Development of a method to cryopreserve Greenshell mussel™ (Perna canaliculus) veliger larvae

Cryopreservation of larvae of Greenshell™ mussel Perna canaliculus, the most cultivated species in New Zealand, can provide flexibility for selective breeding programmes and enhance its global production. In this study, we set out to develop a reliable protocol for freezing D-stage larvae of Greenshell™ mussels that ensured long-term survival for successful rearing of thawed larvae in the hatchery. The effects of different combinations of cryoprotecting agents (CPA), varying CPA equilibration times, larval concentrations per straw as well as different larval development stages (48 h vs 72 h old) were evaluated by assessing the behavioural response (swimming activity, algal consumption), shell size and survival of larvae, up to 4 days post-thawing. The protocol yielding the best larval performances was a combination of the following CPA (final concentrations): 14% ethylene-glycol (EG) + 0.6 M trehalose (TRE) + 1% polyvinyl-pyrrolidone (PVP), prepared with Milli-Q water. Stocking densities ranging from 50,000 to 150,000 larvae per straw (0.25 mL) and a 20 min equilibration time gave the best results, while no significant differences in fitness were found between larvae cryopreserved at 48 h nor 72 h-old. Using the improved cryopreservation protocol, over 50% of previously cryopreserved D-larvae were able to survive after 4 days of rearing, compared with 65% in the unfrozen control. More importantly, about one third of thawed larvae were able to swim and feed, and to potentially develop further. These findings contribute to enhance the selective breeding programmes for this species.     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Freezing African Elephant Semen as a New Population Management Tool

Background

The captive elephant population is not self-sustaining and with a limited number of breeding bulls, its genetic diversity is in decline. One way to overcome this is to import young and healthy animals from the wild. We introduce here a more sustainable alternative method - importation of semen from wild bulls without removing them from their natural habitat. Due to the logistics involved, the only practical option would be to transport cryopreserved sperm. Despite some early reports on African elephant semen cryopreservation, the utility of this new population management tool has not been evaluated.

Methodology/Principal Findings

Semen was collected by electroejaculation from 14 wild African savanna elephant (Loxodonta africana) bulls and cryopreserved using the directional freezing technique. Sperm treatments evaluated included the need for centrifugation, the use of hen or quail yolk, the concentration of glycerol (3%, 5% or 7%) in the extender, and maintenance of motility over time after thawing. Our results suggest that dilution in an extender containing hen yolk and 7% glycerol after centrifugation best preserved post-thaw sperm motility when compared to all other treatments (P≤0.012 for all). Using this approach we were able to achieve after thawing (mean ± SD) 54.6±3.9% motility, 85.3±2.4% acrosome integrity, and 86.8±4.6% normal morphology with no decrease in motility over 1 h incubation at 37°C. Sperm cryopreserved during this study has already lead to a pregnancy of a captive female elephant following artificial insemination.

Conclusions/Significance

With working techniques for artificial insemination and sperm cryopreservation of both African and Asian elephants in hand, population managers can now enrich captive or isolated wild elephant populations without removing valuable individuals from their natural habitat.     

Development of the Acoustically Evoked Behavioral Response in Larval Plainfin Midshipman Fish,Porichthys notatus

The ontogeny of hearing in fishes has become a major interest among bioacoustics researchers studying fish behavior and sensory ecology. Most fish begin to detect acoustic stimuli during the larval stage which can be important for navigation, predator avoidance and settlement, however relatively little is known about the hearing capabilities of larval fishes. We characterized the acoustically evoked behavioral response (AEBR) in the plainfin midshipman fish, Porichthys notatus, and used this innate startle-like response to characterize this species'' auditory capability during larval development. Age and size of larval midshipman were highly correlated (r² = 0.92). The AEBR was first observed in larvae at 1.4 cm TL. At a size ≥1.8 cm TL, all larvae responded to a broadband stimulus of 154 dB re1 µPa or −15.2 dB re 1 g (z-axis). Lowest AEBR thresholds were 140–150 dB re 1 µPa or −33 to −23 dB re 1 g for frequencies below 225 Hz. Larval fish with size ranges of 1.9–2.4 cm TL had significantly lower best evoked frequencies than the other tested size groups. We also investigated the development of the lateral line organ and its function in mediating the AEBR. The lateral line organ is likely involved in mediating the AEBR but not necessary to evoke the startle-like response. The midshipman auditory and lateral line systems are functional during early development when the larvae are in the nest and the auditory system appears to have similar tuning characteristics throughout all life history stages.     

Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method

This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62±4.7% and 62±4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95±3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56±7.2% and 50±6.8% for implantation rate and 40±7.1% and 35±6.5% for offspring rate at birth); but significantly lower than in the fresh group (78±6.6% for implantation rate and 70±7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44±7.2% and 50±6.8%), but significantly higher than in the fresh group (23±6.6%, P < 0.05). However, fetal losses were similar between groups (10±4.4%, 15±4.8% and 8±4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of €0.05 per device.     

Setting tools for the early assessment of the quality of thawed Pacific oyster (Crassostrea gigas) D-larvae

Parameters used to assess the survival of larvae after cryopreservation generally misestimate the damages that prevent larval development. The objectives of the present study were to 1) define the reliability of the survival rate, assessed at 2 and 7 days post fertilization, to estimate Pacific oyster larval quality after thawing, and 2) select complementary tools allowing an early and reliable estimation of their quality. Oyster larvae were reared for 25 h after fertilization at 19 °C and cryopreserved at early D-stage. Then, thawed larvae were incubated in 2-L beakers. At 2 days post fertilization, the survival rate of thawed Pacific oyster larvae was lower than that of fresh larvae for only one experiment (Experiment 3) among the four identical experiments carried out in this work (Experiments 1-4). By contrast, the survival of thawed larvae, as assessed 7 days after fertilization, was lower than that of fresh larvae for the four experiments. These results confirm that the quality of thawed larvae is lower than that of fresh larvae and that the survival rate, estimated 2 days post fertilization, is not adapted to a reliable estimation of the subsequent development ability of thawed larvae. Then, complementary parameters were tested at 2 days: the movement characteristics (Experiments 1 and 2) and the morphologic features (Experiments 3 and 4) of thawed larvae. Compared to values observed on fresh larvae, the percentage of thawed motile larvae was different for only one experiment (Experiment 2) of the two. Compared to control, a reduced Average Path Velocity (VAP) of larvae (determined at the D-larval stage using a CASA-Computer Assisted Sperm Analysis-system) was observed after thawing for both experiments (Experiments 1 and 2), suggesting the ability of larval movement velocity to assess the decrease of the quality of thawed oyster larvae. Using an ASMA (Automated Sperm Morphology Analysis) device, a lower area of thawed larvae was observed, compared to control and for the two experiments (Experiments 3 and 4). By contrast, the Crofton perimeter of thawed larvae was lower than that of control larvae for only one experiment (Experiment 3) and no significant difference of circularity between fresh and thawed larvae was recorded for Experiments 3 and 4. In conclusion, changes in the movement velocity (assessed by CASA) and in the area (measured by ASMA) of D-larvae allow an early and reliable estimation of the quality of thawed Pacific oyster larvae.     

Alterations of Neuromuscular Function after the World's Most Challenging Mountain Ultra-Marathon

We investigated the physiological consequences of the most challenging mountain ultra-marathon (MUM) in the world: a 330-km trail run with 24000 m of positive and negative elevation change. Neuromuscular fatigue (NMF) was assessed before (Pre-), during (Mid-) and after (Post-) the MUM in experienced ultra-marathon runners (n = 15; finish time  = 122.43 hours ±17.21 hours) and in Pre- and Post- in a control group with a similar level of sleep deprivation (n = 8). Blood markers of muscle inflammation and damage were analyzed at Pre- and Post-. Mean ± SD maximal voluntary contraction force declined significantly at Mid- (−13±17% and −10±16%, P<0.05 for knee extensor, KE, and plantar flexor muscles, PF, respectively), and further decreased at Post- (−24±13% and −26±19%, P<0.01) with alteration of the central activation ratio (−24±24% and −28±34% between Pre- and Post-, P<0.05) in runners whereas these parameters did not change in the control group. Peripheral NMF markers such as 100 Hz doublet (KE: −18±18% and PF: −20±15%, P<0.01) and peak twitch (KE: −33±12%, P<0.001 and PF: −19±14%, P<0.01) were also altered in runners but not in controls. Post-MUM blood concentrations of creatine kinase (3719±3045 Ul·¹), lactate dehydrogenase (1145±511 UI·L⁻¹), C-Reactive Protein (13.1±7.5 mg·L⁻¹) and myoglobin (449.3±338.2 µg·L⁻¹) were higher (P<0.001) than at Pre- in runners but not in controls. Our findings revealed less neuromuscular fatigue, muscle damage and inflammation than in shorter MUMs. In conclusion, paradoxically, such extreme exercise seems to induce a relative muscle preservation process due likely to a protective anticipatory pacing strategy during the first half of MUM and sleep deprivation in the second half.     

Embryonic and Larval Development and Early Behavior in Grass Carp,Ctenopharyngodon idella: Implications for Recruitment in Rivers

With recent findings of grass carp Ctenopharyngodon idella in tributaries of the Great Lakes, information on developmental rate and larval behavior is critical to efforts to assess the potential for establishment within the tributaries of that region. In laboratory experiments, grass carp were spawned and eggs and larvae reared at two temperature treatments, one “cold” and one “warm”, and tracked for developmental rate, egg size, and behavior. Developmental rate was quantified using Yi’s (1988) developmental stages and the cumulative thermal units method. Grass carp had a thermal minimum of 13.5°C for embryonic stages and 13.3°C for larval stages. Egg size was related to temperature and maternal size, with the largest eggs coming from the largest females, and eggs were generally larger in warmer treatments. Young grass carp larvae exhibited upward and downward swimming interspersed with long periods of lying on the bottom. Swimming capacity increased with ontogeny, and larvae were capable of horizontal swimming and position holding with gas bladder emergence. Developmental rates, behavior, and egg attributes can be used in combination with physical parameters of a river to assess the risk that grass carp are capable of reproduction and recruitment in rivers.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.     

Inheritance of Diapause in Crosses between the Northernmost and the Southernmost Strains of the Asian Corn Borer Ostrinia furnacalis

The northernmost Harbin strain (N strain) of the Asian corn borer, Ostrinia furnacalis enters facultative diapause as fully grown larvae in response to short daylengths; whereas the southernmost Ledong strain (S strain) exhibits almost no diapause under the same light conditions. In the present study, we examined the inheritance of diapause induction and termination by crossing the two strains under a range of environmental conditions. The N strain showed a typical long-day response with a critical daylength of approximately15.88 h at 22°C, 15.72 h at 25°C and 15.14 h at 28°C, whereas the S strain showed a weak photoperiodic response at 22°C. The F1 progeny also showed a long-day response at 22, 25 and 28°C. However, the critical daylengths in S ♀ × N ♂ crosses were significantly longer than those in N ♀ × S ♂ crosses, indicating a sex linkage in the inheritance of diapause induction, with the male parent having more influence on the following F1 progeny. The incidence of diapause in S ♀ × N ♂ crosses was the same as in the N strain under short daylengths of 11-13 h, indicating that diapause trait is completely dominant over the non-diapause trait. The critical daylength in backcross to N was significantly longer than it was in backcross to S, showing a grandfather gene effect. Whether the inheritance of diapause fits an additive hypothesis or not was dependent on the rearing photoperiod, and the capacity for diapause was transmitted genetically in the manner of incomplete dominance. The duration of diapause for the reciprocal crosses under different diapause-terminating conditions showed different patterns of inheritance. The results in this study reveal that genetic and genetic-environmental interactions are involved in diapause induction and termination in O. furnacalis.     

Cytochrome oxidase activity during diapause and metamorphosis of the Japanese beetle,Popillia japonica Newman

1. Determinations were made on the activity of cytochrome oxidase of individual Japanese beetles during growth, diapause, and metamorphosis. All readings were made on homogenates at a dilution of 1:1,000, except for adult beetles, when the final dilution was 1:10,000. 2. The activity of the enzyme increased during larval growth from a low value of 0.022 in the second instar, to high values ranging from 0.074 to 0.083 in diapause third instar larvae. 3. The high activity of cytochrome oxidase during larval diapause indicates that this condition may be physiologically different from that occurring in the egg or pupal stages of most other insects. 4. During metamorphosis, the activity of cytochrome oxidase follows the characteristic U-shaped curve associated with respiratory metabolism. It thus appears that most of the oxidation occurring in metamorphosing individuals is mediated through the cytochrome system. 5. The activity of cytochrome oxidase is significantly higher in the adult male than it is in the adult female; the values (calculated on the basis of a 1:1,000 dilution) were 0.40 ± 0.028 and 0.25 ± 0.012, respectively.     

Caffeine Impairs Myocardial Blood Flow Response to Physical Exercise in Patients with Coronary Artery Disease as well as in Age-Matched Controls

Background

Caffeine is one of the most widely consumed pharmacologically active substances. Its acute effect on myocardial blood flow is widely unknown. Our aim was to assess the acute effect of caffeine in a dose corresponding to two cups of coffee on myocardial blood flow (MBF) in coronary artery disease (CAD).

Methodology/Principal Findings

MBF was measured with ¹⁵O-labelled H2O and Positron Emission Tomography (PET) at rest and after supine bicycle exercise in controls (n = 15, mean age 58±13 years) and in CAD patients (n = 15, mean age 61±9 years). In the latter, regional MBF was assessed in segments subtended by stenotic and remote coronary arteries. All measurements were repeated fifty minutes after oral caffeine ingestion (200 mg). Myocardial perfusion reserve (MPR) was calculated as ratio of MBF during bicycle stress divided by MBF at rest. Resting MBF was not affected by caffeine in both groups. Exercise-induced MBF response decreased significantly after caffeine in controls (2.26±0.56 vs. 2.02±0.56, P<0.005), remote (2.40±0.70 vs. 1.78±0.46, P<0.001) and in stenotic segments (1.90±0.41 vs. 1.38±0.30, P<0.001). Caffeine decreased MPR significantly by 14% in controls (P<0.05 vs. baseline). In CAD patients MPR decreased by 18% (P<0.05 vs. baseline) in remote and by 25% in stenotic segments (P<0.01 vs. baseline).

Conclusions

We conclude that caffeine impairs exercise-induced hyperaemic MBF response in patients with CAD to a greater degree than age-matched controls.     

Impact of Untreated Obstructive Sleep Apnea on Left and Right Ventricular Myocardial Function and Effects of CPAP Therapy

Background

Obstructive sleep apnea (OSA) has deteriorating effect on LV function, whereas its impact on RV function is controversial. We aimed to determine the effect of OSA and continuous positive airway pressure (CPAP) treatment on left and right ventricular (LV, RV) function using transthoracic echocardiography (TTE) and 2 dimensional speckle tracking (2D ST) analysis of RV deformation capability.

Methods and Results

82 patients with OSA and need for CPAP therapy were prospectively enrolled and underwent TTE at study inclusion and after 6 months of follow up (FU). Multivariate regression analysis revealed an independent association between baseline apical right ventricular longitudinal strain (RV-Sl), BMI and the severity of OSA (apical RV-Sl: P = 0.0002, BMI: P = 0.02). After CPAP therapy, LV functional parameters (LVEF: P<0.0001, LV performance index: P = 0.03, stroke volume: P = 0.042), and apical RV-Sl (P = 0.001) improved significantly. The effect of CPAP therapy was related to severity of OSA (LVEF: AHI 5–14, 66.4±8.8%, 68.5±10.6% [P = ns]; AHI 15–30∶59.8±7.7%, 68.6±9.3% [P = 0.002]; AHI>30∶54.1±12.4%, 68.2±13.6%[P<0.0001]; apical RV-Sl: AHI 5–14: −17.3±8.7%, −16.0±10.8% [P = ns], AHI 15–30: −9.8±6.0%, −15.4±10.9% [P = 0.028], AHI>30: −6.3±5.7%, −17.9±11.2% [P<0.0001]).

Conclusions

OSA seems to have deteriorating effect on LV and RV function. We found a beneficial effect of CPAP on LV and RV functional parameters predominately in patients with severe OSA. 2D speckle tracking might be of value to determine early changes in global and regional right ventricular function.     

Docosahexaenoic acid in diluent for goat semen cryopreservation

The effects of docosahexaenoic acid (DHA) in the diluent for cryopreservation of goat semen on seminal quality and the optimal levels to be used were evaluated. After collection, semen was pooled and physically evaluated, then divided into four aliquots with different DHA levels in the diluent: 0, 10, 20, and 30 ng mL^-1. The semen was cryopreserved in a TK 3000® freezing machine and then thawed for assessment at 37 °C. Sperm motility and vigor, membrane integrity, acrosomal integrity, mitochondrial activity, and sperm chromatin compaction were evaluated after thawing. A completely randomized design was used. For normally distributed variables, ANOVA and regression analysis were used to test for differences between treatments, and for non-parametric data, the Kruskal Wallis test was used at the 5% significance level. There were no differences among groups in terms of membrane integrity, acrosomal integrity, or chromatin compaction. There was a decrease in class I mitochondrial activity with increasing DHA level (P<0.05), but no differences in classes II, III, and IV (P>0.05). The inclusion of 10 to 30 ng mL^-1 of DHA in the diluent did not result in improvements in seminal quality parameters after thawing, with some impairment observed in the mitochondrial activity of the sperm cells.     

Focus: Allergic Diseases and Type II Immunity: Study of the Correlation between HRCT Semi-quantitative Scoring, Concentration of Alveolar Nitric Oxide,and Clinical-functional Parameters of Systemic Sclerosis-induced Interstitial Lung Disease

Introduction: The correlation between alveolar nitric oxide (CANO) and the severity of interstitial lung disease (ILD) evaluated by high resolution computed tomography (HRCT) has not been well demonstrated. Methods: It was a perspective and observational study, including patients with diagnosed systemic sclerosis (SSc). They performed lung function testing (LFT), exhaled nitric oxide (NO) measurements, exercise testing, chest X-ray, and HRCT. Study patients were divided into SSc with ILD (SSc-ILD+) or without ILD (SSc-ILD-). SSC-ILD+ patients were revisited after 6 months and 12 months to complete the study. Results: Thirty-one control subjects and 74 patients with SSc (33 SSc-ILD- and 41 SSc-ILD+) were included. Forty-one SSc-ILD+ patients were followed-up at 6 months and 12 months. Lung functional parameters of patients with SSc-ILD+ were lower than that of SSc-ILD- patients. The level of CANO was significantly higher in SSc-ILD+ than SSc-ILD- patients (8.6 ± 2.5 vs 4.2 ± 1.3 ppb and P<0.01). Warrick and Goldin scores of patients with SSc-ILD+ were respectively 16.5 ± 5.2 and 12.7 ± 4.3. Warrick scores were reduced after 6 and 12 months of follow-up vs at inclusion (12.4 ± 4.3 and 9.1 ± 3.2 vs 16.5 ± 5.2; P<0.05, P<0.01, and P<0.05; respectively). ΔWarrick and ΔGoldin scores were significantly and inversely correlated with ΔFVC, ΔTLC, ΔTLCO, ΔVO₂ max; that was also correlated with ΔCANO (R= 0.783, P<0.01 and R= 0.719 and P<0.05). Conclusion: CANO is a relevant biomarker for the diagnosis of ILD in patients with SSc, especially in combination with HRCT.     

De Novo Lipogenesis and Cholesterol Synthesis in Humans with Long-Standing Type 1 Diabetes Are Comparable to Non-Diabetic Individuals

Background

Synthesis of lipid species, including fatty acids (FA) and cholesterol, can contribute to pathological disease. The purpose of this study was to investigate FA and cholesterol synthesis in individuals with type 1 diabetes, a group at elevated risk for vascular disease, using stable isotope analysis.

Methods

Individuals with type 1 diabetes (n = 9) and age-, sex-, and BMI-matched non-diabetic subjects (n = 9) were recruited. On testing day, meals were provided to standardize food intake and elicit typical feeding responses. Blood samples were analyzed at fasting (0 and 24 h) and postprandial (2, 4, 6, and 8 hours after breakfast) time points. FA was isolated from VLDL to estimate hepatic FA synthesis, whereas free cholesterol (FC) and cholesteryl ester (CE) was isolated from plasma and VLDL to estimate whole-body and hepatic cholesterol synthesis, respectively. Lipid synthesis was measured using deuterium incorporation and isotope ratio mass spectrometry.

Results

Fasting total hepatic lipogenesis (3.91±0.90% vs. 5.30±1.22%; P = 0.41) was not significantly different between diabetic and control groups, respectively, nor was synthesis of myristic (28.60±4.90% vs. 26.66±4.57%; P = 0.76), palmitic (12.52±2.75% vs. 13.71±2.64%; P = 0.65), palmitoleic (3.86±0.91% vs. 4.80±1.22%; P = 0.65), stearic (5.55±1.04% vs. 6.96±0.97%; P = 0.29), and oleic acid (1.45±0.28% vs. 2.10±0.51%; P = 0.21). Postprandial lipogenesis was also not different between groups (P = 0.38). Similarly, fasting synthesis of whole-body FC (8.2±1.3% vs. 7.3±0.8%/day; P = 0.88) and CE (1.9±0.4% vs. 2.0±0.3%/day; P = 0.96) and hepatic FC (8.2±2.0% vs. 8.1±0.8%/day; P = 0.72) was not significantly different between diabetic and control subjects.

Conclusions

Despite long-standing disease, lipogenesis and cholesterol synthesis was not different in individuals with type 1 diabetes compared to healthy non-diabetic humans.     

Prenatal Hypoxia Is Associated with Long-Term Retinal Dysfunction in Rats

Background

Intra-uterine growth restriction (IUGR) has been associated with increased predisposition to age-related complications. We tested the hypothesis that rat offspring models of IUGR would exhibit exacerbated, age-related retinal dysfunction.

Methods

Female Sprague-Dawley rats (maintained at 11.5% O₂ from gestational day 15 to 21 to induce IUGR) and control offspring (maintained at 21% O₂ throughout pregnancy) had retinal function assessed at 2 months (young) and 14 months of age (aged) with electroretinogram (ERG) recordings. Retinal anatomy was assessed by immunofluorescence.

Results

Deficits in rod-driven retina function were observed in aged IUGR offspring, as evidenced by reduced amplitudes of dark-adapted mixed a-wave V_max (by 49.3%, P<0.01), b-wave V_max (by 42.1%, P<0.001) and dark-adapted peak oscillatory potentials (by 42.3%, P<0.01). In contrast to the rod-driven defects specific to aged IUGR offspring, light adapted ERG recordings revealed cone defects in young animals, that were stationary until old age. At 2 months, IUGR offspring had amplitude reductions for both b-wave (V_max by 46%, P<0.01) and peak oscillatory potential (V_max by 38%, P<0.05). Finally, defects in cone-driven responses were further confirmed by reduced maximal photopic flicker amplitudes at 2 (by 42%, P<0.001) and 14 months (by 34%, P = 0.06) and critical flicker fusion frequencies at 14 months (Control: 42±1 Hz, IUGR: 35±2 Hz, P<0.05). These functional changes were not paralleled by anatomical losses in IUGR offspring retinas.

Conclusions

These data support that the developing retina is sensitive to stressors, and that pathways governing cone- and rod-driven function differ in their susceptibilities. In the case of prenatal hypoxia, cone- and rod-driven dysfunction manifest at young and old ages, respectively. We must, therefore, take into account the specific impact that fetal programming might exert on age-related retinal dystrophies when considering related diagnoses and therapeutic applications.