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This practice review examines some of the early evidence, and reporting, of housing market change in England prompted by the Covid-19 pandemic. Taking a transect from central London, through surrou...

The aim was to compare the effect of dimethylacetamide (DMA) and N-methylacetamide (NMA) concentrations on the quality and fertility of post-thaw chicken semen. Ejaculates were obtained from 30 Hi-Line White roosters and processed according to the following treatments: lake pre-freezing extender + 0.1M trehalose (LPF-T) + 6% DMA (control treatment), LPF-T + 9% DMA, LPF-T + 6% NMA, and LPF-T + 9% NMA. Sperm quality (viability, motility, and kinetic traits) was assessed before and after cryopreservation. A total of 15 laying hens per treatment were inseminated to assess fertility and embryo viability. Sperm cryopreserved in presence of DMA had significantly better in vitro quality compared to NMA, showing the highest proportion of viable and progressive motile sperm recovered after thawing. Furthermore, proportion of progressive motile sperm and the VCL, LIN, ALH, and WOB mean values were significantly improved in semen samples frozen/thawed with 6% compared to 9% cryoprotectant concentration. However, the best cryoprotective action on sperm quality played by DMA and the lowest cryoprotectant concentration did not translate into a concomitant advantage in in vivo semen fertility that showed no differences between cryoprotectant and cryoprotectant concentration treatments. Finally, the cryoprotectant DMA and NMA showed an opposite effect on embryo viability in comparison with the effect played on in vitro semen quality, being NMA more efficient than DMA on preserving viable embryos. The present results suggest the urgency to further decrease the cryoprotectant concentration in poultry semen freezing procedures and to assess the specific toxic effect of cryoprotectant on sperm integrity, fertility, and embryo development.

The aim of this study was to compare the effect of two permeant-cryoprotectants, dimethylacetamide (DMA) and N-methylacetamide (NMA) used at different concentrations (0%, 2%, 4%, 6%) on the quality and fertility of post-thaw rooster semen. Ejaculates were processed in 7 treatments: Lake pre-freezing+0.1 M trehalose (LPF-T) (control treatment), LPF-T+2% DMA, LPF-T+4% DMA, LPF-T+6% DMA, LPF-T+2% NMA, LPF-T+4% NMA, LPF-T+6% NMA. Sperm quality [sperm membrane integrity (SMI), motility and kinetic parameters] was assessed before and after cryopreservation. Fertility and embryo viability were recorded. Increasing both DMA and NMA concentration from 2 to 6% improved SMI, total motile sperm, progressive motile sperm (PMS), VCL, VSL and VAP values. PMS recovery rates were significantly the highest in 6% DMA, 4% NMA and 6% NMA treatments. Semen cryopreserved with DMA produced the best fertility and embryo viability at 6%; progressive lower values were recorded at lower concentrations, with no viable embryos at 2%. Semen cryopreserved with NMA showed the best fertility values at 2% and lower values were recorded at higher concentrations; live embryos were found in all NMA treatments. Finally, NMA and DMA showed a similar positive concentration dependent effect of the quality of cryopreserved semen. NMA, not DMA, provided the highest fertility and embryo viability values at the lowest 2%. Therefore, the use of NMA is recommended in order to reduce the cryoprotectant concentration, with a concomitant reduction in the risk of toxicity, providing at the same time the adequate cryoprotective action to obtain viable embryos after artificial insemination of cryopreserved chicken semen.

The ongoing COVID-19 pandemic has brought the issue of housing quality into sharp relief. For many people, home before the pandemic was a place to return to after work, school or a holiday: one of a number of spaces in which life is played out. But COVID-19 has focused daily existence on the home. It has suddenly become more intensely multi-functional: a workspace, a classroom and a leisure space all bundled into one (it was always these things, but never to the exclusion of everywhere else). This situation will not last, but individuals’ and households’ perceptions of the required utility of home may have changed irrevocably. Housing quality, and how to support and achieve that quality, is a perennial debate in planning and housing circles. Regulation and good design feature as answers or antidotes to the worst crimes of largely unfettered speculative development in the UK, the most heinous of which have often been committed by developers of the conversions of commercial buildings to residential use, under deregulated permitted development rights (PDR) in England. In this viewpoint, we bring together past research on regulation and design innovation to provide a brief commentary on likely future debates in this area.

The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ techniques to improve the efficiency of conservation programs. Aim of the present review is to describe all the activities developed for the implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. These activities can be classified into three main topics: (1) identification of species-specific semen freezing/thawing reference procedures; (2) drafting Standard Operative Procedures (SOP) for the implementation of the semen cryobank; (3) storage of semen doses from Italian chicken and turkey breeds to establish the cryobank. Several trials have been developed to identify a specie-specific semen cryopreservation protocol for chickens and turkeys. The major results are reviewed and a final reference protocol described. Taking into consideration the FAO guidelines for cryoconservation of animal genetic resources, SOP were drafted with the aim to provide technical guidance and logistical support on the choice of priority breeds, selection of birds for semen production, infrastructures and storage sites, birds and semen management, cryopreservation process and doses traceability. Lastly, the Italian Semen Cryobank was created. A total of 112 semen doses from 22 cockerels of three breeds, and 74 doses from 12 turkey males of three breeds were stored in the Cryobank. Breed specific semen quality parameters assessed before and after cryopreservation are reported. The described activities provide information and tools useful for the implementation of semen cryobanking in avian species and might be transferred also to other species after appropriate adaptations.HIGHLIGHTS Implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds Drafting Standard Operative Procedures provides technical guidance and logistical support on the design and establishment of the cryobank Semen cryobank is a precious genetic reservoir and could be useful to safeguard genetic variability in small population in vivo conserved

This is the first of two case study chapters. It begins by looking back at Guangdong’s longer history before turning to the province’s contemporary relationship with Feng Shui. That analysis draws on interviews with key informants working in the built environment and is organised into three parts: past uses and attitudes of Feng Shui, current uses and attitudes, and Feng Shui in our three critical domains: domestic, commercial and public spaces. The case illustrates reform pressures on Feng Shui: the extraction of science and pseudoscience from superstition, and the political turn against Feng Shui versus the force of social demand that continues to make it relevant in the planning and design of the built environment. The chapter ends by noting the long overlay of absolute with abstract space, with Feng Shui instrumentalised in pursuit of political control in the past and economic gain today.

Feng Shui and the City examines the past and contemporary influences of Feng Shui on Chinese built environments across three domains: domestic space, spaces of commercial development and the public realm. This opening chapter introduces the meaning and origins of Feng Shui before examining the way that places/spaces everywhere are the products of cultural praxis. Societies imprint their values onto the spaces of their dwelling: the production of space is a social process. From that position, the chapter outlines a perspective on Feng Shui and the City which uses Lefebvre’s (1991) notion of absolute and abstract space: spaces of ‘cosmic naturalness’, ‘symbolic existence’ and ‘everyday life’ versus spaces of domination and control. It is proposed that Feng Shui, as cultural referent, has transitioned from symbolism to an instrument of commodification: a proposition then tested in two cases, which set up a broader discussion of modern Feng Shui’s authenticity.

The development of the large-scale intensive poultry production system caused, in recent past, an almost complete disappearance of native breeds. The Mericanel della Brianza (MB) is an Italian bantam chicken breed characterized by optimal rusticity and a very good attitude to broodiness. The aim of this study was to assess peculiar phenotypic characteristics, breeding performance and genetic makeup of a small MB chicken nucleus under an in situ conservation program at the Poultry Conservation Centre of Local Genetic Resources of the University of Milan since 2010. The number of selected breeders in the nucleus progressively increased over the 5 years time from 22 (8M+14F) in 2010 to 58 (10M+48F) in 2014. FAO guidelines for phenotypic characterization of animal genetic resources were used to describe the productive, the reproductive traits and the performance of the MB chickens. The mean number of eggs per hen per week increased progressively from 1.86 in 2010 to 2.33 in 2014. Overall mean egg weight was 33.8 g (SD 3.71 g and CV 11%) and proportion of fertile eggs was 84%. Genetic screening of breeders was performed in 2010, 2012 and 2014 using the panel of 30 microsatellite loci recommended by ISAG and FAO for biodiversity studies. A total of 120 blood samples from twenty-four families (eight in 2010, six in 2012 and ten in 2014) were included in the study. The Mean Number of Alleles (MNA), the allelic frequencies, the observed (H O ) and expected (H E ) heterozygosity, the Polymorphism Information Content (PIC) and the inbreeding coefficient (F IS ) value were estimated. All markers were polymorphic with the exception of the MCW0034 and MCW0248 loci. These two markers and other 4, not properly amplifying, were excluded from analyses. A total number of 77 alleles (MNA = 3.08) in 25 microsatellites have been detected in the overall population. The genetic diversity within and among families was determined with the Wright's F-statistics fixation index (F ST ). F ST showed similarities among breeders within the nucleus in 2012 and 2014, but disclosed a variation respect to the nucleus of individuals of 2010. The genetic distance observed across years was confirmed by the PCA analysis. Despite the selection applied to decrease the presence of undesired traits (i.e. both morphological and reproductive traits), the genetic variability was conserved in the nucleus population across years.

This paper examines the influence of Feng Shui on urban form and spatial design at multiple levels, from the domestic spaces of the home, through commercial development projects, to the planning and building of cities. It contrasts the ancient power of China’s emperors to directly plan cities according to Feng Shui principles with its indirect influence today, underpinned by cultural and commercial drivers rather than the direct influence of regulation. Although ‘official’ adherence to Feng Shui seems less explicit than it once was, there are signs that it retains a place in the decision-making environment. The paper concludes by advancing a research agenda around the embeddedness of Feng Shui within the cultures of planning regulation and decision making.

Summary The Milanino is an Italian chicken breed included in a conservation project run by the University of Milan. It is characterized by good fertility, heavy body weights, high adaptation ability to adverse climate conditions, and disease resistance. Because of these characteristics, the Milanino could represent an important genetic resource for alternative production systems. This research was aimed at studying the effect of bird density on growth and slaughter performance, as well as stress response in Milanino chickens kept in outdoor pens. One hundred and sixty Milanino chickens were randomly assigned to 2 experimental groups kept at different densities (2 m2/bird and 8 m2/bird) and were slaughtered at 185 days of age. Growth and slaughter performance and stress condition were recorded. The interaction bird density * sex * age significantly affected body weight and an opposite trend was found between females and males: heavier females were found in the high-density group, while heavier males were found in the low-density group. Bird density did not affect carcass weight data. The stress marker (H/L ratio) was significantly higher in birds kept at the higher density (2 m2/bird). In conclusion, the Milanino provided satisfactory growth performance with different rearing density but the lower density, 8 m2/bird, should be preferred to minimize welfare problems for male birds.

Concern for space in new homes in England grew during the mid-2000s, largely as a result of unfavourable floor space comparisons with housing being built elsewhere in Europe. English homes were getting smaller, but space standards in other countries appeared to be preventing the cramming of too many rooms onto shrinking floor plates. Therefore, government in England faced calls to prescribe national space standards as a way of guaranteeing a basic level of domestic functionality and liveability. Strict standards elsewhere were assumed to result in better housing products, albeit in the context of different planning and finance regimes. This paper uses interviews with regulators, architects and house-builders in Turin, Italy, to challenge this assumption and to argue that an appropriate, context-sensitive, balance between flexible regulation and innovations in design (frequently activated by site or space constraints) is often the more effective route to achieving greater functionality and liveability in new housing.

The planning system in England has been subject to dramatic shifts since 2010. The previous government's preference for a strong regional focus in land-use strategy has been substituted with an emphasis on local control and responsibility. This has involved a ‘down-scaling’ of the system, with the revocation of ‘regional spatial strategies’ and a re-emphasis of the primacy of local government plans. It has also brought a ‘localisation’ of plan-making to a neighbourhood scale and a new role for community actors within local development planning. The shift in scale and focus has been accompanied by concerns over the system's ability to achieve broader and longer-term spatial objectives. Drawing on in-depth interviews with senior planning officers, this paper reflects on the risks that reform poses to the achievement of strategic planning objectives, looking in particular at housing delivery.

The aim of this study was to assess the combined effect of dimethylacetamide (DMA) and two non-permeating cryoprotective agents, trehalose and sucrose, on the quality of post-thaw chicken semen. Adult Lohmann roosters (n = 27) were used. Semen was processed according to the following treatments: Lake pre-freezing extender +6% DMA (LPF, control treatment), LPF + 0.1 M trehalose (LPF-T treatment), LPF + 0.1 M sucrose (LPF-S treatment) and LPF + 0.1 M trehalose + 0.1 M sucrose (LPF-TS treatment). Semen was loaded into straws and frozen in nitrogen vapour. Sperm quality (viability, mobility and kinetic parameters) was assessed immediately after thawing (T0) and at 5 (T5), 10 (T10) and 15 min (T15) thereafter. The different cryodiluent combinations significantly affected the kinetic parameters. The presence of trehalose, alone or with sucrose, combined with DMA improved the quality of motion in cryopreserved sperm in comparison to DMA alone (LPF) and DMA with sucrose (LPF-S). In particular, the highest values in linearity (LIN) and wobble (WOB) were measured in the treatment LPF-T. The treatments significantly affected the recovery rate of progressive motile sperm that presented the best value soon after thawing in the LPF-T treatment; moreover, the presence of trehalose, alone (LPF-T) or with sucrose (LPF-TS), significantly improved the recovery rate of progressive motile sperm also at T5 and T10 compared to LPF and LPF-S. The present results show a positive synergic action of DMA and trehalose on motile function of thawed chicken sperm.

Milanino is a heavy Italian chicken breed included in a conservation project of the University of Milan and is an important genetic resource for alternative production systems. This research was aimed to study the effect of the dietary protein concentration on growth, slaughter performance and meat composition in free-range reared Milanino chickens. A total of 120 Milanino chickens were fed on different protein concentrations (HP = 20% CP and LP = 16% CP), reared according to a free-range system and slaughtered at 150 and 180 d of age. Growth, slaughter performance and meat (breast and thigh) composition were recorded. The protein concentration of the diet did not affect the overall Milanino mean body weight recorded in the straight-run group in the whole rearing period. However, the growth rate within sex was significantly different between the dietary treatments: heavier females were found in the HP group from 125 d onwards, while no differences were recorded in male body weights. The protein concentration of the diet did not affect carcass weight data or meat composition. The present results suggest the use of a low-protein diet for rearing straight-run Milanino chickens for long rearing periods. However, in females, a high-protein diet is recommended from 125 d of age onwards.

The global economic crisis impacted significantly on residential development in the UK, stalling some local projects as private companies encountered financial difficulty. In response, government put in place an array of tools for rescuing private schemes, including supplying the Registered Providers (RPs) of social housing with the means to purchase complete or partially complete private homes. What seemed an ideal solution—the private developer avoids bankruptcy and Registered Providers acquire much needed affordable homes—in reality has been complicated by several factors. Drawing on interviews with key actors in England and on examples of successful and unsuccessful transfers of housing stock from the private to the public sector, this review seeks to identify key impediments to this type of transfer, including the inability of RPs to acquire housing stock that does not adhere to particular ‘quality standards’.

This monograph reports on a study comparing the regulation of internal housing space in Italy – since the introduction of broad, generic standards in 1975 – and in England, where there are no universal rules governing internal space. After tracing the evolution of standards in both countries from public health legislation in the late 19th century to specific building and urban codes in Italy today, and a range of standards applied to some social housing in England, it outlines the space characteristics of homes in the two countries, before drawing on a series on interviews with key built environment professionals in the cities of Turin and Manchester, to unpack the ‘conditions’ of space standard regulation in both countries. The purpose of the study has been to explore the politics, practicalities, acceptability and impact of internal space regulation in Italy and England, and to ask why regulatory standards in Italy seem more palatable than they are in England and Wales: the only remaining part of the EU where legal minimum space standard for residential development remain absent. Discussion also centres on how regulatory approaches to delivering housing quality compare with approaches based on local negotiation through planning, and how these are situated in the context of different market drivers, lending and planning cultures. Moving to a regulatory approach in England (with fixed space standards) may be problematic given that such a move would not address the market fundamentals – speculative production and investment consumption – that, alongside land supply constraints, determine the amount of space in homes. In this context, a more effective strategy might be to expose home buyers to more information on internal space, thus influencing market behaviour and thereafter, the types of homes built to supply future demand.

The aim of the present study was to investigate the correlation between the number of ovarian follicles and in vitro embryo development and quality in sheep. Sarda ewe ovaries were classified according to the number of follicles:or=4 (Low), 5-7 (Intermediate), andor=8 (High). IVM, IVF and IVC were performed under standard conditions. Cleavage rate and blastocyst development were assessed 48 h after fertilization and on Days 6, 7 and 8 of culture, respectively. Expanded blastocysts were vitrified; blastocoel re-expansion and hatching rates were assessed at 8, 16 and 72 h post-thawing and hatched blastocysts were analyzed with the TUNEL assay. In a subset of thawed blastocysts the incorporation of amino acids was evaluated. The proportion of ovaries varied significantly among the three groups (ANOVA F=12.20, P=0), and more ovaries (59%) were assigned to the Low group than to the Intermediate (28%; ANOVA F=8.19, P=0.009) and High group (13%; ANOVA F=18.63, P=0), (High vs. Intermediate F=6.31, P=0.020). The three groups statistically differed in the proportion of total blastocysts (chi(2)(2)=22.616, P=0.00), of blastocysts produced on Days 6 (chi(2)(2)=6.829, P=0.033) and 7 (chi(2)(2)=6.810, P=0.033), while no difference was found in the proportion of blastocysts obtained on Day 8 (chi(2)(2)=3.874, P=0.144) of culture after fertilization. A higher proportion of total blastocysts was obtained from the High (44%) compared with the other two groups (Low: 28%, chi(2)(2)=22.629, P=0; Intermediate: 33%, chi(2)(2)=7.266, P=0.007), while the Low and Intermediate groups did not statistically differ either in the total blastocyst output (chi(2)(2)=3.384, P=0.066), nor in the number of blastocysts produced on Days 6 (Low: 7%, Intermediate: 9%; chi(2)(2)=0.874, P=0.35), 7 (Low: 14%, Intermediate: 16%, chi(2)(2)=1.256, P=0.26) and 8 (Low: 6%, Intermediate: 7% chi(2)(2)=0.554, P=0.45) of culture. The High group produced a significantly higher percentage of embryos on Days 6 (High: 13%, Low: 7%; chi(2)(2)=6.840, P=0.009) and 7 (High: 21%, Low: 14%; chi(2)(2)=6.806, P=0.009) of culture post-insemination than the Low group. The three categories did not differ in the blastocoel re-expansion (chi(2)(2)=0.095, P=0.95) and hatching rates (chi(2)(2)=0.754, P=0.68) after 72 h post-warming, in the total number of cells per blastocyst (ANOVA F=1.12, P=0.337) and in the (F=0.46, P=0.639) incorporation of amino acids. The number of TUNEL-positive cells per embryo was higher (ANOVA F=4.32, P=0.022) in the Low group compared to the other groups. In conclusion, high ovarian follicle number enhances in vitro embryo output in sheep, but has no effect on blastocyst quality.

The aim of the present trial was to study the effect of different freezing rates on the survival of cryopreserved rooster semen packaged in straws. Slow and fast freezing rates were obtained keeping straws at different distances in the vapor above the surface of the nitrogen during freezing. Adult Lohmann roosters (n=27) were used. Two experiments were conducted. In Experiment 1, semen was packaged in straws and frozen comparing the distances of 1, 3 and 5cm in nitrogen vapor above the surface of the liquid nitrogen. In Experiment 2, the distances of 3, 7 and 10cm above the surfaces of the liquid nitrogen were compared. Sperm viability, motility and progressive motility and the kinetic variables were assessed in fresh and cryopreserved semen samples. The recovery rates after freezing/thawing were also calculated. In Experiment 1, there were no significant differences among treatments for all semen quality variables. In Experiment 2, the percentage of viable (46%) and motile (22%) sperm in cryopreserved semen was greater when semen was placed 3cm compared with 7 and 10cm in the vapor above the surface of the liquid nitrogen. The recovery rate of progressive motile sperm after thawing was also greater when semen was stored 3cm in the vapor above the surface of the liquid nitrogen. More rapid freezing rates are required to improve the survival of rooster sperm after cryopreservation and a range of distances from 1 to 5cm in nitrogen vapor above the surface of the liquid nitrogen is recommended for optimal sperm viability.

This data article contains supporting information regarding the research article entitled “Combined effect of permeant and non-permeant cryoprotectants on the quality of frozen/thawed chicken sperm”(Mosca et. al., 2016) [1]. The combined effect of the permeant cryoprotectants agent dimethylacetamide and the non-permeant cryoprotectants agent trehalose on the quality of frozen-thawed chicken semen was assessed. In particular, the quantitative dimethylacetamide/trehalose ratio was investigated freezing semen samples according to the following treatments: trehalose 0.1M+0% dimethylacetamide (DMA-0), trehalose 0.1M+3% dimethylacetamide (DMA-3), trehalose 0.1M+6% dimethylacetamide (DMA-6).

The feasibility to accurately se- lect viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess if in vitro-produced embryo quality could be determined by the timing of blastocoelic cavity re-expansion after vitrification, warming, and in vitro culture using sheep as a model. Blastocysts were produced in vitro, vitrified/warmed, and cultured in TCM-199 plus 10% FCS for 72 hr. Embryos were divided into two groups: re-expanded within 8 hr (A) and from 8 to 16 hr (B) of IVC after warming. Fast re-expanded blastocysts showed higher in vitro hatching rates and total cell number calculated on the hatched blastocysts compared with slow re- expanded ones (P

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.

This is a book about love. Every page of this highly enjoyable text expresses the love that its author ‒ an architect, urban planner and three times mayor of Curitiba ‒ feels for cities. It is not ...

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake’s diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P 0.0001), while ATP values significantly decreased after thawing (P 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing. In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezabililty between the two species.

La biodiversità in campo agricolo è essenziale per il sostentamento della popolazione umana e per la tutela della ricchezza degli agro-sistemi. Dal 2008 la Regione Lombardia, in collaborazione con il CNR, ha avviato la creazione della Banca delle Risorse Genetiche Animali Lombarde (LABank) con l'intento di stoccare materiale seminale, ed eventualmente embrioni, di alcune razze locali lombarde di interesse zoo-economico. LABank è stata allestita presso l'Unità Organizzativa di Supporto dell'Istituto di Biologia e Biotecnologia Agraria (IBBA) del CNR al Parco Tecnologico Padano di Lodi. Gli animali donatori sono stati individuati e scelti grazie alla collaborazione dell'Associazione Italiana Allevatori e delle Associazioni Provinciali. L'archiviazione di tutte le informazioni riguardanti i soggetti donatori, il materiale genetico stoccato e la localizzazione dei campioni di LABank viene effettuata attraverso l'utilizzo di un software (Cryoweb). LABank aderisce al Network delle Criobanche delle Risorse Genetiche Animali Italiane al fin di condividere, tramite una banca virtuale, le informazioni relative al materiale genetico di razze di interesse zootecnico crio-conservato come riserva genetica e di creare una rete di istituzioni impegnate nella crio-conservazione di materiale genetico di razze locali.

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation.

The role of melatonin in modulating mammalian reproduction is of particular interest; however, its effects on ovarian follicles and their oocytes still remain to be characterized. This study determined the influence of melatonin treatment on follicular growth patterns and on in vitro oocyte developmental competence. In a first experiment, the effects of melatonin supplementation on follicular dynamics were evaluated using daily transrectal ultrasonographies for 21 days, in 7 multiparous Sarda goats receiving a subcutaneous implant of 18 mg of melatonin and in 5 control untreated does. Melatonin caused more follicular waves (5.2 ± 0.2 versus 4 ± 0.3; P < 0.05) as the waves were shortened at around 2 days when compared with the non-melatonin treated control goats (P < 0.001). Oocyte developmental competence was evaluated in a second experiment by applying procedures for in vitro embryo production. There were no significant differences in the total number of oocytes obtained from 6 control (n = 192) and 7 melatonin-treated (n = 265) goats given follicle stimulating hormone to induce follicular development. Differences in oocyte developmental competence between the two groups became evident after in vitro fertilization and culture; melatonin increased the rate of cleaved oocytes in comparison with control animals (82.5 versus 63.4%; P < 0.001), advanced timing of embryo development and enhanced blastocyst output (31.5 versus 16.3%; P < 0.01). However, blastocyst quality, as evaluated by cryotolerance and gene expression analysis, was not found to be different between the groups. In conclusion, in vivo melatonin treatment is beneficial for increasing ovarian follicle turnover and improving oocyte developmental competence and kinetics of the blastocyst. © 2009 Blackwell Munksgaard.

We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV

The influence of trehalose on European mouflon spermatozoa cryopreservation during the non-breeding season was tested. Semen was frozen in two different extenders: (a) recommended Tris-based ram extender (CTR); (b) CTR extender supplemented with trehalose 0.147 mM (TRH). Sperm viability and acrosome integrity were assessed using propidium iodide and fluorescein isothiocynate labelled Pisum Sativum agglutinin. Trehalose significantly enhanced sperm viability after thawing compared with CTR extender (62.7% vs 51.8%; p < 0.05), whereas no differences were observed on acrosome integrity (42.9% vs 42.1%). Trehalose influence was also evidenced in the in vitro fertility test performed with sheep oocytes matured in vitro. Both fertilization rates (60.9% TRH vs 43.6% CTR; p < 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p < 0.001) were higher for trehalose frozen semen compared with control extender frozen semen. A higher percentage of zygotes resulting from fertilization with trehalose cryopreserved semen presented the first cleavage earlier if compared with the group fertilized with control semen (48.7% vs 31.5%, respectively; p < 0.01). This result was confirmed by embryo kinetic development. Fertilization with trehalose cryopreserved semen leaded to an higher percentage of blastocysts (40.2% vs 27.8% CTR; p < 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p < 0.05). Our data demonstrated that, during mouflon non-breeding season, trehalose extender enhances spermatozoa viability and its in vitro fertilizing capacity, allowing the production of an higher number of blastocysts.

The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.

Our aim was to evaluate if loading prepubertal ovine oocyte with trehalose would impact on their further developmental potential in vitro and if it would improve their survival to vitrification procedures. COCs matured in vitro with (TRH) or without (CTR) 100mM trehalose were tested for developmental potential after in vitro fertilization and culture. Trehalose uptake was measured by the antrone spectrophotometric assay. No differences were recorded between the two experimental groups in fertilization rates (91.1 CTR vs 92.5% TRH), cleavage rates calculated on fertilized oocytes (96.1 CTR vs 95.4% TRH), first cleavage kinetic (56.1 CTR vs 51% TRH), and blastocyst rates (14.3 CTR vs 13.0% TRH). Anthrone assay revealed that in TRH group trehalose concentration/oocyte was 2.6microM. MII oocytes were then vitrified using cryoloops in TCM 199 containing 20% FCS, sucrose 0.5M, 16.5% Me(2)SO, 16.5% EG and plunged in LN(2). After warming, oocytes from TRH and CTR groups were tested for membrane integrity using the propidium iodide (PI)/Hoechst differential staining, and for developmental ability after in vitro fertilization. Trehalose in maturation medium affected membrane resistance (P

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus–oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean ± s.e.m., 89.67 ± 5.74 and 26.69 ± 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90β and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups.

In order to preserve endangered psittacine species, more basic and applied research in reproductive biology is required. Assisted reproductive technologies such as artificial insemination play an important role in parrots species conservation programs to overcome the problem of infertile eggs and male infertility. The aim of this study was to define an effective in vitro protocol in order to standardize the sperm quality evaluation in psittacines, studying Melopsittacus undulatus as model species. Semen was collected from twenty adult males by massage technique from May to June. Sperm concentration was measured by the spectrophotometric method. Sperm quality (sperm membrane integrity (SMI), motility, and kinetic parameters) was assessed on fresh semen. Three different experimental protocols were performed to compare the effects of various processing conditions on SMI, motility, and kinetic parameters. In protocol 1, test was performed by Lake extender with three different pH, 7.4 versus 8.2 versus 8.4, and two different equilibration temperatures after dilution of fresh semen (4°C versus 25°C). In protocol 2, two dilution rates of semen after collection were valuated, 1 : 3 versus 1 : 4, as well as three different semen storage temperatures (4°C versus 25°C versus 38°C) before sperm motility analysis with the computer-assisted sperm analysis (CASA). In protocol 3, two different Makler chamber temperatures (38 versus 41°C) during motility analysis were tested. A significant progressive improvement in spermatozoa motility and kinetic parameters was registered with pH 8.4. Progressive motility and all kinetic parameters were higher at 4°C equilibration temperature. Straightness (STR) kinetic parameter was better with 1 : 4 dilution rate. Total motile sperm was higher in 41°C Makler chamber. In this study, for the first time, the effects of different processing protocols on psittacines seminal quality analysis were investigated. Significant differences conditioning the effectiveness of analysis protocols have been described.