Improving phylogenetic resolution of the Lamiales using the complete plastome sequences of six Penstemon species.

The North American endemic genus Penstemon (Mitchell) has a recent geologic origin of ca. 3.6 million years ago (MYA) during the Pliocene/Pleistocene transition and has undergone a rapid adaptive evolutionary radiation with ca. 285 species of perennial forbs and sub-shrubs. Penstemon is divided into six subgenera occupying all North American habitats including the Arctic tundra, Central American tropical forests, alpine meadows, arid deserts, and temperate grasslands. Due to the rapid rate of diversification and speciation, previous phylogenetic studies using individual and concatenated chloroplast sequences have failed to resolve many polytomic clades. We investigated the efficacy of utilizing the plastid genomes (plastomes) of 29 species in the Lamiales order, including five newly sequenced Penstemon plastomes, for analyzing phylogenetic relationships and resolving problematic clades. We compared whole-plastome based phylogenies to phylogenies based on individual gene sequences (matK, ndhF, psaA, psbA, rbcL, rpoC2, and rps2) and concatenated sequences. We also We found that our whole-plastome based phylogeny had higher nodal support than all other phylogenies, which suggests that it provides greater accuracy in describing the hierarchal relationships among taxa as compared to other methods. We found that the genus Penstemon forms a monophyletic clade sister to, but separate from, the Old World taxa of the Plantaginaceae family included in our study. Our whole-plastome based phylogeny also supports the rearrangement of the Scrophulariaceae family and improves resolution of major clades and genera of the Lamiales.

Effect of high-fat and high-carbohydrate diets on pulmonary O2 uptake kinetics during the transition to moderate-intensity exercise.

Mitochondrial pyruvate dehydrogenase (PDH) regulates the delivery of carbohydrate-derived substrate to the mitochondrial tricarboxylic acid cycle and electron transport chain. PDH activity at rest and its activation during exercise is attenuated following high-fat (HFAT) compared with high-carbohydrate (HCHO) diets. Given the reliance on carbohydrate-derived substrate early in transitions to exercise, this study examined the effects of HFAT and HCHO on phase II pulmonary O2 uptake (V̇o2 p) kinetics during transitions into the moderate-intensity (MOD) exercise domain. Eight active adult men underwent dietary manipulations consisting of 6 days of HFAT (73% fat, 22% protein, 5% carbohydrate) followed immediately by 6 days of HCHO (10% fat, 10% protein, 80% carbohydrate); each dietary phase was preceded by a glycogen depletion protocol. Participants performed three MOD transitions from a 20 W cycling baseline to work rate equivalent to 80% of estimated lactate threshold on days 5 and 6 of each diet. Steady-state V̇o2 p was greater (P < 0.05), and respiratory exchange ratio and carbohydrate oxidation rates were lower (P < 0.05) during HFAT. The phase II V̇o2 p time constant (τV̇o2 p) [HFAT 40 ± 16, HCHO 32 ± 19 s (mean ± SD)] and V̇o2 p gain (HFAT 10.3 ± 0.8, HCHO 9.4 ± 0.7 ml·min(-1·)W(-1)) were greater (P < 0.05) in HFAT. The overall adjustment (effective time constant) of muscle deoxygenation (Δ[HHb]) was not different between diets (HFAT 24 ± 4 s, HCHO 23 ± 4 s), which coupled with a slower τV̇o2 p, indicates a slowed microvascular blood flow response. These results suggest that the slower V̇o2 p kinetics associated with HFAT are consistent with inhibition and slower activation of PDH, a lower rate of pyruvate production, and/or attenuated microvascular blood flow and O2 delivery.

Continuous regional anaesthesia provides effective pain management and reduces opioid requirement following major lower limb amputation.

OBJECTIVES: Postoperative stump pain after major lower limb amputation is a significant impediment to the recovery of amputees. The vast majority of patients require opioid analgesics following surgery, which are associated with opioid-related side-effects. Here, we investigate whether intraoperative placement of a peripheral nerve stump catheter followed by continuous infusion of local anesthetic is as effective at pain control as current analgesic practices. If beneficial, this procedure could potentially reduce post-amputation opioid consumption and opioid-related adverse effects. METHODS: A retrospective chart review was conducted of 198 patients over a 4-year period who had undergone a major lower limb amputation for indications related to peripheral vascular disease. Postoperatively, 102 patients received a perineural catheter were compared to 96 patients who did not. The primary outcomes of this study were the amount of morphine equivalents used in the first 72 hours postoperatively and postoperative pain intensity in the first 24 hours. RESULTS: A total of 198 lower-limb amputations were selected for analyses. Multiple regression analyses indicated that perineural catheter use was associated with a lower cumulative postoperative opioid consumption over the first 72 hours but not postoperative pain scores at 24 hours. Perineural catheter use led to a 40% reduction in opioid use during the first 72 hours postoperatively. Mixed model repeated measures analysis demonstrated that this opioid reduction was consistent over time. Other variables related to total opioid use included age, pre-surgical chronic pain, pre-surgical opioid use, patient-controlled analgesia. CONCLUSIONS: Continuous perineural infusions of local anesthetic are a safe and effective method for reducing post-amputation opioid analgesic medications after major lower limp amputation.

Holding bovine oocytes in the absence of maturation inhibitors: kinetics of in vitro maturation and effect on blastocyst development after in vitro fertilization.

Holding immature oocytes before maturation simplifies the transport of oocytes and aids in scheduling later manipulations. We examined the effect of holding bovine oocytes in the absence of meiotic inhibitors on their subsequent meiotic and developmental competence. Oocytes were matured immediately after recovery (control) or were held in a mixture of 40% TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts, and 20% FBS, at room temperature for 16 to 18 h (EH-held) and then matured. Chromatin status was determined at 0, 10, 14, 18, and 22 h of maturation culture. Oocytes were fertilized in vitro after either 18 or 22-24h maturation. The EH treatment maintained oocytes at the germinal vesicle stage (79.3%, vs. 87.7% for control oocytes at 0 h; P>0.05). Upon culture, held oocytes matured more quickly than did control oocytes. The proportions of mature oocytes were not significantly different between groups at 18 h (EH-held, 80.6% and control, 79.3%); however, after 22 h significantly more EH-held than control oocytes had degenerated (24.1% vs. 4.5%, P<0.0001). Blastocyst development was similar between groups for oocytes fertilized after 18 h maturation (EH-held, 29.6% and control, 27.8%). When oocytes were fertilized after 22-24h maturation, EH-held oocytes yielded lower blastocyst development than did control oocytes (16.5% vs. 29.3%, P<0.05). In conclusion, bovine oocytes may be effectively held in the EH treatment before maturation without adversely affecting meiotic or developmental competence. However, holding affects the kinetics of maturation and this must be taken into account when subsequent manipulations are performed.

Denture cream: an unusual source of excess zinc, leading to hypocupremia and neurologic disease.

BACKGROUND: Chronic, excess zinc intake can result in copper deficiency and profound neurologic disease. However, when hyperzincemia is identified, the source often remains elusive. We identified four patients, one previously reported, with various neurologic abnormalities in the setting of hypocupremia and hyperzincemia. Each of these patients wore dentures and used very large amounts of denture cream chronically. OBJECTIVE: To determine zinc concentration in the denture creams used by the patients as a possible source of excess zinc ingestion. METHODS: Detailed clinical and laboratory data for each patient were compiled. Tubes of denture adhesives were analyzed for zinc content using dynamic reaction cell-inductively coupled plasma-mass spectrometry. Patients received copper supplementation. Copper and zinc levels were obtained post-treatment at varying intervals. RESULTS: Zinc concentrations ranging from about 17,000 to 34,000 mug/g were identified in Fixodent and Poli-Grip denture creams. Serum zinc levels improved in three patients following cessation of denture cream use. Copper supplementation resulted in mild neurologic improvement in two patients who stopped using denture cream. No alternative source of excess zinc ingestion or explanation for hypocupremia was identified. CONCLUSION: Denture cream contains zinc, and chronic excessive use may result in hypocupremia and serious neurologic disease.

Transport of equine ovaries for assisted reproduction.

Use of assisted reproduction to obtain foals from valuable mares post-mortem typically necessitates holding of ovaries during shipment to a laboratory. The present study evaluated whether holding ovaries briefly at a warm ( approximately 30 degrees C) temperature improves meiotic and developmental competence of oocytes, as determined after maturation in vitro and intracytoplasmic sperm injection. Ovaries were packaged in pairs in insulated containers, and held either at 24 or 25-35 degrees C for 4h, followed by cooling. Ovaries in both treatments were held for either a short (mean, 7-7.4h) or long (mean, 20.6-20.7h) duration before oocyte recovery. Control ovaries were collected en masse at the abattoir. The ovary temperature in this treatment slowly decreased to approximately 27 degrees C; oocyte recovery was performed after 3.5-7h total holding. There was no effect of temperature on oocyte meiotic or developmental competence within either treatment time period. Oocytes in the short duration holding group had similar meiotic competence to controls, but had a significantly decreased rate (P<0.05) of blastocyst development. Oocytes in the long duration holding group had decreased (P<0.05) meiotic competence and blastocyst development compared to controls. These findings indicate that storage of equine ovaries for only 7h may decrease blastocyst development, and that longer storage reduces both rate of oocyte maturation and blastocyst development. Further work is needed to determine if there is a critical time before 7h post-mortem by which equine oocytes should be recovered to maximize developmental competence.

Teaching teamwork during the Neonatal Resuscitation Program: a randomized trial.

OBJECTIVE: To add a team training and human error curriculum to the Neonatal Resuscitation Program (NRP) and measure its effect on teamwork. We hypothesized that teams that received the new course would exhibit more teamwork behaviors than those in the standard NRP course. STUDY DESIGN: Interns were randomized to receive NRP with team training or standard NRP, then video recorded when they performed simulated resuscitations at the end of the day-long course. Outcomes were assessed by observers blinded to study arm allocation and included the frequency or duration of six team behaviors: inquiry, information sharing, assertion, evaluation of plans, workload management and vigilance. RESULT: The interns in the NRP with team training group exhibited more frequent team behaviors (number of episodes per minute (95% CI)) than interns in the control group: information sharing 1.06 (0.24, 1.17) vs 0.13 (0.00, 0.43); inquiry 0.35 (0.11, 0.42) vs 0.09 (0.00, 0.10); assertion 1.80 (1.21, 2.25) vs 0.64 (0.26, 0.91); and any team behavior 3.34 (2.26, 4.11) vs 1.03 (0.48, 1.30) (P-values <0.008 for all comparisons). Vigilance and workload management were practiced throughout the entire simulated code by nearly all the teams in the NRP with team training group (100% for vigilance and 88% for workload management) vs only 53 and 20% of the teams in the standard NRP. No difference was detected in the frequency of evaluation of plans. CONCLUSION: Compared with the standard NRP, NRP with a teamwork and human error curriculum led interns to exhibit more team behaviors during simulated resuscitations.

Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence.

The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured, fertilized and cultured in groups at 5microl medium per embryo. In Experiment 3, oocytes were held in the EH treatment, then were matured and fertilized. In Study 3.1, injected oocytes were cultured individually in drop sizes as for Experiment 1; in Study 3.2, groups of 2-7 oocytes were cultured in fixed drop sizes of 5 or 50microl. Blastocyst development rates of individually-cultured embryos were not significantly different among drop sizes in either Experiment 1 or 3 (15-29%). In Experiment 2, blastocyst rates were not significantly different between holding treatments (17-23%). In Experiment 3, for group-cultured oocytes, blastocyst development was not significantly different between 5 and 50microl drops (39 and 27%, respectively). In conclusion, compact-cumulus oocytes may be effectively held in the EH treatment before maturation, and single culture of equine embryos yields acceptable blastocyst development. The greatest blastocyst rate (39%) was obtained with group culture in a 5microl droplet.

Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation.

This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In experiment 2, oocytes were either fixed immediately after recovery, or were cultured for 18 h in the presence or absence of roscovitine. Oocytes cultured in the absence of roscovitine had a significantly higher rate of meiotic resumption (18%) than was found in the other two treatments (0). In experiment 3, oocytes were matured immediately or after 16-18 h culture with roscovitine. Maturation rates were similar between oocytes previously treated with roscovitine (22%) and control oocytes (19%). Mature oocytes were fertilized by intracytoplasmic sperm injection and then cultured, with or without oviductal epithelial cells, for 7.5 days. There was no significant effect of roscovitine treatment on blastocyst development. Development to blastocyst of roscovitine-treated oocytes in DMEM/F-12 + co-culture (37%) was significantly higher than that of control oocytes in DMEM/F-12 without co-culture (14%). These data indicate that equine oocytes with compact cumuli can be held in roscovitine before maturation without any harmful effect on blastocyst formation.

Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection.

Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not different among treatments. In Experiment 2, oocytes were treated as in Experiment 1, but embryos were cultured for 7.5d. Blastocyst development was significantly lower in the cycloheximide-treated group than in controls (7% versus 30%) with the room-temperature group intermediate (16%). In Experiment 3, oocytes were cultured at 38.2 degrees C in medium containing roscovitine, or were held at room temperature in sealed glass vials in a mixture of 40% M199 with Earle's salts, 40% M199 with Hanks' salts, and 20% FBS (EH treatment) for 16-18 h, before maturation, sperm injection, and embryo culture for 7.5d. Blastocyst development of oocytes in the EH treatment was significantly higher than that for roscovitine-treated oocytes (34% versus 12%), but not significantly different from that for controls (25%). Oocytes in the EH treatment did not mature during holding (70% germinal vesicle stage after 18 h holding). Whereas culture with cycloheximide or roscovitine of equine oocytes with expanded cumuli reduced subsequent blastocyst formation, these oocytes could be held in a modified M199 at room temperature overnight without adverse affecting meiotic or developmental competence.

The Canadian National Outcomes Measurement Study in Schizophrenia: overview of the patient sample and methodology.

OBJECTIVE: The Canadian National Outcomes Measurement Study in Schizophrenia (CNOMSS) is a prospective survey of routine clinical practice. METHOD: Patients with schizophrenia or a related disorder were consecutively enrolled from all regions of Canada. Both academic and community psychiatric clinics were included and patients were followed up for 2 years. Clinical and functional status, quality of life, medication and economic costs were assessed at enrollment and monitored throughout the follow-up period. RESULTS: Patients attending an academic clinic tended to be younger and more severely ill than those from community clinics. Both types of sites prescribed atypical neuroleptics to more than three-quarters of the patients. The majority of those enrolled were unemployed and living in poverty. Poor clinical status was associated with poverty. CONCLUSION: The CNOMSS provides demographic, clinical and treatment-related information about a large Canada-wide sample of psychiatric patients. The following three articles in this issue of Acta Psychiatrica Scandinavica explore issues related to medication, quality of life and resource utilization.

Medication decisions and clinical outcomes in the Canadian National Outcomes Measurement Study in Schizophrenia.

OBJECTIVE: To evaluate over a 2-year period, patients from academic/non-academic centres, from each region of Canada, to determine whether location or other variables such as medication type, gender or income was associated with outcome as defined by non-hospitalization and persistence on original treatment. METHOD: A total of 448 patients were recruited from academic and non-academic centres across all provinces of Canada and followed up for 2 years. RESULTS: Patients from British Columbia had significantly lower rates of hospitalization than patients from other provinces. Male patients showed greater symptomatic improvement at 2 years from initial assessment compared to females. Patients on clozapine, risperidone and olanzapine were least likely to be hospitalized. CONCLUSION: There were some regional differences noted in both utilization of types of antipsychotic medications and hospitalization rates. In this sample of stable out-patients over 70% who started on monotherapy with clozapine, risperidone, olanzapine and quetiapine remained on the same medication over the 2-year study period.

Resource utilization in a Canadian national study of people with schizophrenia and related psychotic disorders.

OBJECTIVE: To determine how the use of the newer, so called atypical antipsychotic medications, effects the pharmacoeconomic treatment burden of schizophrenia and related conditions and to provide a clear comparison of the costs and risks associated with these atypical drugs. METHOD: In this 2-year, open-label, prospective study, resource utilization (RU) data were collected on 160 patients with these conditions. A comparison between risks and costs was performed by combining the generalized CNOMSS data on both economic factors and risk assessments. RESULTS: The main findings of the study were that the total adjusted 1- and 2-year costs were lowest for quetiapine. Drug acquisition costs were lowest for risperidone for both the 1- and 2-year cohorts. Clozapine use was predictably associated with the highest overall and medication costs at both 1 and 2 years. CONCLUSION: Treatment with risperidone or quetiapine was associated with the lowest overall costs when compared with olanzapine or clozapine.

Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence.

We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the fibrillar and intermediate configurations decreased significantly, and the proportions of degenerating and homogeneously fluorescent configurations increased. When cultured, 20% of oocytes classified as having fibrillar chromatin resumed meiosis, whereas 82% of intermediate and 81% to 86% of condensed chromatin oocytes did so. Meiotic resumption was higher in oocytes recovered immediately after slaughter, but these oocytes took longer to mature. Duration of maturation significantly affected blastocyst development rates in Cp oocytes recovered after a delay (13% and 38% for oocytes matured 24 and 36 h, respectively). Oocytes recovered after a delay had higher blastocyst development rates than did those collected immediately after slaughter. We conclude that the fibrillar and intermediate chromatin configurations may degenerate during ovary storage, resulting in decreased maturation rates, especially of Cp oocytes. Time of oocyte recovery and duration of maturation significantly affect the rate of blastocyst development. Oocytes with Cp and Ex cumuli have similar developmental competence to the blastocyst stage.

Ammonia emission and deposition in Scotland and its potential environmental impacts.

The main source of atmospheric ammonia (NH3) in Scotland is livestock agriculture, which accounts for 85% of emissions. The local magnitude of emissions therefore depends on livestock density, type, and management, with major differences occurring in various parts of Scotland. Local differences in agricultural activities therefore result in a wide range of NH3 emissions, ranging from less than 0.2 kg N ha(-1) year(-1) in remote areas of the Scottish Highlands to over 100 kg N ha(-1) year-1 in areas with intensive poultry farming. Scotland can be divided loosely into upland and lowland areas, with NH3 emission being less than and more than 5 kg N ha(-1) year(-1), respectively. Many semi-natural ecosystems in Scotland are vulnerable to nitrogen deposition, including bogs, moorlands, and the woodland ground flora. Because NH3 emissions occur in the rural environment, the local deposition to sensitive ecosystems may be large, making it essential to assess the spatial distribution of NH3 emissions and deposition. A spatial model is applied here to map NH3 emissions and these estimates are applied in atmospheric dispersion and deposition models to estimate atmospheric concentrations of NH3 and NH4+, dry deposition of NH3, and wet deposition of NHx. Although there is a high level of local variability, modelled NH3 concentrations show good agreement with the National Ammonia Monitoring Network, while wet deposition is largest at high altitude sites in the south and west of Scotland. Comparison of the modelled NHx deposition fields with estimated thresholds for environmental effects ("critical loads") shows that thresholds are exceeded across most of lowland Scotland and the Southern Uplands. Only in the cleanest parts of the north and west is nitrogen deposition not a cause for concern. Given that the most intense effects occur within a few kilometres of sources, it is suggested that local spatial abatement policies would be a useful complement to traditional policies that mitigate environmental effects based on emission reduction technologies.

Concentrations of ammonia and nitrogen dioxide at roadside verges, and their contribution to nitrogen deposition.

Bimonthly integrated measurements of NO2 and NH3 have been made over one year at distances up to 10 m away from the edges of roads across Scotland, using a stratified sampling scheme in terms of road traffic density and background N deposition. The rate of decrease in gas concentrations away from the edge of the roads was rapid, with concentrations falling by 90% within the first 10 m for NH3 and the first 15 m for NO2. The longer transport distance for NO2 reflects the production of secondary NO2 from reaction of emitted NO and O3. Concentrations above the background, estimated at the edge of the traffic lane, were linearly proportional to traffic density for NH3 (microg NH3 m(-3) = 1 x 10(-4) x numbers of cars per day), reflecting emissions from three-way catalysts. For NO2, where emissions depend strongly on vehicle type and fuel, traffic density was calculated in terms of 'car equivalents'; NO2 concentrations at the edge of the traffic lane were proportional to the number of car equivalents (microg NO2 m(-3) = 1 x 10(-4) x numbers of car equivalents per day). Although absolute concentrations (microg m(-3)) of NH3 were five times smaller than for NO2, the greater deposition velocity for NH3 to vegetation means that approximately equivalent amounts of dry N deposition to road side vegetation from vehicle emissions comes from NH3 and NO2. Depending on traffic density, the additional N deposition attributable to vehicle exhaust gases is between 1 and 15 kg N ha(-1) y(-1) at the edge of the vehicle lane, falling to 0.2-10 kg N ha(-1) y(-1) at 10 m from the edge of the road.

Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection.

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57-59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55-61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

Effect of holding at room temperature on initial chromatin configuration and in vitro maturation rate of equine oocytes.

The relationship of holding time in media at room temperature (approximately 22 degrees C) to initial chromatin configuration and rate of in vitro maturation (IVM) of equine oocytes was determined. Only oocytes having a complete, compact cumulus were used in this study. Oocytes were removed from ovaries 3.5-8 h after slaughter and were put into one of four treatment groups: (1) immediate/fix (IF) = immediate fixation following removal from the ovary; (2) delay/fix (DF) = fixation after oocytes were held 1-4 h in medium at room temperature; (3) immediate/mature (IM) = immediate placement into maturation medium at 5% CO2 at 38.2 degrees C; and (4) delay/mature (DM) = placement into maturation medium at 5% CO2 at 38.2 degrees C after oocytes were held 1-4 h in medium at room temperature. Chromatin configurations in fixed oocytes were classified as fluorescent nucleus (FN), condensed chromatin (CC), fibrillar, intermediate, or fibrous germinal vesicle (GV). Other classifications were Metaphase I, II, or degenerating/abnormal. Oocytes held at room temperature before fixation (DF) had a lower proportion of oocytes in the fibrous GV, fibrillar and intermediate configurations than did oocytes fixed immediately (IF; 1/54, 2% versus 15/51, 30%, respectively, P < 0.001). Oocytes held before fixation tended to have a higher percentage of both the CC and FN configurations than did oocytes fixed immediately (CC: 22/40, 55% versus 11/36, 31%, respectively, P = 0.056; FN: 17/40, 43% versus 10/36, 28%, respectively, P = 0.066). Holding of oocytes did not affect the rate of resumption of meiosis or the rate of degeneration in culture; however, of oocytes resuming meiosis, more oocytes in the delayed than in the immediate maturation group had reached MII by 24h culture (14/15, 93% versus 8/15, 53%, respectively, P = 0.018).

Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa.

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.