Use of assisted reproductive technologies (ARTs) to shorten the generational interval in ruminants: current status and perspectives.

The challenges posed by climate change and increasing world population are stimulating renewed efforts for improving the sustainability of animal production. To meet such challenges, the contribution of genomic selection approaches, in combination with assisted reproductive technologies (ARTs), to spreading and preserving animal genetics is essential. The largest increase in genetic gain can be achieved by shortening the generation interval. This review provides an overview of the current status and progress of advanced ARTs that could be applied to reduce the generation time in both female and male of domestic ruminants. In females, the use of juvenile in vitro embryo transfer (JIVET) enables to generate offspring after the transfer of in vitro produced embryos derived from oocytes of prepubertal genetically superior donors reducing the generational interval and acceleration genetic gain. The current challenge is increasing in vitro embryo production (IVEP) from prepubertal derived oocytes which is still low and variable. The two main factors limiting IVEP success are the intrinsic quality of prepubertal oocytes and the culture systems for in vitro maturation (IVM). In males, advancements in ARTs are providing new strategies to in vitro propagate spermatogonia and differentiate them into mature sperm or even to recapitulate the whole process of spermatogenesis from embryonic stem cells. Moreover, the successful use of immature cells, such as round spermatids, for intracytoplasmic injection (ROSI) and IVEP could allow to complete the entire process in few months. However, these approaches have been successfully applied to human and mouse whereas only a few studies have been published in ruminants and results are still controversial. This is also dependent on the efficiency of ROSI that is limited by the current isolation and selection protocols of round spermatids. In conclusion, the current efforts for improving these reproductive methodologies could lead toward a significant reduction of the generational interval in livestock animals that could have a considerable impact on agriculture sustainability.

Population Genomic History of the Endangered Anatolian and Cyprian Mouflons in Relation to Worldwide Wild, Feral, and Domestic Sheep Lineages.

Once widespread in their homelands, the Anatolian mouflon (Ovis gmelini anatolica) and the Cyprian mouflon (Ovis gmelini ophion) were driven to near extinction during the 20th century and are currently listed as endangered populations by the International Union for Conservation of Nature. While the exact origins of these lineages remain unclear, they have been suggested to be close relatives of domestic sheep or remnants of proto-domestic sheep. Here, we study whole genome sequences of n = 5 Anatolian mouflons and n = 10 Cyprian mouflons in terms of population history and diversity, comparing them with eight other extant sheep lineages. We find reciprocal genetic affinity between Anatolian and Cyprian mouflons and domestic sheep, higher than all other studied wild sheep genomes, including the Iranian mouflon (O. gmelini). Studying diversity indices, we detect a considerable load of short runs of homozygosity blocks (<2 Mb) in both Anatolian and Cyprian mouflons, reflecting small effective population size (Ne). Meanwhile, Ne and mutation load estimates are lower in Cyprian compared with Anatolian mouflons, suggesting the purging of recessive deleterious variants in Cyprian sheep under a small long-term Ne, possibly attributable to founder effects, island isolation, introgression from domestic lineages, or differences in their bottleneck dynamics. Expanding our analyses to worldwide wild and feral Ovis genomes, we observe varying viability metrics among different lineages and a limited consistency between viability metrics and International Union for Conservation of Nature conservation status. Factors such as recent inbreeding, introgression, and unique population dynamics may have contributed to the observed disparities.

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.

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.

Heavy Metal(loid) Accumulation in the Ovarian Tissue of Free-Ranging Queens and Bitches Inhabiting Highly Polluted Urban Environments.

There is strong scientific evidence that exposure to environmental contaminants, such as heavy metal(loid)s (HMs), can impair female reproductive function. Pets, such as cats and dogs, who share the same habitat as humans, may be particularly useful sentinel models for detecting HMs in the ovary. In the present study, we compared the concentration of essential (Ems; Cu, Fe, Mn, Se, and Zn) and non-essential metal(loid)s (NEMs; Al, As, Cd, and Pb) in the ovarian tissues of free-ranging queens and bitches of different ages living in industrialized/highly polluted (south group) and non-polluted (north group) urban areas of the island of Sardinia, Italy. The results showed that both EMs and NEMs were present at detectable concentrations in feline and canine ovaries and their levels varied according to geographical areas and animal age. Among the EMs, Cu was found elevated in older queens and bitches inhabiting the southern area. Cadmium and lead were higher in feline and canine ovaries of older animals from the south compared to those living in the north. In addition, Cd and Pb concentrations increased in individuals of both species living in the south. These findings showed new perspectives for the use of pets as early warning sentinels of environmental pollution by HMs and for the risk of human exposure within a "One Health" approach. Pets may help to study the link between exposure to metals and female reproductive disturbances in mammals.

Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization.

BACKGROUND: Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 µmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 µmol/L) exposure. Therefore, the concentration of 1 µmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes. RESULTS: We found that 1 µmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 µmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes. CONCLUSIONS: Taken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

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.

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.

Raman spectroscopy-based approach to study the female gamete.

In the last years, an increasing interest has emerged on the development of new non-invasive methods for the assessment of oocyte quality in order to improve outcomes of assisted reproductive technologies (ARTs) either in medical or veterinary fields. Raman microspectroscopy (RMS) has been proposed as a promising tool for the examination of the mammalian female gamete and identification of markers of its developmental competence. This technique provides a unique spectral fingerprint indicative of molecular composition of the cell and allows probing subcellular compartments. Studies have been carried out analysing by RMS fixed or living oocytes derived from different animal models. RMS imaging has been successfully applied to discriminate the biochemical changes of the global molecular architecture of mouse oocytes at different stages of maturation and those occurring in different conditions of maturation and oocyte aging. RMS can also detect modifications of specific structural components, including the oocyte zona pellucida and F-actin subcortical cytoskeleton in fresh sheep oocytes and those underwent to vitrification procedures. Finally, the recent application of Coherent anti-Stokes Raman scattering (CARS) microscopy for examination of oocyte lipid component will be briefly discussed. CARS overcomes some limits of RMS providing vibrational and spectral information with higher sensitivity, spatial resolution which is ideal to study living oocytes. This review summarizes the research on RMS approaches for oocyte evaluation showing the high potential use, current limitations and new improvements.

Mitochondrial D-loop Sequence Variability in Three Native Insular Griffon Vulture (Gyps fulvus) Populations from the Mediterranean Basin.

The islands of Sardinia, Crete, and Cyprus are hosting the last native insular griffon populations in the Mediterranean basin. Their states have been evaluated from "vulnerable" to "critically endangered". The sequence analysis of molecular markers, particularly the mtDNA D-loop region, provides useful information in studying the evolution of closely related taxa and the conservation of endangered species. Therefore, a study of D-loop region sequence was carried out to estimate the genetic diversity and phylogenetic relationship within and among these three populations. Among 84 griffon specimens (44 Sardinian, 33 Cretan, and 7 Cypriot), we detected four haplotypes including a novel haplotype (HPT-D) that was exclusively found in the Cretan population with a frequency of 6.1%. When considered as a unique population, haplotype diversity (Hd) and nucleotide diversity (π) were high at 0.474 and 0.00176, respectively. A similar level of Hd and π was found in Sardinian and Cretan populations, both showing three haplotypes. The different haplotype frequencies and exclusivity detected were in accordance with the limited matrilineal gene flow (FST = 0.07097), probably related to the species reluctance to fly over sea masses. The genetic variability we observe today would therefore be the result of an evolutionary process strongly influenced by isolation leading to the appearance of island variants which deserve to be protected. Furthermore, since nesting sites and food availability are essential elements for colony settlement, we may infer that the island's colonization began when the first domestic animals were transferred by humans during the Neolithic. In conclusion, our research presents a first contribution to the genetic characterization of the griffon vulture populations in the Mediterranean islands of Sardinia, Crete and Cyprus and lays the foundation for conservation and restocking programs.

Ovine Granulosa Cells Isolation and Culture to Improve Oocyte Quality.

The functional cooperation between the oocyte and its surrounding granulosa cells is mandatory to oocyte growth and acquisition of developmental competence in mammalian species. Availability of in vitro methods for culture granulosa cells is important in understanding the biology of these cells and the response of maturing oocyte to in vitro culture and co-culture. Using the ovine as a model the ensuing chapter describes methods for primary culture of ovine granulosa cells and their co-culture with theca cells or oocytes that allow to mimic the molecular interactions between somatic cells and maturing oocyte and improve oocyte quality.

Effects of melatonin administration on seminal plasma metabolites and sperm fertilization competence during the non-reproductive season in ram.

The purpose of this study was to investigate the effects of ram melatonin treatment on the sperm quality and metabolite composition of the seminal plasma in the non-breeding season. Four mature rams were treated with 54 mg melatonin in March subcutaneous implants and four untreated rams served as control. At 0, 30, 90 and 120 days semen samples were collected and sperm, separated from seminal plasma, was evaluated for its capacity to fertilize and produce embryos in vitro. Seminal plasma metabolites were extracted and analyzed by capillary electrophoresis/mass spectroscopy. In the resulting electropherograms, the area corresponding to selected metabolites was extracted and quantified. Ram melatonin treatment affected the in vitro fertilization competence of sperm. Blastocyst output increased until 90 days after treatment (27.20 ±â€¯7.35 vs 54.7 ±â€¯4.4% at 0 and 90 days respectively; p < 0.05) while the untreated group did not show statistical differences. In treated rams, the concentration of melatonin in seminal plasma increased from 3.34 ±â€¯1.70 at day 0-9.65 ±â€¯2.89 AU (Arbitrary Units) after 90 days, then decreased to reach the level of the untreated ram after 120 days (p < 0.05). During 90 days after melatonin treatment, an increase (p < 0.05) in seminal plasma concentrations of glutamic acid (6.28 ±â€¯1.53 vs 14.93 ±â€¯1.53 AU at 0 and 90 days respectively), glutamine (16.89 ±â€¯4.65 vs 54.51 ±â€¯4.65 AU), carnitine (22.97 ±â€¯9.81 vs 104.30 ±â€¯9.81 AU), acetyl-carnitine (48.15 ±â€¯17.32 vs 217.69 ±â€¯17.32 AU), choline (1.82 ±â€¯1.55 vs 14.16 ±â€¯1.55 AU) and arginine (1.31 ±â€¯1.08 vs 14.25 ±â€¯1.08 AU) was detected. Tyrosine concentration increased during 30 days from melatonin treatment (12.79 ±â€¯3.93 vs 27.08 ±â€¯3.04 AU) but at 90 days its levels were similar to the untreated group. In conclusion, melatonin treatment during the non-breeding season improves the concentration of several metabolites in seminal plasma and sperm fertilization competence in Sarda breed ram.

A recovery time after warming restores mitochondrial function and improves developmental competence of vitrified ovine oocytes.

The aim of the present study was to assess the ability of vitrified/warmed oocyte to recover from vitrification-induced damages after warming. In vitro matured, vitrified/warmed ovine oocytes were assessed for developmental competence, mitochondrial activity and distribution, ATP, ROS and catalase levels during 6 h of in vitro culture using fresh oocytes as control. ATP content in vitrified oocytes was lower than control during 4 h of post warming culture (p < .01). Vitrified oocytes were able to fill this gap only after 6 h of post-warming incubation. Moreover, mitochondrial activity was significantly lower (p < 0.01) in vitrified oocytes compared to controls, and this difference was maintained up to 2 h of incubation. Then the activity increased and at 4 h it was higher compared to controls (p < 0.01). These oocytes showed an increasing rate of clustered distribution of mitochondria which was lower than controls during the first 4 h of post warming culture (p < 0.01). ROS level was significantly higher at 0 h in vitrified compared to control oocytes and this difference was maintained also at 2 h and 6 h of incubation (p < 0.01). Catalase level was higher in vitrified oocytes than controls (p < 0.01) during the entire culture period. Cleavage and blastocyst rates were lower in vitrified oocytes compared to control ones during the two first time point of incubation period (p < .01), indeed they increased significantly from 0 to 4 h of incubation post warming (p < 0.01). The study demonstrated that vitrified/warmed oocytes need an extra time to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, time of damage recovery after vitrification could be used to standardize the vitrification protocols and to improve the developmental competence of vitrified/warmed oocytes.

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.

The First Mitogenome of the Cyprus Mouflon (Ovis gmelini ophion): New Insights into the Phylogeny of the Genus Ovis.

Sheep are thought to have been one of the first livestock to be domesticated in the Near East, thus playing an important role in human history. The current whole mitochondrial genome phylogeny for the genus Ovis is based on: the five main domestic haplogroups occurring among sheep (O. aries), along with molecular data from two wild European mouflons, three urials, and one argali. With the aim to shed some further light on the phylogenetic relationship within this genus, the first complete mitochondrial genome sequence of a Cypriot mouflon (O. gmelini ophion) is here reported. Phylogenetic analyses were performed using a dataset of whole Ovis mitogenomes as well as D-loop sequences. The concatenated sequence of 28 mitochondrial genes of one Cypriot mouflon, and the D-loop sequence of three Cypriot mouflons were compared to sequences obtained from samples representatives of the five domestic sheep haplogroups along with samples of the extant wild and feral sheep. The sample included also individuals from the Mediterranean islands of Sardinia and Corsica hosting remnants of the first wave of domestication that likely went then back to feral life. The divergence time between branches in the phylogenetic tree has been calculated using seven different calibration points by means of Bayesian and Maximum Likelihood inferences. Results suggest that urial (O. vignei) and argali (O. ammon) diverged from domestic sheep about 0.89 and 1.11 million years ago (MYA), respectively; and dates the earliest radiation of domestic sheep common ancestor at around 0.3 MYA. Additionally, our data suggest that the rise of the modern sheep haplogroups happened in the span of time between six and 32 thousand years ago (KYA). A close phylogenetic relationship between the Cypriot and the Anatolian mouflon carrying the X haplotype was detected. The genetic distance between this group and the other ovine haplogroups supports the hypothesis that it may be a new haplogroup never described before. Furthermore, the updated phylogenetic tree presented in this study determines a finer classification of ovine species and may help to classify more accurately new mitogenomes within the established haplogroups so far identified.

Differences in the Kinetic of the First Meiotic Division and in Active Mitochondrial Distribution between Prepubertal and Adult Oocytes Mirror Differences in their Developmental Competence in a Sheep Model.

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development.

Phylogeny and patterns of diversity of goat mtDNA haplogroup A revealed by resequencing complete mitogenomes.

We sequenced to near completion the entire mtDNA of 28 Sardinian goats, selected to represent the widest possible diversity of the most widespread mitochondrial evolutionary lineage, haplogroup (Hg) A. These specimens were reporters of the diversity in the island but also elsewhere, as inferred from their affiliation to each of 11 clades defined by D-loop variation. Two reference sequences completed the dataset. Overall, 206 variations were found in the full set of 30 sequences, of which 23 were protein-coding non-synonymous single nucleotide substitutions. Many polymorphic sites within Hg A were informative for the reconstruction of its internal phylogeny. Bayesian and network clustering revealed a general similarity over the entire molecule of sequences previously assigned to the same D-loop clade, indicating evolutionarily meaningful lineages. Two major sister groupings emerged within Hg A, which parallel distinct geographical distributions of D-loop clades in extant stocks. The pattern of variation in protein-coding genes revealed an overwhelming role of purifying selection, with the quota of surviving variants approaching neutrality. However, a simple model of relaxation of selection for the bulk of variants here reported should be rejected. Non-synonymous diversity of Hg's A, B and C denoted that a proportion of variants not greater than that allowed in the wild was given the opportunity to spread into domesticated stocks. Our results also confirmed that a remarkable proportion of pre-existing Hg A diversity became incorporated into domestic stocks. Our results confirm clade A11 as a well differentiated and ancient lineage peculiar of Sardinia.

Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida.

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in ß-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.

Haplotype affinities resolve a major component of goat (Capra hircus) MtDNA D-loop diversity and reveal specific features of the Sardinian stock.

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity.We identified haplotype groups overrepresented in Sardinian goats as a result of founder effects. We found that breeders maintain diversity of matrilines most likely through equalization of the reproductive potential. Moreover, the relevant amount of inter-farm mtDNA diversity found does not increase proportionally with distance. Our results illustrate the effects of breeding practices on the composition of maternal gene pool and identify mtDNA types that may be considered in projects aimed at retrieving the maternal component of the oldest breeds of Sardinia.

Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization.

Parthenogenetic activation of the mammalian oocyte constitutes an essential step to a number of oocyte- or embryo-related technologies. Mammalian parthenotes are useful tools for studying the roles of paternal and maternal genomes in early mammalian development and are considered potential candidates for an ethical source of embryonic stem cells. We investigated the in vitro developmental competence of pre-implantation ovine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) together with the expression of a panel of fourteen genes at different times of development. IVF and PA embryos showed similar developmental competence. No differences in gene expression were observed between PA and IVF two cell-stage embryos, while PA morulae showed a significantly higher expression of IGF2. At the blastocyst stage, parthenotes exhibited up-regulation of TP-1, CDC2, and IGF2 transcripts and significantly lower levels of AQP3, ATP1A1, H2A.Z, hsp90beta, and OCT4, while NANOG, BAX, CCNB1, CDH1, GAPDH, and IGF2R displayed similar expression patterns in the two groups. Our study indicates that oocyte parthenogenetic activation does not impair in vitro pre-implantation development to the blastocyst stage, but affects the gene expression status of the embryo after the activation of its own genome.