Cryopreservation of rabbit semen: effectiveness of different permeable and non-permeable cryoprotectants on post-thaw sperm quality and reproductive performances

Rabbit breeding for meat production is based mainly on artificial insemination (AI) programs. In order to obtain many of potential advantages of AI, improvement of the storage of rabbit semen is necessary. Therefore, meat rabbit farming would greatly benefit if semen could be stored after collection and subsequently used for AI without affecting fertility. The rabbit sperm can be stored by refrigeration (in liquid or solid extenders) or freezing. The use of frozen semen would provide more practical advantages for the commercial rabbit industry, as cryopreserved semen could potentially maintain the functionality of the sperm cells for months or even years by facilitating the samples transport in time and space. Cryopreservation has been widely used in the cattle industry, less used in other livestock species, such as pigs, sheep, poultry and rabbit. Therefore, a large number of protocols aimed to cryopreserve the rabbit semen have been developed by many researchers. Each of these protocols has involved in the study of some aspect that can affect the success of the rabbit sperm cryopreservation, such as the composition of the freezing medium, nature of cryoprotectant (CPA) and its concentration, freezing conditions and cooling and warming temperatures. The choice of the CPA is certainly one of the most important aspect for an effective freezing protocol for rabbit semen. The CPAs are chemical compounds included in freezing extenders to reduce the physical and chemical stresses resulting from the cooling, freezing, and thawing of sperm cells. The main biophysical factor that can cause cell destruction during the cryopreservation process is the formation of intracellular ice crystals that can be avoided by increasing the cellular dehydration process, by means of a CPA in the freezing solution. Hence, the addition of the permeable CPAs is a necessary step in the freezing protocol of rabbit sperm, because they minimize cellular injury during cryopreservation. Once CPA has been added to a sperm suspension, a period of time is required for the CPA to permeate the cells. This is called the equilibration time, which varies according to the nature and concentration of the CPA. So the efficacy of the CPAs depends on their type, their concentrations and the exposure time of sperm cells before freezing (equilibration time). Nevertheless, the paradox is that the permeable CPAs themselves could have a toxic effect on sperm (membrane destabilization, protein and enzymes denaturation) related directly to the concentration used and the time of cell exposure. Therefore, it is necessary the addition of a non-permeable cryoprotectant to offset the cryodamage caused by permeable CPA. Different combination of permeable and non-permeable CPAs have been tested for the cryopreservation of rabbit semen, but none of them has given optimal results to consider one of these as the suitable CPA for freezing rabbit semen. Therefore the process of rabbit sperm cryopreservation still suffers for a lack of standardized freezing protocols. In this thesis were conducted three different researches, that have as common denominator to find an effective freezing protocol for rabbit semen, by studying of the effects of different permeable and non-permeable CPAs. The first study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different CPA concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the semen post-thaw quality. After establishing the best protocols for each CPA, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. The animals used for this study, were 32 rabbit bucks and 342 does. Semen was collected using an artificial vagina and the ejaculates were pooled (4 ejaculates/pool). Pooled semen samples were diluted to a ratio 1:1 (v:v) with a freezing medium composed of Tris-citrate-glucose (TCG) containing 8, 12, or 16% DMA or DMSO (all combined with 2% sucrose as a non-permeating CPA) to give final concentrations of 1% sucrose and 4%, 6% or 8% DMA or DMSO. The diluted semen was loaded in 0.25 mL plastic straws and equilibrated for 5, 15 or 45 min before freezing in liquid nitrogen vapor (5 cm above the liquid nitrogen surface). The variables assessed after thawing were sperm motility, viability, osmotic-resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the CPA concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 minutes. These freezing protocols were selected to compare the two CPAs in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group), were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Conception rates and numbers of total born were similar respectively for the DMSO-frozen (79.8% and 7.7±0.3 young per kindling) and fresh semen (81.6% and 8.6±0.3) yet higher (P≤0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7±0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the CPA (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the CPA is highly influenced by the concentration of CPA used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm. The second study was designed to identify the most effective non-permeable CPA for the cryopreservation of rabbit semen by comparing the effects of different concentrations of low-density lipoproteins (LDL) on post-thaw sperm quality with those of whole egg yolk or sucrose. The performance of the non-permeable CPAs identified as the most effective was assessed in vivo by determining fertility and prolificacy rates. In this study 32 rabbit bucks and 90 does were used. Pooled semen samples were diluted to a ratio of 1:1 (v:v) in freezing extender (TCG and 16% DMSO as permeable CPA) containing as non-permeable CPAs 6, 8, 10 or 15% LDL from egg yolk, 0.1 M sucrose, or 15% egg yolk. The semen was loaded in 0.25 mL straws and frozen in liquid nitrogen vapor. After thawing, we determined sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Our results clearly revealed a significant effect of LDL concentration on semen quality. Also, at an optimal concentration of 10%, motility and acrosome integrity were improved over the values recorded for egg yolk (P<0.05). Based on the in vitro data, 3 groups of does (n = 30 each) were inseminated with fresh semen or semen frozen using sucrose or 10% LDL. Sucrose led to a significantly higher conception rate (86.7%) than LDL (66.7%) and reproductive performance was similar to that observed for fresh semen. Our findings indicate the markedly better performance of sucrose in vivo as a non-permeable CPA for the cryopreservation of rabbit semen. The aim of the third study was to evaluate the effect of addition of Ficoll 70 into the cryopreservation medium containing sucrose and DMSO on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (> 80%) were used in these experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% DMSO and 2% sucrose (control) or in the same medium enriched with 4% of Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged into liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-Fluos) and by electron microscopy. Better cryoprotective effect was observed when the Ficoll 70 was added, compared to the semen cryopreserved with sucrose and DMSO only. The higher values (P˂0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P˂0.05) of acrosome intact sperm was found in the Ficoll compared to control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, this study showed that the addition of the Ficoll 70 might improve several characteristic of rabbit spermatozoa measured in vitro following freezing/thawing. These researches were important because we have been able to find effective cryopreservation protocols, that showed similar reproductive performances compared to use of fresh semen. These findings provide direction for future studies designed to address the possibility of using doses of frozen semen for the artificial insemination of rabbits in commercial farms. Moreover, the use of frozen semen will be attractive to the establishment of a gene bank from national or endangered rabbit breeds as gene resources.Attualmente, i sistemi di allevamento intensivi per la produzione di carne di coniglio si basano principalmente su programmi di inseminazione artificiale (IA). Pertanto, l’allevamento intensivo del coniglio da carne potrebbe usufruire di molti vantaggi se il seme potesse essere conservato dopo la raccolta e successivamente utilizzato per l’IA senza compromettere la fertilità. Due sono le tecnologie disponibili per la conservazione del seme di coniglio: la refrigerazione (in diluenti liquidi o solidi) e il congelamento. L'uso di seme congelato fornirebbe vantaggi più pratici per l'allevamento del coniglio da carne, in quanto, esso potrebbe potenzialmente mantenere la funzionalità delle cellule spermatiche per mesi o anni, facilitando il trasporto dei campioni nel tempo e nello spazio. La crioconservazione del seme è stata ampiamente utilizzata nel settore bovino, meno in altre specie animali, come i suini, ovini, polli e conigli. Pertanto, un gran numero di protocolli di congelamento per il seme di coniglio sono stati sviluppati da molti ricercatori. Diversi sono i fattori che possono influenzare il successo del congelamento del seme, quali la composizione del diluente di congelamento, la natura del crioprotettore (CPA) e la sua concentrazione, le condizioni di congelamento, e le temperature di raffreddamento e scongelamento. La scelta del CPA è sicuramente uno degli aspetti più importanti per ottenere un efficace protocollo di congelamento per il seme di coniglio. I CPA sono molecole, che vengono aggiunte nel diluente di congelamento, per ridurre gli stress fisici e chimici derivanti dal raffreddamento, congelamento e scongelamento degli spermatozoi. Infatti, il principale fattore biofisico che può causare la distruzione cellulare, durante il processo di crioconservazione, è la formazione dei cristalli di ghiaccio intracellulari. La loro formazione può essere evitata aumentando il processo di disidratazione cellulare, mediante l’aggiunta di un CPA nella soluzione di congelamento. Il CPA richiede un periodo di tempo necessario affinché esso penetri nello spermatozoo. Questo tempo è definito tempo di equilibratura, che varia in funzione della natura e della concentrazione del CPA. Quindi l'efficacia di un CPA dipende dalla natura chimica, dalla concentrazione e dal tempo ottimale con cui le cellule spermatiche si espongono ad esso prima del congelamento. Tuttavia, il paradosso è che i CPA permeabili potrebbero avere un effetto tossico sugli spermatozoi (causando la destabilizzazione della membrana e/o la denaturazione delle proteine e degli enzimi). Quest’effetto tossico dipende dalla concentrazione del CPA utilizzata e il tempo di esposizione delle cellule spermatiche con esso. Pertanto, per mitigare questi effetti tossici, è necessaria l'aggiunta di un CPA non-permeabile. Per la crioconservazione del seme di coniglio sebbene, siano stati studiati diversi CPA permeabili e non-permeabili, nessuno di essi ha riportato risultati soddisfacenti. Pertanto il congelamento del seme di coniglio è ancora carente di uno specifico ed efficace protocollo. In questa tesi sono state condotte tre ricerche, che hanno avuto come comune denominatore quello di trovare un protocollo di congelamento efficace per il seme di coniglio, studiando gli effetti di diversi CPA permeabili e non-permeabili. Il primo studio ha avuto come scopo quello di identificare un protocollo di congelamento adatto per il seme di coniglio confrontando gli effetti di diverse concentrazioni di due CPA permeabili (dimetilacetammide (DMA) e dimetilsolfossido (DMSO)) e diversi tempi di equilibratura sulla qualità post-scongelamento del seme. Dopo aver stabilito i migliori protocolli per ogni CPA, la loro efficacia è stata confrontata in vivo, esaminando la capacità fertilizzante del seme congelato con quello fresco. In questa ricerca sono stati utilizzati 32 maschi e 342 femmine. Il seme è stato raccolto mediante una vagina artificiale e costituiti pool di seme (4 eiaculati/pool). I pool sono stati diluiti nel rapporto 1:1 (v:v), con un diluente di congelamento composto da TCG (Tris-acido citrico-glucosio) contenente l’8, il 12 o il 16% di DMA o DMSO, rispettivamente (tutti contenenti il 2% di saccarosio come CPA non-permeabile), ottenendo una concentrazione finale del 4, 6 o 8% di DMA o DMSO, e l’1% di saccarosio. Il seme diluito è stato aspirato in straws da 0.25 mL. Le straws sono state equilibrate a 5°C per 5, 15 o 45 minuti prima di essere congelate sui vapori di azoto (a 5 cm dal livello di azoto). Le variabili valutate dopo lo scongelamento sono state: motilità, vitalità, resistenza osmotica, integrità acrosomiale e integrità del DNA. La concentrazione del CPA e il tempo di equilibratura hanno influenzato le variabili considerate. I migliori risultati sulla qualità post-scongelamento sono stati ottenuti utilizzando il 6% di DMA o l’8% di DMSO e un tempo di equilibratura di 45 minuti. Questi protocolli di congelamento sono stati selezionati per comparare i due CPA in una prova di inseminazione artificiale. Tre gruppi da 114 coniglie (28 nullipare e 86 pluripare in ciascun gruppo) sono state inseminate con seme fresco e seme congelato utilizzando i due migliori protocolli. Il tasso di concepimento e il numero di coniglietti nati sono stati simili tra il seme congelato con il DMSO (79.8% e 7.7±0.3) e il seme fresco (81.6% e 8.6±0.3) e sono stati significativamente più alti rispetto al seme congelato utilizzando la DMA (47.4% e 6.7±0.4). Inoltre, il numero di coniglietti nati vivi ottenuti da seme congelato con DMSO, pur essendo inferiore rispetto a quello registrato con il seme fresco, è stato più alto rispetto a quello ottenuto utilizzando la DMA (P<0.05). Lo stato fisiologico delle coniglie (nullipare o pluripare) non ha avuto alcuna influenza sui risultati di fertilità e di prolificità. I nostri risultati indicano che la sopravvivenza degli spermatozoi congelati di coniglio, con DMSO o DMA rispettivamente, è stata fortemente influenzata dalla concentrazione del CPA utilizzato e dal tempo di equilibratura. Secondo i nostri dati di fertilità e prolificità in vivo, emerge che il DMSO è più efficace rispetto alla DMA per la crioconservazione degli spermatozoi di coniglio. Il secondo studio ha avuto come obiettivo quello di identificare il CPA non-permeabile più efficace per il congelamento del seme di coniglio, comparando gli effetti di diverse concentrazioni di lipoproteine a bassa densità (LDL), del tuorlo d’uovo intero e del saccarosio sulla qualità post-scongelamento del seme. I CPA non-permeabili che sono risultati più efficaci nel preservare la qualità post-scongelamento in vitro sono stati successivamente valutati in vivo, determinando le performance riproduttive (tasso di concepimento, fertilità, numero di coniglietti nati e numero di coniglietti nati vivi) e comparandole con il seme fresco. In questo studio sono stati utilizzati 32 maschi e 90 femmine. I pool di seme sono stati diluiti con un rapporto di 1:1 (v:v) usando un diluente di congelamento composto da TCG, contenente il 16% di DMSO come CPA permeabile e il 6, 8, 10 o 15% di LDL estratte dal tuorlo d’uovo, 0.1 M di saccarosio o il 15% di tuorlo d’uovo come CPA non-permeabile. Il seme diluito è stato aspirato in straws da 0.25 mL e congelato sui vapori d’azoto. Dopo lo scongelamento sono state valutate le seguenti variabili: motilità, vitalità, resistenza-osmotica, integrità dell’acrosoma e del DNA. I risultati ottenuti in vitro hanno rivelato un effetto significativo della concentrazione di LDL sulla qualità post-scongelamento del seme. La concentrazione ottimale è stata quella del 10%, mostrando valori di motilità e integrità dell’acrosoma migliori rispetto a quelli del tuorlo d’uovo (P<0.05). Sulla base dei risultati ottenuti in vitro il 10% di LDL e il saccarosio sono stati scelti come i migliori CPA non-permeabili, e utilizzati per una prova in vivo. Tre gruppi da 30 coniglie sono stati inseminati con seme fresco o con seme congelato utilizzando il saccarosio e il 10% di LDL, rispettivamente. Il tasso di concepimento ottenuto con il seme congelato con il saccarosio (86.7%) è stato significativamente più alto (P<0.05) rispetto a quello del seme congelato con le LDL (66.7%). Le performance riproduttive ottenute con il saccarosio sono state simili a quelle ottenute con il seme fresco. I nostri risultati indicano che, marcatamente in vivo, il saccarosio è il miglior CPA non-permeabile per il congelamento del seme di coniglio. Lo scopo del terzo studio è stato quello di valutare l’effetto dell’aggiunta del Ficoll 70, nel diluente di congelamento che conteneva saccarosio e DMSO, sulla qualità del seme di coniglio post-scongelamento. Il Ficoll, polimero di elevato peso molecolare, aumenta la viscosità del mezzo e per questo potrebbe proteggere meglio gli spermatozoi durante il processo di congelamento. Per questa sperimentazione sono stati utilizzati 4 maschi e 86 femmine. Sono stati utilizzati solo quegli eiaculati che hanno mostrato una buona motilità iniziale (>80%). I pool di seme sono stati diluiti con i seguenti medium di congelamento: 1) diluente commerciale (DMRS; Minitube, Germany) contenente il 16% di DMSO e il 2% di saccarosio (controllo), 2) Minitube contenente il 16% di DMSO, il 2% di saccarosio e il 4% di Ficoll 70 (Ficoll). Il seme diluito è stato aspirato in straws da 0.25 mL e congelato sui vapori di azoto liquido. La qualità del seme fresco e scongelato è stata valutata in vitro usando il Computer Assisted Semen Analysis system (CASA), sonde fluorescenti (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) e mediante microscopia elettronica. Un migliore effetto crioprotettivo è stato osservato dopo l’aggiunta del Ficoll 70 rispetto al seme congelato con il solo saccarosio e DMSO. I maggiori valori (P<0.05) di motilità totale e progressiva degli spermatozoi subito dopo lo scongelamento e 30 minuti di incubazione a 37°C sono ottenuti nel seme congelato con il Ficoll. Inoltre, il numero maggiore (P<0.05) di spermatozoi con acrosoma integro sono stati ritrovati nel gruppo trattato con il Ficoll rispetto al controllo. Nessuna differenza significativa è stata osservata per il tasso di fertilità e numero di nati tra il seme congelato e il seme fresco. Questo studio ha dimostrato che l’aggiunta del Ficoll 70 potrebbe migliorare la qualità del seme di coniglio post-scongelamento. Queste ricerche sono state importanti, in quanto siamo stati in grado di trovare protocolli di crioconservazione efficaci, che hanno mostrato performance riproduttive simili a quelle del seme fresco. Queste scoperte forniscono un contributo fondamentale per l’utilizzo di dosi di seme congelato da destinare all’IA negli allevamenti intensivi del coniglio da carne. Inoltre, l’utilizzo del seme congelato potrebbe essere di grande importanza per la creazione di una banca genetica per razze di coniglio nazionali o in via d’estinzione.Dottorato di ricerca internazionale in Benessere, biotecnologia e qualità delle produzioni animali - Welfare, biotechnology and quality of animal productions (XXVI ciclo - XXVI cycle

The Use of Ovarian Fluid as Natural Fertilization Medium for Cryopreserved Semen in Mediterranean Brown Trout: The Effects on Sperm Swimming Performance

In the context of the “Life—Nat.Sal.Mo” project, obtaining an effective semen cryopreservation protocol was an important milestone that allowed the implementation of the first European cryobank of native Mediterranean brown trout (S. cettii) inhabiting Molise rivers (Italy). The main use of our semen cryobank is represented by its practical application in artificial reproduction practices aimed at maximizing the genetic variability of the offspring and reducing the genetic introgression in the native trout populations in the project area. Thus, the choice of the most suitable activation/fertilization medium represents the last key step in the development of artificial reproduction protocols using cryopreserved semen. Therefore, the aim of the present study was to investigate the effect of ovarian fluid as a natural activation media of spermatozoa on the post-thaw sperm swimming performance of Mediterranean trout, comparing it with D-532 and a mixed solution of 50% D-532 and 50% ovarian fluid. Our results suggest that the presence of ovarian fluid alone or in combination with D-532 in the artificial microenvironment of reproduction represents a key factor to increase the success of fertilization when the frozen semen of Mediterranean brown trout is used

The role of semen cryobanks for protecting endangered native salmonids: Advantages and perspectives as outlined by the LIFE Nat.Sal.Mo. project on Mediterranean brown trout (Molise region – Italy)

The Mediterranean brown trout is one of the most endangered freshwater results clearly showed the efficiency of the freezing procedure used, both in vitro and in vivo. In fact, we recorded satisfactory values of post-thaw sperm motility and viability that ranged from 40% to 80%, and excellent fertilization rate in vivo, which ranged from 64% to 81% species. A complicated network of climate and human influences has severely harmed its biodiversity. The introduction of alien trout is one of the most serious threats to native populations’ intraspecific diversity. In Molise region (south-Italy) an important conservation program (LIFE Nat.Sal.Mo project) has recently been proposed to preserve the genetic integrity of native Mediterranean trout. This project, alongside safeguarding and re-establishing the habitats’ usefulness aims to restore the genetic integrity of the autochthonous population. This is one of the major goals, and it is accomplished by employing frozen wild breeder semen in conjunction with proper fertilization techniques to carry out artificial reproduction to enhance genetic diversity in the progeny and maintain fitness within self-sustaining populations. In this regard, the implementation of the first European semen cryobank has played a strategic role for conserving extant genomic diversity of native population. The goal of this review is to outline the procedures developed and guidelines established for the creation of a Mediterranean trout sperm cryobank. Here, we specifically provide an overview of some of the main challenges associated with the implementation of semen cryobank, the results achieved, the prospects for restoring genetic integrity in native populations, and lastly, future views for hatchery management to preserve the wild biodiversity of native salmonid species. During the project timeframe 1,683 semen doses, from 150 native breeders were stored inside the cryobank. Our results clearly showed the efficiency of the freezing procedure used, both in vitro and in vivo. In fact, we recorded satisfactory values of post-thaw sperm motility and viability that ranged from 40% to 80%, and excellent fertilization rate in vivo, which ranged from 64% to 81%

C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein δ (C/EBPδ), Bmi1, and ΔNp63α identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPδ and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPδ inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27Kip1 and p57Kip2. These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPδ, but not ΔNp63α, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPδ is recruited to the chromatin of positively (p27Kip1 and p57Kip2) and negatively (p16INK4A and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity

Anatomical and Functional Effects of Oral Administration of Curcuma Longa and Boswellia Serrata Combination in Patients with Treatment-Naïve Diabetic Macular Edema

Anti-vascular endothelial growth factor nowdays represents the standard of care for diabetic macular edema (DME). Nevertheless, the burden of injections worldwide has created tremendous stress on the healthcare system during the COVID-19 pandemic. The aim of this study was to investigate the effects of the oral administration of Curcuma longa and Boswellia serrata (Retimix(®)) in patients with non-proliferative diabetic retinopathy (DR) and treatment-naïve DME < 400 μm, managed during the COVID-19 pandemic. In this retrospective study, patients were enrolled and divided into two groups, one undergoing observation (Group A, n 12) and one receiving one sachet a day of Retimix(®) (Group B, n 49). Best-corrected visual acuity (BCVA) and central macular thickness (CMT) measured by spectral-domain optical coherence tomography were performed at baseline, then at one and six months. A mixed-design ANOVA was calculated to determine whether the change in CMT and BCVA over time differed according to the consumption of Retimix(®). The interaction between time and treatment was significant, with F (1.032, 102.168) = 14.416; η(2) = 0.127; p < 0.001, indicating that the change in terms of CMT and BCVA over time among groups was significantly different. In conclusion, our results show the efficacy of Curcuma longa and Boswellia serrata in patients with non-proliferative DR and treatment-naïve DME in maintaining baseline CMT and BCVA values over time

Overview of Turkey Semen Storage: Focus on Cryopreservation – A Review

Abstract This review updates the current state of technologies available for turkey semen storage (hypothermic- liquid storage and cryopreservation), with special attention paid to cryopreservation. Liquid semen can be stored for up to 24 or 48 h at temperatures around 5°C, while cryopreservation allows long-term storage at -196°C. The possibility of using frozen turkey semen for artificial insemination (AI) would have practical benefits for turkey production. Reported fertility rates in response to AI using frozen/thawed semen range from 15.8 to 84.3%. Unsatisfactory fertility may be attributed to an inability of turkey spermatozoa to successfully survive the freezing/thawing process, and this, along with the high variability observed, makes this technique unacceptable for commercial breeding programs. There is therefore a need to standardize the whole freezing and thawing process to improve the post-thaw quality of turkey semen and minimize variability in results. Finding an efficient freezing protocol for turkey semen will allow for the creation of a sperm cryobank, improving current prospects for the commercial use of frozen turkey semen and also for the long-term conservation of the genetic diversity of this bird

Sarcopenia: age-related skeletal muscle changes from determinants to physical disability.

Human aging is characterized by skeletal muscle wasting, a debilitating condition which sets the susceptibility for diseases that directly affect the quality of life and often limit life span. Sarcopenia, i.e. the reduction of muscle mass and/or function, is the consequence of a reduction of protein synthesis and an increase in muscle protein degradation. In addition, the capacity for muscle regeneration is severely impaired in aging and this can lead to disability, particularly in patients with other concomitant diseases or organ impairment. Immobility and lack of exercise, increased levels of proinflammatory cytokines, increased production of oxygen free radicals or impaired detoxification, low anabolic hormone output, malnutrition and reduced neurological drive have been advocated as being responsible for sarcopenia. It is intriguing to notice that multiple pathways converge on skeletal muscle dysfunction, but the factors involved sometimes diverge to different pathways, thus intersecting at critical points. It is reasonable to argue that the activity of these nodes results from the net balance of regulating mechanisms, as in the case of the GH/IGF-1 axis, the testosterone and Cortisol functions, the pro- and anti-inflammatory cytokines and receptors. Both genetic and epigenetic mechanisms operate in regulating the final phenotype, the extent of muscle atrophy and reduction in strength and force generation. It is widely accepted that intervention on lifestyle habits represents an affordable and practical way to modify on a large scale some detrimental outcomes of aging, and particularly sarcopenia. The identification of the molecular chain able to reverse sarcopenia is a major goal of studies on human aging

Zootechnical Brown Trout (Salmo trutta L. 1758) Ovarian Fluid Fails to Upregulate the Swimming Performances of Native Mediterranean Brown Trout (Salmo cettii Rafinesque, 1810) Sperm in the Biferno River

In external fertilizer fish, ovarian fluid (OF) seems to play a key role in fertilization success, improving spermatozoa swimming performance. These OF/sperm interaction mechanisms are frequently species-specific and/or population-specific and could decrease the risk of genetic introgression of wild populations from introduced or escaped zootechnical individuals. The Mediterranean brown trout (Salmo cettii) is threatened by genetic introgression with strains of domestic brown trout (Salmo trutta) that were introduced for recreational purposes. The aim of our study was to test if native S. cettii females, rather than zootechnical S. trutta, produce OF with a greater ability to upregulate the sperm motility of conspecific males. Thus, we compared the sperm swimming performances of males inhabiting the Biferno River (Molise region—Southern Italy) activated in native S. cettii vs. zootechnical S. trutta female’s OFs. In our study, native females’ OFs (20% diluted), compared to spring water, has the ability to significantly boost the sperm performance of the autochthonous males, while zootechnical S. trutta fails. These preliminary results suggest that OF-sperm interactions could potentially influence or direct the hybridization mechanisms involving the native Mediterranean trout inhabiting the Biferno River and the domestic lineage of brown trout introduced in the past

Semen cryopreservation for the Mediterranean brown trout of the Biferno River (Molise-Italy): comparative study on the effects of basic extenders and cryoprotectants

This study was designed to optimize the semen freezing protocol of the native Mediterranean brown trout inhabiting the Molise rivers through two experiments: an in vitro analysis of the effects of two basic extenders combined with three cryoprotectants on post-thaw semen quality; and an in vivo test to assess the fertilization and hatching rate. Semen was diluted at a ratio of 1:3 in a freezing medium composed of a glucose extender (A) or mineral extender (B). Each basic component contained 10% dimethylsulfoxide, dimethylacetamide or methanol. The post-semen quality was evaluated considering motility, duration of motility, viability and DNA integrity. The basic extender and cryoprotectant were shown to have significant effects on these variables, and the best results were obtained using extender A or B combined with dimethylsulfoxide (P &lt; 0.05). These freezing protocols were selected for fertilization trials in vivo. Fertilization and hatching rates were significantly higher in fresh semen. No significant differences were observed in frozen semen using extender A or B, although higher percentages of eyed eggs and hatching rates were recorded using extender A. According to our in vitro and in vivo results, the glucose-based extender and dimethylsulfoxide emerged as the best combination for an effective cryopreservation protocol for semen of this trou

Pathogenesis of tendinopathies: inflammation or degeneration?

The intrinsic pathogenetic mechanisms of tendinopathies are largely unknown and whether inflammation or degeneration has the prominent role is still a matter of debate. Assuming that there is a continuum from physiology to pathology, overuse may be considered as the initial disease factor; in this context, microruptures of tendon fibers occur and several molecules are expressed, some of which promote the healing process, while others, including inflammatory cytokines, act as disease mediators. Neural in-growth that accompanies the neovessels explains the occurrence of pain and triggers neurogenic-mediated inflammation. It is conceivable that inflammation and degeneration are not mutually exclusive, but work together in the pathogenesis of tendinopathies