Effect of storage time and cryoprotectant concentrations on the fertilization rate and hatching rate of cryopreserved sperm in red seabream (Pagrus major Temminck & Schlegel, 1843)

This study examined the effects of storage time and cryoprotectant concentrations on the post-thaw sperm of red seabream, Pagrus major. Sperm treated with 12%, 15%, 18% and 21% DMSO were cryopreserved for 10, 30, 60 and 360 days, and fertilization and hatching rates were analysed. For all groups, there were no differences in the fertilization rates and hatching rates between sperm cryopreserved for < 60 days and fresh sperm (98.8 +/- 0.8%, 96.4 +/- 1.3%). However, for sperm cryopreserved for 360 days, both fertilization rates (88.6 +/- 3.0% to 7.0 +/- 1.9%) and hatching rates (79.4 +/- 7.2% to 3.3 +/- 0.8%) decreased drastically. Furthermore, the cryoprotectant concentrations affected sperm quality significantly (P < 0.05). When cryopreserved for 360 days, sperm treated with 15% DMSO obtained the best results compared with other concentrations. We suggest that 15% DMSO may be an effective cryoprotectant for long-term sperm cryopreservation of red seabream.

Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF

STUDY QUESTION. Are significant abnormalities in outward (K+) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER. Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY. Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K+ channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K+ channels in human spermatozoa or the incidence and functional consequences of K+ channel defects. STUDY DESIGN, SIZE AND DURATION. Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS. Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca2+ influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K+ signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE. Patch clamp electrophysiology was used to assess outward (K+) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca2+]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION. For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS. These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized.

Seasonal variability in the fertilization rate of women undergoing assisted reproduction treatments

The objective of this study was to evaluate whether seasonality affects human-assisted reproduction treatment outcomes. For this, 1932 patients undergoing intracytoplasmic sperm injection (ICSI) were assigned to a season group according to the day of oocyte retrieval: winter (n = 435), spring (n = 444), summer (n = 469) or autumn (n = 584). Analysis of variance was used to compare the ICSI outcomes. The fertilization rate was increased during the spring (winter: 67.9%, spring: 73.5%, summer: 68.7% and autumn: 69.0%; p < 0.01). In fact, a nearly 50% increase in the fertilization rate during the spring was observed (odds ratio 1.45, confidence interval 1.20-1.75; p < 0.01). The oestradiol concentration per number of oocytes was significantly higher during the spring (winter: 235.8 pg/mL, spring: 282.1 pg/mL, summer: 226.1 pg/mL and autumn: 228.7 pg/mL; p = 0.030). This study demonstrates a seasonal variability in fertilization after ICSI, where fertilization is higher during the spring than at any other time.

Apical leaf necrosis as a defence mechanism against pathogen attack: effects of high nutrient availability on onset and rate of necrosis

An outdoor experiment was conducted to increase understanding of apical leaf necrosis in the presence of pathogen infection. Holcus lanatus seeds and Puccinia coronata spores were collected from two adjacent and otherwise similar habitats with differing long-term N fertilization levels. After inoculation, disease and necrosis dynamics were observed during the plant growing seasons of 2003 and 2006. In both years high nutrient availability resulted in earlier disease onset, a higher pathogen population growth rate, earlier physiological apical leaf necrosis onset and a reduced time between disease onset and apical leaf necrosis onset. Necrosis rate was shown to be independent of nutrient availability. The results showed that in these nutrient-rich habitats H. lanatus plants adopted necrosis mechanisms which wasted more nutrients. There was some indication that these necrosis mechanisms were subject to local selection pressures, but these results were not conclusive. The findings of this study are consistent with apical leaf necrosis being an evolved defence mechanism.

Scallop size does not predict amount or rate of induced sperm release

Within populations of broadcast spawning marine invertebrates such as scallops, larger animals typically have larger gonads. Presumably, this means those larger males have more sperm to release than small males. However, there has never been a direct test of whether larger males actually release more sperm, at a higher rate, during spawning. To address this, we compared the allometry of induced sperm release with that of reproductive investment (gonad weight) in ripe males of 2 species of scallops, Chlamys bifrons and Chlamys asperrima. We did not find that larger scallops released more sperm or released it faster than small scallops, and were able to reject the hypothesis that instantaneous sperm release was related to body size in the same way as gonad weight. Consequently, we speculate that if larger broadcast spawning males do release more sperm, they may do so by spawning on more occasions within a reproductive season.

Influence of fertilization on the growth of radicchio "Rosso di Chioggia" cultivated in two different environments

Este trabajo evalúa la influencia de la fertilización en radicchio tipo "Rosso de Chioggia" (precocidad media) sobre algunos índices de crecimiento. Se realizaron ensayos durante dos campañas en Rovigo (Italia) y una en Mendoza (Argentina), aplicándose dosis crecientes de NPK, identificadas como N0P0K0, N1P1K1, N2P2K2, en Mendoza y en Rovigo, además, N3P2K2. Durante el cultivo se calcularon índices de crecimiento como: relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), specific leaf area (SLA), leaf weigh ratio (LWR), crop growth ratio (CGR), leaf area index (LAI) and leaf area duration (LAD). En Mendoza, el CGR estuvo fuertemente influenciado por NAR desde el trasplante hasta alcanzar 776 grados días (GDD); desde 1052 a 1653 GDD el CGR fue afectado por el LAI el cual aumentó marcadamente debido a las condiciones ambientales favorables. Entre los 1052 y 1653 GDD el incremento del LAI determinó una reducción en la eficiencia fotosintética. En Rovigo, la tendencia de los índices fue disímil en los dos años, encontrándose respuestas diferentes en LAR y en SLA. En el segundo año, el CGR siempre arrojó valores más altos, mientras que NAR no difirió en ninguno de los años. En la segunda mitad del ciclo, CGR estuvo fuertemente asociado a una menor eficiencia fotosintética, debido a la formación de la cabeza. Valores elevados de LAI indicaron una extensión del ciclo, retrasando la formación de la cabeza. Las plantas alcanzaron la madurez comercial con LWR entre 0,35 - 0,40 g g-1. En ambos ambientes, no se observó claramente el efecto de la fertilización sobre los índices; si bien las dosis más altas mostraron mayor actividad de crecimiento en las etapas tempranas.

Inferior field loss increases rate of falls in older adults with glaucoma

PURPOSE: To examine the visual predictors of falls and injurious falls among older adults with glaucoma. METHODS: Prospective falls data were collected for 71 community-dwelling adults with primary open-angle glaucoma, mean age 73.9 ± 5.7 years, for one year using monthly falls diaries. Baseline assessment of central visual function included high-contrast visual acuity and Pelli-Robson contrast sensitivity. Binocular integrated visual fields were derived from monocular Humphrey Field Analyser plots. Rate ratios (RR) for falls and injurious falls with 95% confidence intervals (CIs) were based on negative binomial regression models. RESULTS: During the one year follow-up, 31 (44%) participants experienced at least one fall and 22 (31%) experienced falls that resulted in an injury. Greater visual impairment was associated with increased falls rate, independent of age and gender. In a multivariate model, more extensive field loss in the inferior region was associated with higher rate of falls (RR 1.57, 95%CI 1.06, 2.32) and falls with injury (RR 1.80, 95%CI 1.12, 2.98), adjusted for all other vision measures and potential confounding factors. Visual acuity, contrast sensitivity, and superior field loss were not associated with the rate of falls; topical beta-blocker use was also not associated with increased falls risk. CONCLUSIONS: Falls are common among older adults with glaucoma and occur more frequently in those with greater visual impairment, particularly in the inferior field region. This finding highlights the importance of the inferior visual field region in falls risk and assists in identifying older adults with glaucoma at risk of future falls, for whom potential interventions should be targeted. KEY WORDS: glaucoma, visual field, visual impairment, falls, injury

Use of Bayesian MUNE to show differing rate of loss of motor units in subgroups of ALS

Objectives To evaluate differences among patients with different clinical features of ALS, we used our Bayesian method of motor unit number estimation (MUNE). Methods We performed serial MUNE studies on 42 subjects who fulfilled the diagnostic criteria for ALS during the course of their illness. Subjects were classified into three subgroups according to whether they had typical ALS (with upper and lower motor neurone signs) or had predominantly upper motor neurone weakness with only minor LMN signs, or predominantly lower motor neurone weakness with only minor UMN signs. In all subjects we calculated the half life of MUs, defined as the expected time for the number of MUs to halve, in one or more of the abductor digiti minimi (ADM), abductor pollicis brevis (APB) and extensor digitorum brevis (EDB) muscles. Results The mean half life of MUs was less in subjects who had typical ALS with both upper and lower motor neurone signs than in those with predominantly upper motor neurone weakness or predominantly lower motor neurone weakness. In 18 subjects we analysed the estimated size of the MUs and demonstrated the appearance of large MUs in subjects with upper or lower motor neurone predominant weakness. We found that the appearance of large MUs was correlated with the half life of MUs. Conclusions Patients with different clinical features of ALS have different rates of loss and different sizes of MUs. Significance: These findings could indicate differences in disease pathogenesis.

Quantitative studies of lower motor neuron degeneration in amyotrophic lateral sclerosis : Evidence for exponential decay of motor unit numbers and greatest rate of loss at the site of onset

Objective: To use our Bayesian method of motor unit number estimation (MUNE) to evaluate lower motor neuron degeneration in ALS. Methods: In subjects with ALS we performed serial MUNE studies. We examined the repeatability of the test and then determined whether the loss of MUs was fitted by an exponential or Weibull distribution. Results: The decline in motor unit (MU) numbers was well-fitted by an exponential decay curve. We calculated the half life of MUs in the abductor digiti minimi (ADM), abductor pollicis brevis (APB) and/or extensor digitorum brevis (EDB) muscles. The mean half life of the MUs of ADM muscle was greater than those of the APB or EDB muscles. The half-life of MUs was less in the ADM muscle of subjects with upper limb than in those with lower limb onset. Conclusions: The rate of loss of lower motor neurons in ALS is exponential, the motor units of the APB decay more quickly than those of the ADM muscle and the rate of loss of motor units is greater at the site of onset of disease. Significance: This shows that the Bayesian MUNE method is useful in following the course and exploring the clinical features of ALS. 2012 International Federation of Clinical Neurophysiology.

Rate of torque and electromyographic development during anticipated eccentric contraction is lower in previously strained hamstrings

Background: Hamstring strain injuries are prevalent in sport and re-injury rates have been high for many years. Whilst much focus has centred on the impact of previous hamstring strain injury on maximal eccentric strength, high rates of torque development is also of interest, given the important role of the hamstrings during the terminal swing phase of running. The impact of prior strain injury on myoelectrical activity of the hamstrings during tasks requiring high rates of torque development has received little attention. Purpose: To determine if recreational athletes with a history of unilateral hamstring strain injury, who have returned to training and competition, will exhibit lower levels of myoelectrical activity during eccentric contraction, rate of torque development and impulse 30, 50 and 100ms after the onset of myoelectrical activity or torque development in the previously injured limb compared to the uninjured limb. Study design: Case-control study Methods: Twenty-six recreational athletes were recruited. Of these, 13 athletes had a history of unilateral hamstring strain injury (all confined to biceps femoris long head) and 13 had no history of hamstring strain injury. Following familiarisation, all athletes undertook isokinetic dynamometry testing and surface electromyography assessment of the biceps femoris long head and medial hamstrings during eccentric contractions at -60 and -1800.s-1. Results: In the injured limb of the injured group, compared to the contralateral uninjured limb rate of torque development and impulse was lower during -600.s-1 eccentric contractions at 50 (RTD, injured limb = 312.27 ± 191.78Nm.s-1 vs. uninjured limb = 518.54 ± 172.81Nm.s-1, p=0.008; IMP, injured limb = 0.73 ± 0.30 Nm.s vs. uninjured limb = 0.97 ± 0.23 Nm.s, p=0.005) and 100ms (RTD, injured limb = 280.03 ± 131.42Nm.s-1 vs. uninjured limb = 460.54.54 ± 152.94Nm.s-1,p=0.001; IMP, injured limb = 2.15 ± 0.89 Nm.s vs. uninjured limb = 3.07 ± 0.63 Nm.s, p<0.001) after the onset of contraction. Biceps femoris long head muscle activation was lower at 100ms at both contraction speeds (-600.s-1, normalised iEMG activity (x1000), injured limb = 26.25 ± 10.11 vs. uninjured limb 33.57 ± 8.29, p=0.009; -1800.s-1, normalised iEMG activity (x1000), injured limb = 31.16 ± 10.01 vs. uninjured limb 39.64 ± 8.36, p=0.009). Medial hamstring activation did not differ between limbs in the injured group. Comparisons in the uninjured group showed no significant between limbs difference for any variables. Conclusion: Previously injured hamstrings displayed lower rate of torque development and impulse during slow maximal eccentric contraction compared to the contralateral uninjured limb. Lower myoelectrical activity was confined to the biceps femoris long head. Regardless of whether these deficits are the cause of or the result of injury, these findings could have important implications for hamstring strain injury and re-injury. Particularly, given the importance of high levels of muscle activity to bring about specific muscular adaptations, lower levels of myoelectrical activity may limit the adaptive response to rehabilitation interventions and suggest greater attention be given to neural function of the knee flexors following hamstring strain injury.

Impact of previous hamstring strain injury on rate of torque and EMG development during eccentric contraction.

Background: Hamstring strain injuries (HSIs) are prevalent in sport and re-injury rates have been high for many years. Whilst much focus has centred on the impact of previous hamstring strain injury on maximal eccentric strength, high rates of torque development is also of interest, given the important role of the hamstrings during the terminal swing phase of gait. The impact of prior strain injury on neuromuscular function of the hamstrings during tasks requiring high rates of torque development has received little attention. The purpose of this study is to determine if recreational athletes with a history of unilateral hamstring strain injury, who have returned to training and competition, will exhibit lower levels of eccentric muscle activation, rate of torque development and impulse 30, 50 and 100ms after the onset of electromyographical or torque development in the previously injured limb compared to the uninjured limb. Methods: Twenty-six recreational athletes were recruited. Of these, 13 athletes had a history of unilateral hamstring strain injury (all confined to biceps femoris long head) and 13 had no history of hamstring strain injury. Following familiarisation, all athletes undertook isokinetic dynamometry testing and surface electromyography assessment of the biceps femoris long head and medial hamstrings during eccentric contractions at -60 and -1800.s-1. Results: In the injured limb of the injured group, compared to the contralateral uninjured limb rate of torque development and impulse was lower during -600.s-1 eccentric contractions at 50 (RTD, p=0.008; IMP, p=0.005) and 100ms (RTD, p=0.001; IMP p<0.001) after the onset of contraction. There was also a non-significant trend for rate of torque development during -1800.s-1 to be lower 100ms after onset of contraction (p=0.064). Biceps femoris long head muscle activation was lower at 100ms at both contraction speeds (-600.s-1, p=0.009; -1800.s-1, p=0.009). Medial hamstring activation did not differ between limbs in the injured group. Comparisons in the uninjured group showed no significant between limbs difference for any variables. Conclusion: Previously injured hamstrings displayed lower rate of torque development and impulse during eccentric contraction. Lower muscle activation was confined to the biceps femoris long head. Regardless of whether these deficits are the cause of or the result of injury, these findings have important implications for hamstring strain injury and re-injury and suggest greater attention be given to neural function of the knee flexors.