Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation

In vitro penetration (IVP) of swine oocytes by homologous spermatozoa was evaluated in two experiments using four boars as semen donors. In experiment 1, the IVP rate and the number of penetrating spermatozoa (PSP) were compared using three co-incubation systems for vitrified oocytes and fresh sperm: (1) 35 mL petri dishes in a CO₂ incubator, (2) 35 mL petri dishes in bags (submarine system) and (3) glass flasks partially submerged in water bath with the same gas mixture used for the bag system. Mean PSP was 8.2 ± 10.1 and the IVP rate was 90.5%. The PSP differed across all systems (P = 0.0006): 15.5 ± 0.5 for flasks, 6.3 ± 0.4 for CO₂, and 3.9 ± 0.4 for bags. The IVP rate for flasks (95.0%) was greater (P = 0.01) than for CO₂ and bags (90.8% and 85.0%, respectively), but it did not differ between flasks and CO₂ for three boars (P > 0.05). In experiment 2, co-incubation was done as described for glass flasks in experiment 1. The IVP rate and PSP were compared for cryopreserved oocytes: either vitrified in open pulled straws (OPS), or frozen in cryotubes. Mean PSP was 5.4 ± 6.5 and IVP rate was 89.6%. Both PSP and IVP rate were greater (P < 0.0001) for oocytes frozen in cryotubes (7.0 ± 0.3% and 95.8%, respectively) than those frozen in OPS (3.7 ± 0.3% and 83.4%, respectively), with no differences found for three boars (P > 0.05). In summary, successful IVP of swine oocytes by homologous spermatozoa can be achieved using gametes incubated in glass flasks and oocytes frozen in cryotubes.     

Betacarotene supplementation increases ovulation rate without an increment in LH secretion in cyclic goats

This study aimed to evaluate the effects of betacarotene (BC) supplementation on ovulation rate (OR) and luteinizing hormone (LH) secretion in adult goats during the breeding season. Additionally, total ovarian activity (TOA) comprising the total number of ultrasonographically detectable antral follicles (AF) and corpora lutea (OR) was also assessed. In early October, adult goats [n = 22, 3.5 years of age, 7/8 Sannen-Alpine; 26°N, 103°W at 1117 m.a.s.l.] were randomly assigned to: (i) BC group (BCG), orally supplemented with 50 mg of BC/goat/day [n = 10; live weight (LW) = 45.9 ± 2.0 kg, body condition score (BCS; range: 0-emaciated to 5-obese) = 3.0 ± 0.1], and (ii) control group (CONT) [n = 12; LW = 46.2 ± 2.0 kg, BCS = 3.0 ± 0.1]. All animals received a basal diet of alfalfa hay, corn silage and corn grain, with free access to water and mineral salts. The whole experimental period spanned 34 days before and 17 days after ovulation. On day 23 of the experiment, estrus was synchronized with progestin-releasing intravaginal sponges; 36 h prior to estrus, an intensive blood sampling (every 15 min for 6 h) was performed to determine mean LH concentrations, pulsatility (LH-PULSE) and area under the curve (LH-AUC) for serial LH concentrations. Afterwards, by the end of the luteal phase (i.e., 17 days after the onset of estrus), an ultrasonographic scanning was performed to evaluate OR and TOA [AF + OR]. The average LW and BCS did not differ (p > 0.05) during the experimental period. BC-supplemented goats showed an increase in OR (3.4 ± 0.2 versus 2.8 ± 0.2; p < 0.05) and exhibited lower (p < 0.05) serum LH concentrations, LH-AUC and LH-PULSE compared to CONT. A positive correlation was recorded between OR and LW (r² = 0.42, p < 0.05) and BCS (r² = 0.47, p < 0.05). In addition, AF (5.0 ± 0.6 versus 3.4 ± 0.6) and TOA (8.4 ± 0.6 versus 6.2 ± 0.6) were greater (p < 0.05) in the BC-supplemented group than CONT. Supplementation with BC enhanced ovarian follicular development and ovulation rate in adult female goats under decreased photoperiods through LHRH-independant pathways or direct effects of BC on ovarian function.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development,cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR^®, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg ± 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Assessment of two thawing processes of cryopreserved human sperm in pellets

In this study, we evaluated the effects of the thawing methodology on sperm function after cryopreservation in pellets. We compared the use of two thawing procedures: method (1) maintaining pellet for 10 min in air at room temperature, then another 10-min period in air at 37 °C followed by dilution in a thawing medium; and method (2) immersing the pellets directly in thawing medium at 37 °C for 20 min. This procedure leads to a higher rate of temperature increase and a dilution of the glycerol present in the freezing medium. We analyzed the effect of the thawing procedure on sperm motility, viability, membrane lipid packing disorder, acrosome status, reactive oxygen species (ROS) level and sperm chromatin condensation. This study revealed a positive effect of the M2 thawing methodology on sperm parameters. The percentage of spermatozoa with fast-linear movement is increased (M1: 17.26% vs. M2: 28.05%, p < 0.01), with higher viability (M1: 37.81% vs. M2: 40.15%, p < 0.01) and less acrosome damage (M1: 40.44% vs. M2: 35.45%, p = 0.02). We also detected an increase in the percentage of viable spermatozoa with low membrane lipid disorder (M1: 31.36% vs. M2: 33.17%, p = 0.03) and a reduction in chromatin condensation (44.62 vs. 46.62 arbitrary units, p = 0.02). Further studies will be necessary to evaluate the possible clinical applications.     

Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes

A critical stage in the optimization of in vitro maturation (IVM) is the selection of good quality oocytes. There exists a relationship between the size of the cumulus investment and the in vitro developmental ability of the cumulus–oocyte complex (COC), which provides a basis for the selection of the COCs. This study was designed to evaluate the effect of the number of cumulus cell layers which enclose the oocytes, on the in vitro maturation, cytoplasm quality and cumulus expansion of the ovine oocytes. Ovaries were obtained from an abattoir and transported to the laboratory within 1–2 h, at 37 °C. Oocytes (n = 535) were recovered by means of an aspiration pump (set at a flow rate of 10 mL H₂O/min), with a disposable 20 G needle attached. Oocytes were divided into four classes (classes I to IV – with more than 5, 3–4, 1–2 and no cumulus cell layers, respectively) and separately cultured in a TCM199 medium for 24 h. The morphology of oocytes was evaluated following in vitro culture (IVC) to assess cumulus expansion, cytoplasm quality (score I with a homogenous cytoplasm and II with granulated cytoplasm) and nuclear maturation stage. The percentage of maximum cumulus expansion for classes I to III oocytes were 53.0 ± 1.0, 36.3 ± 2.2 and 16.3 ± 1.8% respectively. The rate of meiotic resumption of oocytes in classes I to IV were 77.0 ± 2.7, 77.2 ± 1.9, 53.0 ± 2.1 and 2.7 ± 1.1% respectively. The proportion of oocytes with a cytoplasm quality I in oocyte classes I to IV were 62.8 ± 1.5, 59.4 ± 1.2, 36.4 ± 2.1 and 0.5 ± 1.1%, respectively. Results showed that the presence of ≥3 cumulus cell layers in the COC prior to IVM led to a better (p < 0.05) cumulus expansion, meiotic resumption and cytoplasmic maturation rate. Thus the morphological grading of immature ovine oocytes may be an appropriate selection criterion regarding their developmental ability.     

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p = 0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6–38.0%) and the frequency of genetically normal/balanced gametes (34.3–62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R = 0.4524, p = 0.2604; AB: R = 0.5238, p = 0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.     

Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity – Correlation with body mass index

Irisin was recently identified as cleavage product of fibronectin type III domain containing 5 (FNDC5) and shown to increase energy expenditure in mice and humans and therefore was discussed as potential treatment option in obesity. However, the regulation of irisin under conditions of severely altered body weight such as anorexia nervosa and obesity remains to be investigated. We analyzed circulating irisin levels over a broad spectrum of body weight in 40 patients with anorexia nervosa (mean body mass index, BMI 12.6 ± 0.7 kg/m²), normal weight controls (22.6 ± 0.9 kg/m²) and obese patients with BMI of 30–40 (36.9 ± 1.2 kg/m²), 40–50 (44.9 ± 1.1 kg/m²) and >50 (70.1 ± 2.7 kg/m², n = 8/group). Correlation analyses were performed between irisin and different body indices, parameters of body composition and hormones involved in various homeostatic processes. Obese patients showed higher circulating irisin levels compared to normal weight and anorexic patients (p < 0.05) resulting in a correlation of irisin with body weight (r = 0.47, p < 0.01) and BMI (r = 0.50, p < 0.001). Plasma irisin was also positively correlated with fat mass (r = 0.48, p < 0.01), body cell mass (r = 0.45, p < 0.01) and fat free mass (r = 0.40, p < 0.05). Insulin levels were positively correlated with irisin (r = 0.45, p < 0.01), whereas circulating ghrelin, cortisol, thyroid-stimulating hormone or C-reactive protein were not (p > 0.05). These data indicate that circulating irisin is affected under conditions of altered BMI with highest levels in severely obese patients. The increase of irisin under conditions of obesity may indicate a physiological function to improve glucose tolerance which is often impaired in obese subjects.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Serum sialic acid changes in non-insulin-dependant diabetes mellitus (NIDDM) patients following bitter melon (Momordica charantia) and rosiglitazone (Avandia) treatment

Diabetes mellitus is associated with an increase in sialic acid concentration along with other complications. Sialic acid changes in NIDDM patients were investigated following bitter melon (55 ml/24 h) and rosiglitazone (4 mg/24 h) treatment. A total of 25 patients of both sexes were used in each experimental group. Patients following bitter melon treatment showed no significant difference of serum sialic acid (57.95±4.90 vs. 57.6±5.56 mg/dl, p=0.17) and serum glucose concentration (93.7±9.63 vs. 88.35±6.31 mg/dl, p=0.78) as compared to control subjects. However, the concentration of total cholesterol was significantly high in these patients as compared to control subjects (192±14.23 vs. 170.6±15.1 mg/dl, p<0.03) but within normal range (160–200 mg/dl), suggesting the significant hypoglycemic and lipid-lowering properties of bitter melon. The patients following rosiglitazone treatment showed a significant increase of serum sialic acid concentration (60.2±5.80 vs. 57.6±5.56 mg/dl, p=0.01) along with glucose (112±6.2 vs. 88.35±6.31 mg/dl, p<0.04) and total cholesterol concentration (216.45±20.2 vs. 170.6±15.1 mg/dl, p<0.01) as compared to control subjects. In addition six of the patients had retinopathy, two of whom were suffering also from myocardial infarction and they still had a higher serum sialic acid (61.05±1.20 mg/dl), glucose (187±2.11 mg/dl), total cholesterol (239.10±5.04 mg/dl) and triglyceride (183±4.14 mg/dl) concentration, indicating a poor response of these patients to rosiglitazone. Comparison of serum sialic acid concentration of patients, following bitter melon and rosiglitazone treatment revealed no significant difference but the study showed that bitter melon could be more effective in the management of diabetes and its related complications as compared to rosiglitazone.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Fire service instructor's undergarment choice to reduce Interleukin-6 and minimise physiological and perceptual strain

Fire Service Instructors frequently experience high levels of physiological and perceptual strain during live fire exposures. Instructors are also at risk of cardiovascular illnesses, with cardiac death being the greatest cause of fire fighter death. Current practice for UK instructors is to select undergarment type based on personal preference, between a boiler suit (BOILER) and a wicking base layer (WBL). Research suggests that shorts and t-shirt (SHORTS) may also be a beneficial alternative undergarment choice. The UK South East Fire Service requested an investigation to identify if undergarment selection can lessen the strain experienced by instructors, and reduce the acute inflammatory response to fire exposures. Eight males completed three 45 min sessions in a heat chamber (49.5±1.4 °C and 16.9±4.3% RH) whilst performing intermittent walking. At the end of heat exposure change in heart rate was not effected by garment type (p=0.061, ηp²=0.373). Change in rectal temperature was different between garments (p=0.009, η_p²=0.271), with trends suggesting that BOILER resulted in a greater change (1.03±0.60 °C) than SHORTS (0.76±0.37 °C, p=0.589, d=0.21) and WBL (0.72±0.33 °C, p=0.545, d=0.25). Interleukin-6 post exposure was greater for BOILER (6.96±0.28 pg mL⁻¹) than both SHORTS (6.59±0.30 pg mL⁻¹, p=0.043, d=0.42) and WBL (6.45±0.43 pg mL⁻¹, p=0.031, d=0.51). Overall, undergarment type had little impact on physiological or perceptual strain. However, wearing WBL or SHORTS may reduce the inflammatory response, and consequently decrease the risk of cardiovascular events.     

Dogs with Leishmania chagasi infection have semen abnormalities that partially revert during 150 days of Allopurinol and Amphotericin B therapy

The goal of the present study was to characterize the semen quality of dogs naturally infected with Leishmania chagasi, and treated with Allopurinol and Amphotericin B. Eight naturally infected and eight non-infected dogs were selected. Following semen collection, progressive motility, vigor, concentration and sperm morphology were evaluated. The seminal patterns in the treated animals were evaluated at the beginning (d0) and at days 30 (d30), 60 (d60) and 150 (d150) of treatment. The progressive motility at d0 (35.7 ± 22.3%) was less than that of the control group (77.8 ± 7.1%) (P < 0.05). The vigor was similar to the control group throughout the treatment (P > 0.05). The number of sperm/mL, sperm/ejaculate and sperm/kg of body weight was similar among groups (P > 0.05). The percentages of normal spermatozoa of infected and treated animals were similar throughout the treatment and to the control group (69.1 ± 8.7%) at d60 (37.5 ± 11.2%) and d150 (48.3 ± 10.8%) (P > 0.05), but smaller at d0 (22.7 ± 10.5%) and d30 (28.8 ± 15.9%) (P < 0.05). A greater percentage of acrosome damage was observed in the control group (3.1 ± 2.3%) compared to the d60 (0.1 ± 0.2%) (P < 0.05). The infected dogs had a greater percentage of principal piece defects at d60 (37.0 ± 6.3%) than the control group (16.8 ± 7.3%) (P < 0.05); and greater percentages of detached normal heads at d0 (28.7 ± 19.7%) and d30 (18.5 ± 18.5%) than the control group (0.4 ± 0.5%) (P < 0.05). This reduction in semen quality of the infected animals is suggestive of an epididymal dysfunction. Due to this poor semen quality, caution is recommended when using infected male dogs for reproductive purposes.     

Clinical utility of decorin in follicular fluid as a biomarker of oocyte potential

This study investigated the concentration of decorin (DCN) in mature follicular fluid and the existence in the granulosa cells. It also investigated whether DCN is useful as a biomarker for outcomes of assisted reproductive technology (ART). A retrospective cohort study was performed involving 130 oocytes of 88 patients treated with ART because of unexplained infertility. The concentration of DCN in the follicular fluid (F-DCN) was 39.26 ng/ml (median value); it was higher than that in serum. F-DCN of the oocytes fertilized by intracytoplasmic sperm injection (ICSI) was significantly lower than that of oocytes that were not fertilized (33.24 ng/ml vs 40.18 ng/ml; P = 0.043). When a cut-off level of 34.5 ng/ml was set according to the receiver-operating characteristic curve, the fertilization rate of the oocytes from the follicles in which F-DCN was lower than the cut-off level tended to be good compared to that of the oocytes with F-DCN higher than the cut-off level (P = 0.052). DCN is less likely to be produced by the granulosa cells (GCs), because it was not detected in GCs by immunostaining and Western blot analysis. F-DCN has a possibility to be a biomarker indicating the quality of oocytes collected from the corresponding follicle.     

Oviposition model of Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae)

The longevity and fecundity of Cnaphalocrocis medinalis were investigated at temperatures of 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, and 35.0 °C. Adult longevity and fecundity were significantly influenced by temperature. Longevity decreased with increasing temperature: it was highest at 15.0 °C (23.6 ± 3.51 days) and lowest at 35.0 °C (6.4 ± 0.48 days). Fecundity was highest at 25.0 °C (170.5 ± 45.54 eggs) and lowest at 17.5 °C (11.0 ± 3.68 eggs). The oviposition period was longest at 20.0 °C (8.0 ± 1.09 days) and shortest at 35.0 °C (2.3 ± 0.48 days). The oviposition model presented in this study consisted of two reproductive components (total fecundity and age-specific cumulative oviposition rate) and a survival component (age-specific survival rate). The relationship between adult developmental rate and temperature was described by the Lactin 2 model (r² = 0.98, p < 0.0001). The age-specific survival rate was well explained by a sigmoid function (r² = 0.97, p < 0.00001). The age-specific cumulative oviposition rate was best described by the three-parameter Weibull function (r² = 0.99, p < 0.00001). Temperature-dependent fecundity was estimated using the Briere-2 model (r² = 0.94, p < 0.007). Daily egg production of C. medinalis in relation to adult age and temperature was estimated.     

Increased plasma levels of osteopontin are associated with activation of the renin–aldosterone system and with myocardial and coronary microvascular damage in dilated cardiomyopathy

In patients with dilated cardiomyopathy (DCM) abnormal myocardial blood flow (MBF) has been associated with coronary microvascular dysfunction. The aim of this study was to test the hypothesis that osteopontin (OPN) plasma levels could be associated with the activation of the renin–aldosterone system (RAS) in these patients and be involved in mediating myocardial and coronary damage. In 66 patients with idiopathic left ventricular dysfunction of variable severity the plasma levels of OPN were correlated with biomarkers of systemic metabolism, RAS activation, myocardial dysfunction and with clinical indexes of left ventricle (LV) function and perfusion obtained by 2D-echocardiography and PET. As compared to controls, patients showed a significant increase of inflammatory markers (OPN: 508 ± 30.8 ng/ml vs. 426.9 ± 16.4, p < 0.05 and interleukin (IL)-6: 1.71 ± 0.29 pg/ml vs. 0.38 ± 0.03 pg/ml, p < 0.001) and of indexes of cardiac damage. OPN levels were significantly correlated with the extent of microvascular dysfunction (MBF at rest: p = 0.01; during dipyridamole: p = 0.0003) and with plasma renin activity (PRA) (r = 0.26, p = 0.04). Both in patients with milder or more severe LV dysfunction lower MBF values were associated with higher OPN levels and PRA. These results suggest a interdependent role of RAS and vascular inflammation in cardiomyopathy.     

HDL-associated dehydroepiandrosterone fatty acyl esters: Enhancement of vasodilatory effect of HDL

ObjectiveDehydroepiandrosterone (DHEA) and high-density lipoprotein (HDL) are both vascular relaxants. In the circulation, HDL transports DHEA fatty acyl esters (DHEA-FAEs), which are naturally occurring lipophilic derivatives of DHEA. We studied in isolated rat mesenteric arteries whether HDL-associated DHEA-FAE improves the vasodilatory effect of HDL.Methods and resultsTo prepare DHEA-FAE-enriched HDL, we incubated DHEA with human plasma. After incubation, HDL was isolated, purified, and added in cumulative doses (0.1–125 μg/ml) to noradrenaline-precontracted rat arterial rings. DHEA-FAE-enriched HDL caused a dose-dependent relaxation (maximal 43 ± 4%), which was significantly stronger than the effect of HDL from the control incubation without addition of DHEA (25 ± 2%, p < 0.001). When plasma incubation of DHEA was carried out in the presence of lecithin:cholesterol acyltransferase (LCAT) inhibitor, the relaxation response to HDL (25 ± 3%) did not differ from the control HDL (p = 0.98). Pretreatment of the arterial rings with nitric oxide synthase (NOS) antagonist impaired the relaxation response to DHEA-FAE-enriched HDL (43 ± 4% vs. 30 ± 3%, p = 0.008). Similar experiments were performed with 17β-estradiol (E₂). Compared to control HDL, E₂-FAE-enriched HDL induced slightly but non-significantly stronger relaxation.ConclusionsDHEA-FAE-enriched HDL was a stronger vasodilator than native HDL, and vascular relaxation was in part mediated by NOS, suggesting that DHEA-FAE may improve HDL's antiatherogenic function.