temperatures that the tropical climate provides to most of Brazil and the effects of thermal stress on reproductive processes, the objective of the present study was to analyze, in the warmer months of 2016, conception rates of Nelore bovine embryos in Acre state. For this purpose, oocytes were aspirated (ultrasound-guided follicular aspiration), matured, fertilized with Nelore bull semen, cultured for 6 days, and then the embryos were transferred to crossbred recipients. Pregnancy diagnosis was performed 30 and 60 days after embryo transfer. Meteorological data were obtained at www.inmet.gov.br to generate temperature-humidity index (THI). The data from the conception rates and periods of the year were submitted to the chi-square test at 5% probability to verify independence. Regression analysis was used to verify the relationship between THI and gestation rate. There was a strong relationship between conception rates and THI values, verified by an increase in conception rates as THI values were reduced and a decrease when THI reached the highest value. Our findings demonstrated a negative effect of heat stress in conception rates of crossbred cows in northern Brazil.

Portuguese recommendations for the prevention, diagnosis and management of primary osteoporosis - 2018 update.

BACKGROUND: Advances in osteoporosis (OP)case definition, treatment options, optimal therapy duration and pharmacoeconomic evidence in the national context motivated the Portuguese Society of Rheumatology (SPR) to update the Portuguese recommendations for the diagnosis and management of osteoporosis published in 2007. METHODS: SPR bone diseases' working group organized meetings involving 55 participants (rheumatologists, rheumatology fellows and one OP specialist nurse) to debate and develop the document. First, the working group selected 11 pertinent clinical questions for the diagnosis and management of osteoporosis in standard clinical practice. Then, each question was investigated through literature review and draft recommendations were built through consensus. When insufficient evidence was available, recommendations were based on experts' opinion and on good clinical practice. At two national meetings, the recommendations were discussed and updated. A draft of the recommendations full text was submitted to critical review among the working group and suggestions were incorporated. A final version was circulated among all Portuguese rheumatologists before publication and the level of agreement was anonymously assessed using an online survey. RESULTS: The 2018 SPR recommendations provide comprehensive guidance on osteoporosis prevention, diagnosis, fracture risk assessment, pharmacological treatment initiation, therapy options and duration of treatment, based on the best available evidence. They attained desirable agreement among Portuguese rheumatologists. As more evidence becomes available, periodic revisions will be performed. Target audience and patient population: The target audience for these guidelines includes all clinicians. The target patient population includes adult Portuguese people. Intended use: These recommendations provide general guidance for typical cases. They may not be appropriate in all situations - clinicians are encouraged to consider this information together with updated evidence and their best clinical judgment in individual cases.

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 µm), BL-I (50 µm) and association of drugs (ROS 6.25 µm and BL-I 25 µm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL-I and ROS+BL-I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post-thawing embryos. Embryos from ROS+BL-I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process.

Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 µm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.

Avaliação da migração das células progenitoras após terapia da tendinite equina / Migration evaluation of progenitor cells after treatment of equine tendinitis

A terapia celular vem sendo utilizada com resultados promissores no tratamento da tendinite equina, entretanto ainda existem dúvidas quanto à persistência e ao comportamento dessas células quando implantadas no local da lesão, e quanto à sua migração para outros focos inflamatórios. O objetivo deste estudo foi avaliar a marcação das células-tronco mesenquimais (CTMs) com nanocristal antes e após o implante em lesões tendíneas experimentais do tendão flexor digital superficial (TFDS) de equinos, bem como observar a possibilidade de migração das CTMs marcadas para outro foco de lesão, o membro contralateral do mesmo animal. Para isso, foi realizada a indução de lesão experimental no TFDS em ambos os membros torácicos de cinco equinos e, após sete dias, foram implantadas as CTMs autólogas marcadas com o nanocristal Qtracker 655 em um dos membros dos animais. Após sete dias do implante, foi realizada a biópsia tendínea para posterior avaliação histopatológica, utilizando-se microscopia com fluorescência. Também foi realizado o teste de viabilidade celular antes e após a incubação com o nanocristal. As CTMs marcadas e injetadas no tecido tendíneo mantiveram sua fluorescência sete dias após seu implante, e não ocorreu migração para o membro contralateral. O uso do nanocristal para a marcação das CTMs derivadas da medula óssea equina mostrou-se efetivo pelo fato de essa nanopartícula não ter alterado a viabilidade celular e por ela ter permanecido ativa durante o período implantado...

Cell therapy has been used with promising results in the treatment of equine tendinitis. However, there are still doubts about the persistence and behavior of these cells implanted in the injured tissue and their migration to other inflamed sites. The aim of this study was to evaluate the labeling of mesenchymal stem cells (MSCs) with nanocrystals before and after implantation in experimental tendinitis of the superficial digital flexor tendon (SDFT) of horses, observing the migration possibility of MSCs marked to another lesion, performed on the contralateral limb of the same animal. An experimental lesion was induced in SDFT in both forelimbs of five horses, and after seven days autologous MSCs labeled with Qtracker(r) 655 were implanted in one member of the animals. Tendon biopsy was performed for subsequent histopathological evaluation using fluorescence microscopy seven days after the implant. Cell viability test was also performed before and after incubation with the cell labeling kit. MSCs labeled and injected into the tendon tissue maintained their fluorescence seven days after their implantation and there was no migration to the contralateral limb. The use of nanocrystals for labeling MSCs was effective because it does not alter cell viability and remains active during the experimental period...

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration.

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing.

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from M0 were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5°C under an atmosphere of 5% CO(2) with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 ± 4.8 and 57.3 ± 4.8, respectively, mean ± SEM), or among M0 (62.3 ± 5.7%), M3 (56.9 ± 6.0%), and M6 (66.5 ± 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture.

Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium.

The objective was to evaluate the parthenogenetic activation of domestic cat oocytes. Cumulus-oocyte complexes matured for 36 h were subjected to three protocols of parthenogenetic activation: Group 1 - ionomycin + cycloheximide; Group 2 - ionomycin + roscovitine; and Group 3 - ionomycin + strontium. As a control, a fourth group of oocytes were cultured in the absence of any activation agent. In all groups, embryos were cultured in SOFaa for 72 h after activation and evaluated for activation rate, cleavage, and embryonic development using Hoechst33342. There were no significant differences among the three treated groups for rates of activated oocytes (70.1 +/- 4.3, 75.5 +/- 4.7, and 61.9 +/- 7.2%, for Treatments 1, 2, and 3 respectively; mean +/- SEM), or cleavage (48.1 +/- 5.9, 47.4 +/- 3.8, and 33.3 +/- 6.8%). However, activation and cleavage rates were higher (P < 0.05) than those in the control group (35.5 +/- 6.4 and 11.8 +/- 4.0%). There were no significant differences among treatment groups for proportion of embryos with 2-10 cells, 10-16 cells, and morulas. In the Control group, the embryo production rate was lower (P < 0.05), although the activation rate was high. The authors concluded that all three treatments effectively induced parthenogenetic activation of domestic cat oocytes. However, to optimize the use of strontium and roscovitine, a dose response and the effect of the presence of Ca(++) in the medium requires further study.

Viability and cell cycle analysis of equine fibroblasts cultured in vitro.

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD((R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

Use of a Piezo drill for intracytoplasmic sperm injection into cattle oocytes activated with ionomycin associated with roscovitine.

Intracytoplasmic sperm injection (ICSI) consists of the introduction, by micromanipulation, of a single sperm into the cytoplasm of a mature egg. This technique is particularly advantageous when only a few sperm are available for fertilization, representing an important tool in preserving genetic material, especially from poorly fertile males. The results from ICSI in cattle are very often unsatisfactory and difficult to reproduce. Thus, the goal of this study was to evaluate the effect of the use of a Piezo drill (PD) and oocyte activation with ionomycin + roscovitine (I + R) during ICSI in cattle oocytes. After in vitro maturation (24 h), cumulus complex oocytes were divided into four groups: G1 - the ICSI was performed without the use of a PD and the oocyte was activated with I + R; G2 - the ICSI was performed with the use of the PD and activation with I + R; G3 - the ICSI was performed with the use of the PD, but without activation and G4 - parthenogenetic control, treated with I + R, but without sperm injection. The presumptive zygotes were cultured for 7 days and evaluated on day 3 for cleavage rate and on day 7 for blastocyst formation. Embryo production by standard in vitro fertilization in the laboratory was 78% for cleavage (117/150) and 35% for blastocyst formation (41/150). The cleavage rates obtained in G1, G2 and G4 were similar (66.7%, 71.6% and 66.3%, respectively), demonstrating the beneficial effect of oocyte activation. However, in G3, despite the presence of the sperm and the electric stimulation of a PD, the cleavage rates were significantly lower (17.5%) compared with the groups that used chemical activation, even in the absence of sperm (G4). Despite the beneficial effects of activation, this stimulus alone, or in the absence of the PD, was not sufficient for adequate morulae formation (13.4%, 37.9%, 0.0% and 13.5% for G1, G2, G3 and G4, respectively). Only in G2, when the PD was used followed by artificial activation, blastocysts were obtained (14.7%). These results indicate that cattle oocytes must be activated after ICSI to produce viable embryos.

Ultrastructural characteristics of non-matured and in vitro matured oocytes collected from pre-pubertal and adult domestic cat ovaries.

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4 degrees C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38 degrees C in a humidified environment of 5% O(2), 5% CO(2) and 90% N(2). Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.

Effect of progesterone and ionomycin on domestic cat sperm motility patterns and acrosome reaction.

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 microg/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 microg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mum, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 microm seems to be more suitable to trigger AR in domestic cat sperm.

Aspiration of equine oocytes from immature follicles after treatment with equine pituitary extract (EPE) alone or in combination with hCG.

This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25mg EPE and treated with 2500 IU hCG, (2) superovulation with 25mg EPE, and (3) control (no exogenous treatment). Treatments 7 days after spontaneous ovulation; and all the follicles >10mm were aspirated 24h after the largest follicle achieved a diameter of 27-30 mm for control group, and most follicles reached 22-27 mm for the EPE alone treatment. To the group EPE+hCG, when the follicles reached 22-27 mm, hCG was administered, 24h before OPU. Superovulation increased the number of follicles available for aspiration. The total number of follicles available for aspiration was 61 in the EPE/hCG group, 63 in the EPE group and 42 in the control. The proportion of follicles aspirated varied from 63.5% to 73.8%. Oocyte recovery rate ranged from 15.0% to 16.7% and the proportion of mares that yielded at least one oocyte was 70% (7/10) in the EPE/hCG, 60% (6/10) in the EPE alone and 50% (5/10) in control group. The EPE/hCG treatment had a higher proportion of follicles with expanded granulose cells (64.4%) than the control (3.3%; p<0.05) and the EPE treatment (25.0%). The intervals from spontaneous ovulation to aspiration were similar for all treatments (11-12 days). However, superovulatory treatment significantly increased the aspiration to ovulation interval from 15+/-4 days for control to 27+/-15 days for EPE (p<0.05) and to 23+/-13 days for EPE/hCG treatment with commensurate increases in the time between spontaneous ovulations.

Viability of OPS vitrified sheep embryos after direct transfer.

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Clinical, immunological and histological aspects of an uncommon type II reaction in patients with lepromatous leprosy.

This study reports three cases of an unusual leprotic reaction characterized by superficial bullous ulcerative cutaneous lesions associated with high fever, malaise and oedema in patients with leprosy. Two patients responded to thalidomide treatment, with regression of the symptoms and skin ulcers. The third patient responded to thalidomide plus prednisone. Analysis of the ulcerated skin lesions showed dermal oedema with mononuclear cell infiltrate enriched for gammadelta-positive T lymphocytes and an increased number of Mycobaterium leprae bacilli within capillary endothelium. In contrast, gammadelta+ cells were decreased in or absent from the blood. Tumour necrosis factor-alpha and interleukin-6 were raised in the serum of the patients at the onset of the reaction. After the episode, cytokine levels and the percentage of gammadelta+ cells in the blood returned to normal. These cases characterize an uncommon leprotic reaction with clinical similarities to type II reaction and may indicate a significant role for gammadelta+ T cells in its pathogenesis.

Increased Langerhans cell accumulation after mycobacterial stimuli.

AIMS: To evaluate the role of Langerhans cells (LCs) in the local activation of leprosy lesions. LCs, acting as tolerance inducers and immune stimuli, are dendritic cells recently implicated in cutaneous homeostasis. The role of LCs in the defence against mycobacterial infection remains poorly understood. METHODS AND RESULTS: The number and distribution of CD1a+ skin cells and HLA-DR and intercellular adhesion molecule (ICAM)-1 expression were analysed in leprosy skin lesions and in delayed-type hypersensitivity (DTH) tests. The results showed a high number of LCs in tuberculin and lepromin tests, in tuberculoid lesions and in the epidermis and dermis during type I and II reactions. In multibacillary lesions, however, the number of LCs was consistently low in comparison with other groups. Increased numbers of LCs were accompanied by marked HLA-DR and ICAM-1 expression, suggesting a strong relationship between these immunological events. CONCLUSIONS: CD1a+ cells are implicated in the local immunological events taking place after mycobacterial stimuli and may account for the local activation of all types of reactional episodes in leprosy.