Search

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×106 sperm) and bull (2 mg CLC/120×106 sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.

This study outlines advancements in mangrove monitoring using Earth Observation (EO) technologies, highlighting a transition from historical cartography to modern, near-real-time monitoring systems. Mangroves, critical for biodiversity, climate regulation, and coastal protection, face threats from climate change and human activities. The need for accurate and recurrent mapping tools is emphasized, addressing the limitations of current global datasets like the Global Mangrove Watch (GMW) by incorporating high-resolution data and field measurements. The French National Research Institute for Sustainable Development (IRD) has pioneered mangrove monitoring since the 1990s, with key methodologies including the use of Sentinel-2 and Pleiades satellite imagery for high-resolution mapping, texture-based analysis, and the incorporation of LiDAR data for accurate biomass estimation. Historical cartography is contrasted with contemporary monitoring efforts, focusing on the integration of multi-source data and the importance of localized ground-truthing. This approach enhances the capabilities of earth observation for carbon stock assessments and supports informed decision-making in conservation and climate policy. The work also highlights the need for further integration of local knowledge and advanced EO sensor data to refine carbon sequestration models and improve mangrove management strategies.

The seasonal cycle of the Antarctic sea ice extent is strongly asymmetric, with a relatively slow increase after the summer minimum followed by a more rapid decrease after the winter maximum. This cycle is intimately linked to the seasonal cycle of the insolation received at the top of the atmosphere, but sea ice processes as well as the exchanges with the atmosphere and ocean may also play a role. To quantify these contributions, a series of idealized sensitivity experiments have been performed with an eddy-permitting (1/4∘) NEMO-LIM3 (Nucleus for European Modelling of the Ocean–Louvain-la-Neuve sea ice model version 3) Southern Ocean configuration, including a representation of ice shelf cavities, in which the model was either driven by an atmospheric reanalysis or coupled to the COSMO-CLM2 regional atmospheric model. In those experiments, sea ice thermodynamics and dynamics as well as the exchanges with the ocean and atmosphere are strongly perturbed. This perturbation is achieved by modifying snow and ice thermal conductivities, the vertical mixing in the ocean top layers, the effect of freshwater uptake and release upon sea ice growth and melt, ice dynamics, and surface albedo. We find that the evolution of sea ice extent during the ice advance season is largely independent of the direct effect of the perturbation and appears thus mainly controlled by initial state in summer and subsequent insolation changes. In contrast, the melting rate varies strongly between the experiments during the retreat, in particular if the surface albedo or sea ice transport are modified, demonstrating a strong contribution of those elements to the evolution of ice coverage through spring and summer. As with the advance phase, the retreat is also influenced by conditions at the beginning of the melt season in September. Atmospheric feedbacks enhance the model winter ice extent response to any of the perturbed processes, and the enhancement is strongest when the albedo is modified. The response of sea ice volume and extent to changes in entrainment of subsurface warm waters to the ocean surface is also greatly amplified by the coupling with the atmosphere.

Queerness and transidentity have had a durable relationship with cyberpunk aesthetics, reflected in both cultural works and academic reflections. There is an evident attraction in worlds where technological prowess allows one to evolve beyond one’s corporeal body, or to change it at will. The noir explorations of themes such as discrimination and sex work are also strong sources of resonance with common trans and queer experiences. Culturally, this aesthetic is indissociable from its urban component, with the serendipity, diversity, and density of the city often at the centre of the stories told. This reflects the observed tendency of queer people to congregate in urban centres, where minorities are present in sufficient numbers to create social communities with freer explorations around gender. This appeared to be one of the central aesthetic currents of the global trans community until the early twenty-first century. However, we now observe a change with the rise of more rural aspirations, exemplified by the recent popularity of the cottagecore aesthetic. There seems to be a growing desire to retreat from society at large and isolate in tight-knit homogeneous communities, replacing co-spatiality with online connectedness. This could be the result of multiple factors, among which two seem central: the assimilation of many queers linked to the dissolution of greater federated queer communities, and the increased prioritisation of online interactions. Drawing from human geography, literary analysis and a corpus from Tumblr (a microblogging network) this article thus seeks to explore how aspirational and representational depictions of queerness evolved in tandem with its physical and online practices, transforming the historically close relationship between queerness and urbanity.

Uncertain or inaccurate parameters in sea ice models influence seasonal predictions and climate change projections in terms of both mean and trend. We explore the feasibility and benefits of applying an ensemble Kalman filter (EnKF) to estimate parameters in the Los Alamos sea ice model (CICE). Parameter estimation (PE) is applied to the highly influential dry snow grain radius and combined with state estimation in a series of perfect model observing system simulation experiments (OSSEs). Allowing the parameter to vary in space improves performance along the sea ice edge but degrades in the central Arctic compared to requiring the parameter to be uniform everywhere, suggesting that spatially varying parameters will likely improve PE performance at local scales and should be considered with caution. We compare experiments with both PE and state estimation to experiments with only the latter and have found that the benefits of PE mostly occur after the data assimilation period, when no observations are available to assimilate (i.e., the forecast period), which suggests PE's relevance for improving seasonal predictions of Arctic sea ice.

Objectives: A novel beta coronavirus has been identified as responsible for the 2019 coronavirus infection (Covid-19). Clinical presentations range from asymptomatic cases to acute respiratory distress syndrome with fatal outcome. Such a broad spectrum of disease expression calls for an investigation of immune response characteristics. Methods: We identified subjects admitted for Covid-19 in whom a large panel of immunological markers were measured, including B- and T- and NK-lymphocyte phenotypes, T-lymphocyte subpopulation cells and plasma cytokines. Patients were divided according to symptom severity during hospitalisation, in those with uncomplicated and complicated infection. Differences between groups were analyzed. Results: Seventeen patients were included (mean age: 83 years; 9 women; mean delay of symptoms onset: 4 days). Six had uncomplicated infection, while 11 developed complicated forms during hospitalization. CD10 + B lymphocyte levels were inversely correlated with clinical severity (5.8% vs 2.0%, p = 0.04) and CD10+ levels above 3% were independently associated with uncomplicated forms [Odds Ratio 0.04 (CI 0.002-0.795, p = 0.034)]. TNF-alpha, IL-1, Il-6 and Il-8 measurements upon admission differed between patients who died and those who survived (p < 0.01 for all comparisons). Conclusions: In a population of elderly patients recently infected with Covid-19, CD10 + B cell levels were inversely correlated with clinical severity. Cytokine values upon admission were highly predictive of fatal outcome during hospitalisation. These findings could explain differences in the clinical presentation and allow rapid identification of patients at risk for complications.

Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p

An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents. The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. • A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters. • The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult. • The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content.

The purpose of this research is to evaluate the creasing of non-paper sheet materials, such as plastics and metals, to facilitate origami-based compliant mechanism design. Although it is anticipated that most origami-based design will result from surrogate folds (indirect methods of replacing the crease), it is valuable to provide information that may help in more direct approaches for origami-based design in materials other than paper. Planar sheets of homogeneous material are considered as they maintain the principles fundamental to origami (flat initial state, low cost, readily available). The reduced stiffness along the axis of the crease is an enabling characteristic of origami. Hence a metric based on the deformation of the crease compared to the deformation of the panels enables engineering materials to be evaluated based on their ability to achieve the "hinge-like" behavior observed in folded paper. Advantages of both high and low values of this metric are given. Testing results (hinge indexes, residual angles, localized hinge behavior and cyclic creasing to failure) are presented for various metals and polymers. This methodology and subsequent findings are provided to enable origami-based design of compliant mechanisms.

Urban green roofs offer environmental and social benefits and provide resources for urban wildlife; however, how birds use green roofs remains poorly studied in Mediterranean cities. Here, we develop a 1-year study in Madrid, Spain, recording the birds that use both an urban green roof and the adjacent conventional roofs throughout the four seasons. We recorded a total of 17 bird species in the area, of which 8 use the green roof surveyed. The most common species detected in both types of roofs was the feral pigeon ( Columba livia ), which exploited the green roof permanently, in contrast with the other species observed, which only appeared occasionally. We also found that more species visited the green roof in the central hours of the day and a higher richness of species in the spring. Moreover, we detect that most of the species were residents all year in the area and have granivorous or granivorous-omnivorous feeding habits. The main behaviour observed in the green roof was feeding, while no reproduction of any species was confirmed. We conclude that green roofs offer birds much-needed resources in metropolitan areas, promoting greener, more connected and more biodiverse cities., Competing Interests: We declare we have no competing interests., (© 2024 The Author(s).)

The 2013 seismic sequence at the Castor injection platform offshore Spain, including three earthquakes of magnitude 4.1, occurred during the initial filling of a planned Underground Gas Storage facility. The Castor sequence is one of the most important cases of induced seismicity in Europe and a rare example of seismicity induced by gas injection into a depleted oil field. Here we use advanced seismological techniques applied to an enhanced waveform dataset, to resolve the geometry of the faults, develop a greatly enlarged seismicity catalog and record details of the rupture kinematics. The sequence occurred by progressive fault failure and unlocking, with seismicity initially migrating away from the injection points, triggered by pore pressure diffusion, and then back again, breaking larger asperities loaded to higher stress and producing the largest earthquakes. Seismicity occurred almost exclusively on a secondary fault, located below the reservoir, dipping opposite from the reservoir bounding fault., (© 2021. The Author(s).)

The U rhynchophylla, U tomentosa, Isatis indigotica Fortune, Voacanga Africana, herbal constituents, fungal extracts from Aspergillus duricaulis culture media, include spirooxindoles, polyphenols or bridged spirocyclic alkaloids. Their constituents exhibit specific and synergistic multiple neuroprotective properties including inhibiting of Aβ fibril induced cytotoxicity, NMDA receptor inhibition in mice models of Alzheimer's disease (AD). The pioneering research from Woodward to Waldmann has advanced the synthesis of spirocyclic alkaloids. Furthermore, the elucidation of the genetic analysis, biochemical pathways that links strictosidine to the alkaloids akuammicine, stemmadenine, tabersonine, catharanthine, will now enable the biotechnological generation, also stimulate synthesis of related bridged spirocyclic alkaloids for medicinal investigations. From the value of spirocyclic structures as multi target dementia leads, we hypothesise that simpler Lipinski-like natural/synthetic alkaloid analogues may likewise be discovered that provide neurocognitive enhancing activities against dementia and AD., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

A chronic model of acute myocardial infarction was developed to study the mechanisms involved in adverse postinfarction ventricular remodeling. In an acute myocardial infarction (AMI), the left circumflex coronary artery of New Zealand White rabbits (n = 9) was occluded by ligature for 1 h, followed by reperfusion. A specific care protocol was applied before, during, and after the intervention, and the results were compared with those of a sham operated group (n = 7). After 5 weeks, programmed stimulation and high-resolution mapping were performed on isolated and perfused hearts using the Langendorff technique. The infarct size determined by 2,3,5-triphenyltetrazolium chloride inside of the area at risk (thioflavin-S) was then determined. The area at risk was similar in both groups (54.33% (experimental infarct group) vs. 58.59% (sham group), ns). The infarct size was 73.16% as a percentage of the risk area. The experimental infarct group had a higher inducibility of ventricular arrhythmias (100% vs. 43% in the sham group, p = 0.009). A reproducible chronic experimental model of myocardial infarction is presented in which the extent and characteristics of the lesions enable the study of the vulnerability to develop ventricular arrhythmias because of the remodeling process that occurs during cardiac tissue repair.

Lactoferrin (LF) is a multifunctional protein occurring in many biological secretions including milk. It possesses iron binding/transferring, antibacterial, antiviral, antifungal, anti-inflammatory and anti-carcinogenic properties. These functional properties intimately depend on the structural integrity of LF especially its higher order conformation. LF is primarily extracted from bovine milk and it is subsequently added into many commercial products such as nutritional supplements, infant formula, cosmetics and toothpaste. LF is sensitive to denaturation induced by temperature and other physicochemical stresses. Hence, the extraction, powder formation processes of LF and processing parameters of LF-containing products have to be optimized to minimise its undesired denaturation. This review documents the advances made on structure-function relationships and discusses the effectiveness of methods used to preserve the structure of LF during thermal processing. Oral delivery, as the most convenient way for administering LF, is also discussed focusing on digestion of LF in oral, gastric and intestinal stages. The effectiveness of methods used to deliver LF to intestinal digestion stage in structurally intact form is also compared. Altogether, this work comprehensively reviews the fate of LF during thermal processing and digestion, and suggests suitable means to preserve its structural integrity and functional properties. Scope of review The manuscript aims at providing a comprehensive review of the latest publications on four aspects of LF: structural features, functional properties, nature and extent of denaturation and gastrointestinal digestion. It also analyses how these publications benefit food and pharmaceutical industries.

Children with autism spectrum disorder (ASD) in public education settings experience difficulties with transitions during classroom routines, which can result in challenging behavior. Single-subject research supports techniques for transitions, but school-based approaches often require resources and training unavailable in low-resource districts, limiting implementation. We developed and evaluated the Schedules, Tools, and Activities for Transitions (STAT) program, a short-term, manualized intervention of behavioral supports to support daily routine transitions for students with ASD (K-5) in underresourced districts. We utilized a multisite, cluster-randomized, group comparison design (immediate treatment versus waitlist) with matched pairs ( n = 150 students, 57 educators). Data indicated (a) no group differences for academic engagement or classroom independence, and (b) an advantage for STAT in reducing challenging behavior and increasing teacher fidelity. Results show preliminary support for an intervention that is feasible and perceived as sustainable in real-world settings.

In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models provided accurate codeine quantification in water and human plasma with high prediction accuracy (Q 2 ). In addition, the employment of codeine-d 6 as the internal standard further improved the accuracy of the model, by increasing the Q 2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predictions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm -1 assigned to C-N stretching, C-H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 μM (209.55 ng mL -1 ) and 1.39 μM (416.12 ng mL -1 ), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantification and reproducibility because the competition of the codeine and codeine-d 6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any fluctuations in laser fluence. It is our belief that this may open up new exciting opportunities for testing SERS in real-world samples and applications which would be an area of potential future studies.

Bovine lactoferrin (LF b ) powders were produced using spray drying and freeze drying. Industrially obtained fresh liquid-LF b was used as starting material. The antioxidant capacity, solubility in water, moisture sorption behaviour, the extent of denaturation and changes in the secondary structural features of spray-dried (SDLF b ) and freeze-dried bovine lactoferrin (FDLF b ) powders were determined. The residual moisture content, water activity, particle size and amorphous/crystalline nature of the SDLF b and FDLF b were also measured. Results showed that both SDLF b and FDLF b powders had negligible denaturation and conformation changes compared to the liquid-LF b . Both SDLF b and FDLF b showed type II sorption behaviour with almost identical monolayer moisture content. The SDLF b powders were amorphous in nature with >98% solubility in water. The antioxidant activity of SDLF b was similar to that of the liquid-LF b while it was ∼6% less in FDLF b . Based on the residual moisture content, water activity, solubility and preservation of secondary structure of LF b in resultant powders, a spray drying process with 180°C inlet and 95°C outlet temperature was found to produce similar or better quality LF b powders compared to the ones produced through a freeze drying process., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Histidine is a key component of a number of enzymatic mechanisms, and undertakes a myriad of functionalities in biochemical systems. Its computational modelling can be problematic, as its capacity to take on a number of distinct formal charge states, and tautomers thereof, is difficult to capture by conventional techniques. We demonstrate a means for recovering the experimental Raman optical activity (ROA) spectra of histidine to a high degree of accuracy. The resultant concordance between experiment and theory is of particular importance in characterising physically insightful quantities, such as band assignments. We introduce a novel conformer selection scheme that unambiguously parses snapshots from a molecular dynamics trajectory into a smaller conformational ensemble, suitable for reproducing experimental spectra. We show that the "dissimilarity" of the conformers within the resultant ensemble is maximised and representative of the physically relevant regions of molecular conformational space. In addition, we present a conformer optimisation strategy that significantly reduces the computational costs associated with alternative optimisation strategies. This conformer optimisation strategy yields spectra of equivalent quality to those of the aforementioned alternative optimisation strategies. Finally, we demonstrate that microsolvated models of small molecules yield spectra that are comparable in quality to those obtained from ab initio calculations involving a large number of solvent molecules.

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (°Cmin(-1)) from -6°C to -100°C using a programmable freezer: slow (-15°Cmin(-1), S), medium (-40°Cmin(-1), M) or fast (-60°Cmin(-1), F). After thawing (50°C, 12s), the quality was highest (P<0.05) in C and M samples and lowest in S and F samples. F samples presented the lowest litter sizes (P≤0.05) and fertility whilst M samples exhibited the highest values. In conclusion, the freezing rate affects both the quality and the fertilising ability of frozen-thawed rabbit spermatozoa, with both slow (-15°Cmin(-1)) and fast (-60°Cmin(-1)) freezing rates being detrimental for the quality and fertilising ability.

Cooling sperm to and equilibrating the sperm at 5 °C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 °C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 °C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P<0.01). However, reducing the time sperm were held at 5 °C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P<0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 °C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 °C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 °C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 °C for less than 45 minutes will affect sperm fertilizing ability., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Glycerol (11%; v:v) is the cryoprotectant most often used for the cryopreservation of rooster sperm. However, chicken breeds differ in the resistance of their sperm to the cryopreservation process and endangered or local breeds usually present low fertilizing ability when conventional sperm cryopreservation protocols are used. The objective of this study was to optimize the protocol for the cryopreservation of the sperm from the endangered breed "Gallina Valenciana de Chulilla". For this purpose, 10 pools of semen from 43 roosters of this breed were cryopreserved using 8%, 7%, 6%, or 4% glycerol, and the sperm quality was determined immediately after thawing and in the insemination doses. Lohmann Brown Classic laying hens (n = 40) were used for the insemination trials. The sperm quality after cryopreservation progressively decreased as the glycerol concentration was reduced (P < 0.01); samples frozen using 4% glycerol exhibited the lowest quality (38% total motile sperm and 49% live sperm), and samples frozen using 8% glycerol exhibited the highest quality (67% total motile sperm and 66% live sperm). These differences were also observed after the glycerol was removed (P < 0.01). However, the sperm fertilizing ability was similar for all the treatments (23%-30% fertilized eggs), and increased as the glycerol concentration decreased. In conclusion, semen from roosters frozen using 4% glycerol exhibited lower sperm quality but similar fertilizing ability compared with samples processed using higher glycerol concentrations. These results may provide useful information for developing cryopreservation protocols for other breeds., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10(6) sperm) and bull (2 mg CLC/120×10(6) sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.

Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-β-cyclodextrin/120 × 10(6) sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P <0.05), even in semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P <0.05). Thus, CLC-treated sperm showed a higher tolerance to high glycerol concentrations (5%) in terms of sperm viability (59.19% ± 3) than non-treated sperm (45.58% ± 3; P<0.05). Therefore, it could be necessary to modify the freezing extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1mg CLC/120 × 10(6)sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The use of frozen-thawed (FT) sperm for the artificial insemination of pigs is rare. Treating boar sperm with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances the penetration of immature oocytes in vitro, and this phenomenon has been positively correlated with in vivo fertilisation ability in pigs. The objective of this study was to compare the in vivo fertilising ability of boar sperm treated with 0 (control) or 1mg CLC/120×10(6) sperm (CLC) prior to freezing. The fertilising ability of the FT sperm was compared in hormonally treated (equine/human chorionic gonadotropin hormones; eCG/hCG) weaned sows inseminated once (cervical insemination) at the following fixed-times after hCG administration: 37h (experiment 1) or 30h (experiment 2). In experiment 1, both treatments exhibited similar fertility rates of 67.7 and 55.9% for the control and CLC, respectively (P>0.05); however, the CLC group had a smaller litter size (11.3±0.9) than the control group (13.6±0.8) (P<0.05). In experiment 2, the pregnancy and farrowing rates were 65.2 and 66.7% for the control and CLC groups, respectively, and the litter size was 12.9±1 and 11.3±1 for the control and CLC groups, respectively, which were similar (P>0.05) for both treatments. These results indicate that the timing of insemination developed for the FT control sperm may not be suitable for CLC-treated sperm and that CLC-treated sperm may benefit from a shorter time interval between the hCG treatment and insemination. Moreover, acceptable results can be obtained with FT boar sperm with a single artificial insemination performed 30h after hCG treatment, which is the time interval recommended for fresh sperm., (Copyright © 2013 Elsevier B.V. All rights reserved.)