Phenotype transition from wild mouflon to domestic sheep.

The domestication of animals started around 12,000 years ago in the Near East region. This "endless process" is characterized by the gradual accumulation of changes that progressively marked the genetic, phenotypic and physiological differences between wild and domesticated species. The main distinctive phenotypic characteristics are not all directly attributable to the human-mediated selection of more productive traits. In the last decades, two main hypotheses have been proposed to clarify the emergence of such a set of phenotypic traits across a variety of domestic species. The first hypothesis relates the phenotype of the domesticated species to an altered thyroid hormone-based signaling, whereas the second one relates it to changes in the neural crest cells induced by selection of animals for tameness. These two hypotheses are not necessarily mutually exclusive since they may have contributed differently to the process over time and space. The adaptation model induced by domestication can be adopted to clarify some aspects (that are still controversial and debated) of the long-term evolutionary process leading from the wild Neolithic mouflon to the current domestic sheep. Indeed, sheep are among the earliest animals to have been domesticated by humans, around 12,000 years ago, and since then, they have represented a crucial resource in human history. The aim of this review is to shed light on the molecular mechanisms and the specific genomic variants that underlie the phenotypic variability between sheep and mouflon. In this regard, we carried out a critical review of the most recent studies on the molecular mechanisms that are most accredited to be responsible for coat color and phenotype, tail size and presence of horns. We also highlight that, in such a complicate context, sheep/mouflon hybrids represent a powerful and innovative model for studying the mechanism by which the phenotypic traits related to the phenotypic responses to domestication are inherited. Knowledge of these mechanisms could have a significant impact on the selection of more productive breeds. In fact, as in a journey back in time of animal domestication, the genetic traits of today's domestic species are being progressively and deliberately shaped according to human needs, in a direction opposite to that followed during domestication.

Do natural catastrophic events and exceptional climatic conditions also affect parasites?

Parasites and parasitologists have always lived together in good and bad luck in a sort of forced marriage. In recent times bad luck certainly prevailed, because of increasing man-made emergencies such as wars, chemical disasters, but also because of natural disasters, amplified by climate change, that condition more and more parasite­host equilibrium. The symposium at the National Congress of the Italian Society for Parasitology, was a first occasion for Italian parasitologists to reason about 'disaster parasitology' and researchers' responsibilities. Extreme weather events and their impacts on parasites' epidemiology are illustrated, comparing disasters that recently occurred in Italy with literature data. In particular, the Sardinian Island was hit subsequently by fires and floods exacerbating the effects on ecosystems and parasite­host-relationships. Examples of Cryptosporidium outbreaks in man and Fasciola hepatica infections in various hosts after heavy rains are reviewed and effects of droughts on pasture borne parasites, such as gastro-intestinal nematodes of ruminants are discussed. Heavy rains may also cause dissemination of toxic substances released accidentally from chemical plants as happened e.g. in Milan province (IT) after the dioxin hazard. The overlapping effects of strictly man-made disasters with climate change dependent extreme weather events is further challenging the understanding of what are the consequences of disasters on ecosystems and parasite epidemiology.GIS applications combined with AI programs may help to face the complex challenges, allowing the collection and analysis of spatial/temporal data at whatever level desired. Examples illustrated in the article suggest their employment also in a more systematic, prevention-oriented manner.

Correlation between wing bone microstructure and different flight styles: The case of the griffon vulture (gyps fulvus) and greater flamingo (phoenicopterus roseus).

Flying is the main means of locomotion for most avian species, and it requires a series of adaptations of the skeleton and of feather distribution on the wing. Flight type is directly associated with the mechanical constraints during flight, which condition both the morphology and microscopic structure of the bones. Three primary flight styles are adopted by avian species: flapping, gliding, and soaring, with different loads among the main wing bones. The purpose of this study was to evaluate the cross-sectional microstructure of the most important skeletal wing bones, humerus, radius, ulna, and carpometacarpus, in griffon vultures (Gyps fulvus) and greater flamingos (Phoenicopterus roseus). These two species show a flapping and soaring flight style, respectively. Densitometry, morphology, and laminarity index were assessed from the main bones of the wing of 10 griffon vultures and 10 flamingos. Regarding bone mineral content, griffon vultures generally displayed a higher mineral density than flamingos. Regarding the morphology of the crucial wing bones involved in flight, while a very slightly longer humerus was observed in the radius and ulna of flamingos, the ulna in griffons was clearly longer than other bones. The laminarity index was significantly higher in griffons. The results of the present study highlight how the mechanics of different types of flight may affect the biomechanical properties of the wing bones most engaged during flight.

Postnatal pituitary and follicular activation: a revisited hypothesis in a sheep model.

The importance of postnatal pituitary activation as regards female reproductive development is not yet understood. By taking advantage of the experimental model developed in a previous study, i.e. ewe lambs expressing markedly different ovarian phenotypes at 50 days of age, we designed this study to determine whether differences found in ovarian status during the early prepubertal period are due to different patterns of postnatal pituitary activation, and to assess whether these differences have long lasting effects on subsequent reproductive performance. Results showed that ewe lambs with high antral follicle count (AFC) at 50 days of age had significantly lower plasma FSH concentrations and higher anti-Mullerian hormone (AMH) concentrations during the first 9 weeks of age compared with low AFC ewe lambs (P<0.0001). With a longitudinal experiment we showed that a high AFC in the early prepubertal period is associated with consistently higher AMH concentrations and numbers of antral follicles up to the postpubertal period, and with higher pregnancy rates in the first breeding season. In addition, the effect of age in decreasing AMH concentrations was more marked in the low AFC group. Results of the present study demonstrate that ewe lambs undergo different patterns of postnatal pituitary activation. A high AFC at 50 days of age indicates an advanced phase of ovarian maturation, which was accompanied by constantly higher AMH concentrations up to the postpubertal period, a greater ovarian response to FSH stimulation and by higher pregnancy rates at first mating, as compared with the low AFC group.

Melatonin deprival modifies follicular and corpus luteal growth dynamics in a sheep model.

This study assessed the effect of melatonin deprival on ovarian status and function in sheep. Experimental procedures were carried out within two consecutive breeding seasons. Animals were divided into two groups: pinealectomised (n=6) and sham-operated (n=6). The completeness of the pineal gland removal was confirmed by the plasma concentration of melatonin. Ovarian status was monitored by ovarian ultrasonography for 1 year to study reproductive seasonality. Follicular and corpus luteal growth dynamics were assessed during an induced oestrous cycle. As the effects of melatonin on the ovary may also be mediated by its antioxidant properties, plasma Trolox equivalent antioxidant capacity (TEAC) was determined monthly for 1 year. Pinealectomy significantly extended the breeding season (310±24.7 vs 217.5±24.7 days in controls; P<0.05). Both pinealectomised and sham-operated ewes showed a well-defined wave-like pattern of follicle dynamics; however, melatonin deficiency caused fewer waves during the oestrous cycle (4.3±0.2 vs 5.2±0.2; P<0.05), because waves were 1 day longer when compared with the controls (7.2±0.3 vs 6.1±0.3; P<0.05). The mean area of the corpora lutea (105.4±5.9 vs 65.4±5.9 mm(2); P<0.05) and plasma progesterone levels (7.1±0.7 vs 4.9±0.6 ng/ml; P<0.05) were significantly higher in sham-operated ewes compared with pinealectomised ewes. In addition, TEAC values were significantly lower in pinealectomised ewes compared with control ones. These data suggest that melatonin, besides exerting its well-known role in the synchronisation of seasonal reproductive fluctuations, influences the growth pattern of the follicles and the steroidogenic capacity of the corpus luteum.

In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences.

BACKGROUND: In vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries may avoid side effects connected to hyperstimulation during IVF procedures, including the risk of cancer recurrence. In humans, the scarce availability of immature oocytes limits morphological studies. The monovular ovine may represent an experimental model for IVM studies. METHODS: To assess if the scarce developmental competence of prepubertal oocytes (PO) is related to morphological changes we analyzed, by light and transmission electron microscopy, cumulus-oocyte-complexes (COCs) from lambs (30-40 days old) and sheep (4-6 years old) at sampling and after 7 h, 19 h, 24 h of IVM. Meiotic progression was determined at the same time points. RESULTS: At sampling, the germinal vesicle (GV) of PO was round and centrally or slightly eccentrically located, whereas in adult oocytes (AO) it was irregularly shaped and flattened against the oolemma. PO, differently from AO, showed numerous trans-zonal projections. Organelles, including cortical granules (CGs), were more abundant in AO. After 7 h, the percentage of AO that underwent GVBD-MI transition increased significantly. In PO, the oolemma was juxtaposed to the ZP; in AO, it showed several spikes in correspondence of cumulus cells (CC) endings. In PO, organelles and isolated CGs were scattered in the ooplasm. In AO, groups of CGs were also present under the oolemma. After 19 h, PO underwent GVBD-MI transition; their oolemma showed several spikes, with CC projections retracted and detached from the ZP. AO underwent MI-MII transition; their oolemma regained a round shape. CGs were located beneath the plasmalemma, arranged in multiple, continuous layers, sometime discontinuous in PO. After 24 h, both groups reached the MII-stage, characterized by a regular oolemma and by expanded CCs. PO showed CGs distributed discontinuously beneath the oolemma, while AO showed a continuous monolayer of CGs. CONCLUSIONS: Even if PO were able of reaching morphological maturation after 24 h of IVM, our ultrastructural analysis allowed detecting the presumptive sequence of cytoplasmic alterations connected with the delay of nuclear maturation, that might explain the reduced developmental competence of such oocytes. Data from the sheep model are of interest for zootechny, and provide an experimental basis for improving human IVM technology.

Predictive value of antral follicle count and anti-Müllerian hormone for follicle and oocyte developmental competence during the early prepubertal period in a sheep model.

Circulating anti-Müllerian hormone (AMH) and antral follicle count (AFC) are addressed as suitable markers of oocyte quantity and quality during adulthood. To investigate whether AFC and circulating AMH could predict follicle development and oocyte quality during the prepubertal period we used 40-day-old ewe lambs with high, intermediate and low AFC (≥30, 16-29 and≤15 follicles respectively). The analysis of the response to the exogenous FSH ovarian reserve test showed a positive correlation between AFC, AMH plasma levels, total follicle number and the number of large follicles (≥3mm) grown after exogenous FSH administration. The incorporation of abattoir-derived oocytes collected from ovaries with different AFC in an in vitro embryo production system showed that a high AFC can predict oocyte quality in prepubertal ovaries, reflecting an ovarian status suitable for follicular development. The histological quantification of the ovarian reserve evidenced that AFC was not predictive of differences in either the number of healthy follicles or the size of the primordial follicle pool in prepubertal ovaries. Further studies are needed to investigate the implication on the reproductive performance of the significant inter-individual differences found in the present study in AFC and circulating AMH in the early prepubertal period.

First Gammaherpesvirus detection in a free-living Mediterranean bottlenose dolphin.

Recently, herpes viruses have been detected in different cetacean species from the Atlantic and in Mediterranean striped dolphins (Stenella coeruleoalba). While pathogens such as cetacean morbillivirus have been widely studied following recent epizootics, herpesvirus (HV) distribution and pathogenic effects in cetaceans are still understudied. This study reports the first molecular identification of a Gammaherpesvirus in the genital mucosa of a free-living Mediterranean bottlenose dolphin (Tursiops truncatus) stranded off the coast of central Italy. Sequenced herpesviral PCR product was closely related to other HVs recently isolated in the genital mucosa of various cetacean species.

Adaptive Response to Gillnets Bycatch in a North Sardinia Mediterranean Shag (Gulosus aristotelis desmarestii) Population.

Mediterranean Shag (Gulosus aristotelis desmarestii) is a seabird endemic to the Mediterranean and Black Seas, recently included in the IUCN list of threatened Species. Most of the reproductive colonies are hosted in Sardinia and surrounding islets. Bycatch in fishing nets is one of the most significant threats for this population. Our work aimed to assess alterations in the sex ratio caused by bycatch and to study the adaptive response of the population to a skewed adult sex ratio. The sex ratio of Mediterranean Shags found drowned in the gillnets near the colonies and that of the nestlings of the Corcelli (northeast Sardinia) colony was determined using the sex-linked polymorphism of the gene Chromobox-Helicase-DNA-binding 1. The data of the shags found drowned in gillnets evidenced a high mortality rate (83.3%; p < 0.001) and a larger size of males (35% heavier than females, p < 0.05) compared to females, supporting the theory that heavier individuals are able to forage at great depths. With 64.8% of the nestlings being male, the sex ratio of nestlings was statistically different from parity (p < 0.05). Furthermore, it was related to the brood size. In one- and two-chick broods, 73% and 70% of nestlings, respectively, were males, while in three-chick broods, only 33% were males. Our data identify the higher rate of male shags drowned in gillnets as a factor causing an alteration of the sex ratio in the Mediterranean Shag population. According to the Sex Allocation Theory, an adaptive adjustment of sex made by adult females restores the Mendelian sex ratio in the population.

Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model.

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12-14 years) and young adult ewes (4-5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E(2)) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E(2) and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.

Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner.

Cryopreservation harms spermatozoa at different levels and thus impairs their fertilizing ability. The role of melatonin in protecting spermatozoa from different kind injuries has been widely reported. Thus, this study tested whether the addition of melatonin to ram semen freezing extender could exert a protective effect and ameliorate postthawing sperm function. Melatonin was added to recommended ram extender to yield five different final concentrations: 0.001, 0.01, 0.1, 1, and 10 mm. A control group without melatonin supplementation was included. Spermatozoa viability, motility parameters, and intracellular ATP concentrations were evaluated both before and after cryopreservation, while DNA integrity and in vitro fertilizing ability were evaluated only after thawing. Obtained results showed that the concentration of 1 mm melatonin led to higher viability rates, higher percentages of total motile and progressive motile spermatozoa, higher percentages of spermatozoa with average rapid and medium velocity, higher intracellular ATP concentrations, and higher DNA integrity among semen frozen in control and melatonin-supplemented extenders (P<0.05). In addition, results obtained after the IVF test showed that at 1 mm concentration, melatonin led to a faster first embryonic division and to higher total cleavage rates compared to the other experimental groups (P<0.05). No difference in embryo output was observed among the six experimental groups. In conclusion, the addition of melatonin to ram semen freezing extender protected spermatozoa during cryopreservation in a dose-dependent manner. These results are likely to be mediated by its well-known antioxidant properties, even if a direct action of the indolamine cannot be ruled out.

Anthropogenic Drivers Leading to Population Decline and Genetic Preservation of the Eurasian Griffon Vulture (Gyps fulvus).

Human activities are having increasingly devastating effects on the health of marine and terrestrial ecosystems. Studying the adaptive responses of animal species to changes in their habitat can be useful in mitigating this impact. Vultures represent one of the most virtuous examples of adaptation to human-induced environmental changes. Once dependent on wild ungulate populations, these birds have adapted to the epochal change resulting from the birth of agriculture and livestock domestication, maintaining their essential role as ecological scavengers. In this review, we retrace the main splitting events characterising the vultures' evolution, with particular emphasis on the Eurasian griffon Gyps fulvus. We summarise the main ecological and behavioural traits of this species, highlighting its vulnerability to elements introduced into the habitat by humans. We collected the genetic information available to date, underlining their importance for improving the management of this species, as an essential tool to support restocking practices and to protect the genetic integrity of G. fulvus. Finally, we examine the difficulties in implementing a coordination system that allows genetic information to be effectively transferred into management programs. Until a linking network is established between scientific research and management practices, the risk of losing important wildlife resources remains high.

Seasonal Effect on Developmental Competence, Oxidative Status and Tubulin Assessment of Prepubertal Ovine Oocyte.

The reproductive seasonality of domestic animals is often manipulated in order to have more reproductive periods for commercial purposes related to the production of milk and meat. It is scientifically proven that such an alteration of the reproductive activity in sheep entails a deterioration in oocyte quality, leading to an inability to generate embryos. Since oocytes obtained from prepubertal ewes can be incorporated into an in vitro embryo production system and considering that their quality is crucial to the success of in vitro procedures, the aim of this work was to investigate the effect of seasons on the quality of prepubertal ovine oocytes collected in autumn and spring. Ovaries were collected from a local slaughterhouse from 30-40-day-old suckling lambs during both seasons. Following 24 h of in vitro maturation, oocytes developmental competence, reactive oxygen species (ROS) intracellular levels, and mitochondrial activity were evaluated, and a tubulin assessment was performed. The results on embryo production, as a percentage of first divisions and number of blastocysts obtained, were significantly higher in oocytes collected in the spring. Mitochondrial activity in oocytes was higher, and ROS production significantly lower, in spring than in autumn. Tubulin PTMs (tyrosinated and acetylated α-tubulin) showed a higher immunoreactivity in oocytes collected in spring compared with autumn sampling. Our data showed that seasons may affect the developmental competence, energetic status, and tubulin assessment of oocytes recovered from prepubertal ewes. Therefore, special care should be taken when choosing the period of the year for prepuberal ovine oocytes collection aimed at in vitro embryo reproduction programs.

Genetic characterization and implications for conservation of the last autochthonous Mouflon population in Europe.

Population genetic studies provide accurate information on population structure, connectivity, and hybridization. These are key elements to identify units for conservation and define wildlife management strategies aimed to maintain and restore biodiversity. The Mediterranean island of Sardinia hosts one of the last autochthonous mouflon populations, descending from the wild Neolithic ancestor. The first mouflon arrived in Sardinia ~ 7000 years ago and thrived across the island until the twentieth century, when anthropogenic factors led to population fragmentation. We analysed the three main allopatric Sardinian mouflon sub-populations, namely: the native sub-populations of Montes Forest and Mount Tonneri, and the reintroduced sub-population of Mount Lerno. We investigated the spatial genetic structure of the Sardinian mouflon based on the parallel analysis of 14 highly polymorphic microsatellite loci and mitochondrial D-loop sequences. The Montes Forest sub-population was found to harbour the ancestral haplotype in the phylogeny of European mouflon. We detected high levels of relatedness in all the sub-populations and a mitochondrial signature of hybridization between the Mount Lerno sub-population and domestic sheep. Our findings provide useful insights to protect such an invaluable genetic heritage from the risk of genetic depletion by promoting controlled inter-population exchange and drawing informed repopulation plans sourcing from genetically pure mouflon stocks.

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes.

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5'-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 µm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV = 4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 µmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5'-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.

Involvement of E-cadherin in early in vitro development of adult and juvenile sheep embryos.

The oocyte-to-embryo transition in mammals depends on maternal proteins and transcripts, which accumulate during oocyte differentiation. The aim of the present study was to examine the role of the junctional proteins beta-catenin and E-cadherin during preimplantation in vitro embryo development in sheep, comparing the competence of adult and prepubertal oocytes. We analysed the concentration of beta-catenin and E-cadherin in immature and in vitro-matured oocytes. There was a significant increase in E-cadherin concentration after 24 h of in vitro maturation and this was lower in prepubertal oocytes than in adult ones. We therefore studied the expression and distribution of E-cadherin during the major transition from maternal to embryonic genome. E-cadherin distribution and localisation in sheep was age- and developmental-stage dependent and was related to developmental kinetics. In fact, in adults, the majority of embryos showed the proper distribution of E-cadherin just beneath the membrane surfaces of all blastomeres and the percentage of embryos with this distribution increased with the increase in cell number during development. On the contrary, and regardless of their developmental stage, the majority of prepubertal embryos showed an uneven distribution of the protein, often associated with the occurrence of cellular fragmentation. In conclusion, our results suggest that E-cadherin plays a pivotal role during preimplantation embryo growth in sheep and may be one of the possible cytoplasmic factors involved in the reduced developmental competence of prepubertal female gametes.

Morphological features and microtubular changes in vitrified ovine oocytes.

Cryobanking of oocytes collected from prepubertal donors may supply a virtually unlimited number of female gametes for both basic research and commercial applications. Prepubertal oocytes show some structural and functional limitations compared to the adult ones that may impair their ability to recover damages from cryopreservation. In oocytes, the meiotic spindle is acutely sensitive to temperature deviation, but capable of regeneration following cryopreservation. In the present work, we studied the effects of vitrification and post-warming incubation on the microtubular cytoskeleton and the tubulin post-translational modifications (tyrosination and acetylation) in prepubertal and adult oocytes. Obtained results showed that prepubertal oocytes are more affected by vitrification-induced injuries than adult ones. In fact, prepubertal oocytes showed more severe alterations of the meiotic spindle conformation and a higher percentage of parthenogenetic activation compared to adult ones. Moreover, in the adult oocytes the equilibrium between tyrosinated and acetylated α-tubulin was restored after 4 h of post-warming incubation. Diversely, in prepubertal oocytes the imbalance between tyrosinated and acetylated α-tubulin was increased during post-warming incubation. Our study shows that prepubertal oocytes react differently to the insults provoked by vitrification compared to adult oocytes, showing an impaired ability to recover from vitrification-induced injuries. In the evaluation of oocyte ability to recover from vitrification-induced injuries, tubulin post-translational modifications represent an important indicator for assessing oocyte quality.

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model.

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.

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population.

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December - March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 microl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique.

Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model.

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.