Effect of genotyping density on the detection of runs of homozygosity and heterozygosity in cattle.

Runs of homozygosity (ROHom) are contiguous stretches of homozygous regions of the genome. In contrast, runs of heterozygosity (ROHet) are heterozygosity-rich regions. The detection of these two types of genomic regions (ROHom and ROHet) is influenced by the parameters involved in their identification and the number of available single nucleotide polymorphisms (SNP). The present study aimed to test the effect of chip density in detecting ROHom and ROHet in the Italian Simmental cattle breed. A sample of 897 animals were genotyped at low-density (50k SNP; 397 individuals), medium-density (140k SNP; 348 individuals), or high-density (800k SNP; 152 individuals). The number of ROHom and ROHet per animal (nROHom and nROHet, respectively) and their average length were calculated. ROHom or ROHet shared by more than one animal and the number of times a particular SNP was inside a run were also computed (SNPROHom and SNPROHet). As the chip density increased, the nROHom increased, whereas their average length decreased. In contrast, the nROHet decreased and the average length increased as the chip density increased. The most repeated ROHom harbored no genes, whereas in the most repeated ROHet four genes (SNRPN, SNURF, UBE3A and ATP10A) previously associated with reproductive traits were found. Across the three datasets, 31 SNP, located on Bos taurus autosome (BTA) 6, and 37 SNP (located on BTA21) exceeded the 99th percentile in the distribution of the SNPROHom and SNPROHet, respectively. The genomic region on BTA6 mapped the SLIT2, PACRGL, and KCNIP4 genes, whereas 19 and 18 genes were mapped on BTA16 and BTA21, respectively. Interestingly, most of genes found through the ROHet analysis were previously reported to be related to health, reproduction, and fitness traits. The results of the present study confirm that the detection of ROHom is more reliable when the chip density increases, whereas the ROHet trend seems to be the opposite. Genes and Quantitative Trait Loci (QTL) mapped in the highlighted regions confirm that ROHet can be due to balancing selection, thus related to fitness traits, health, and reproduction, whereas ROHom are mainly involved in production traits. The results of the present study strengthened the usefulness of these parameters in analyzing the genomes of livestock and their biological meaning.

Investigation of phenotypic, genetic and genomic background of Milk spectra in Sarda dairy sheep.

Aim of this study was to analyse the genetic background of milk Fourier transform infrared (FTIR) spectra in dairy sheep. Individual milk FTIR spectra, with 1060 wavenumbers each, were available for 793 adult Sarda breed ewes genotyped at 45,813 SNP. The absorbance values of each wavenumber was analysed using a linear mixed model that included dim class, parity and lambing month as fixed effects and flock-test date and animal as random effects. The model was applied to estimate variance components and heritability and to perform a genome-wide association study for each wavenumber. Average h2 of wavenumbers absorbance was 0.13 ± 0.08, with the largest values observed in the regions associated with the characteristic bonds of carbonylic and methylenic groups of milk fat (h2 = 0.57 at 1724-1728 cm-1; and h2 = 0.34 at 2811-2834 cm-1, respectively). The absorbance values of wavenumbers were moderately correlated with the estimated heritabilities. After the Bonferroni correction, a total of nine markers were found to be significantly associated with 32 different wavenumbers. Of particular interest was the SNP s63269.1, mapped on chromosome 2, that was found to be associated with 27 wavenumbers. Genes previously found to be related to traits of interest (e.g. disease resistance, milk yield and quality, cheese firmness) are located close to the significant SNP. As expected, the heritability estimated for the absorbance of each wavenumbers seems to be associated with the related milk components.

Fetal Growth and Osteogenesis Dynamics during Early Development in the Ovine Species.

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

Analysis of runs of homozygosity of cattle living in different climate zones.

Aim of this study was to analyze the distribution and characteristics of runs of homozygosity in Bos taurus taurus and Bos taurus indicus breeds, as well as their crosses, farmed all around the world. With this aim in view, we used single-nucleotide polymorphisms (SNP) genotypes for 3,263 cattle belonging to 204 different breeds. After quality control, 23,311 SNPs were retained for the analysis. Animals were divided into seven different groups: 1) continental taurus, 2) temperate taurus, 3) temperate indicus, 4) temperate composite, 5) tropical taurus, 6) tropical indicus, and 7) tropical composite. The climatic zones were created according to the latitude of the breeds' country of origin: i) continental, latitude ≥ 45°; ii) temperate, 45°< Latitude >23.26°; iii) tropics, latitude ≤ 23.26°. Runs of homozygosity were computed as 15 SNPs spanning in at least 2 Mb; number of ROH per animal (nROH), average ROH length (meanMb), and ROH-based inbreeding coefficients (FROH) were also computed. Temperate indicus showed the largest nROH, whereas Temperate taurus the lowest value. Moreover, the largest meanMb was observed for Temperate taurus, whereas the lowest value for Tropics indicus. Temperate indicus breeds showed the largest FROH values. Genes mapped in the identified ROH were reported to be associated with the environmental adaptation, disease resistance, coat color determinism, and production traits. Results of the present study confirmed that runs of homozygosity could be used to identify genomic signatures due to both artificial and natural selection.

Domestication and evolution of cattle originated different modern breeds in different places worldwide. The interaction between natural and artificial selection and the adaptation to environment shaped the genome, and the three different types of cattle here considered (taurus, indicus, and composite) may harbor different selection signatures. To study the difference among types and region of origin (tropics, temperate, and continental zones), Regions of Homozygosity (ROH) were used. ROH are continuous homozygous chromosomal segments identical by descendent, which characteristics can give information about inbreeding occurrence and natural and artificial selection. Moreover, it had been investigated which genes were mapped in these regions, and if interesting differences pertaining to environmental adaptation or fitness in general, could be observed.

Embryo biotechnologies in sheep: Achievements and new improvements.

To date, large-scale use of multiple ovulation and embryo transfer (MOET) programmes in ovine species is limited due to unpredictable results and high costs of hormonal stimulation and treatment. Therefore, even if considered reliable, they are not fully applicable in large-scale systems. More recently, the new prospects offered by in vitro embryo production (IVEP) through collection of oocytes post-mortem or by repeated ovum pick-up from live females suggested an alternative to MOET programmes and may be more extensively used, moving from the exclusive research in the laboratory to field application. The possibility to perform oocytes recovery from juvenile lambs to obtain embryos (JIVET) offers the great advantage to significantly reduce the generation interval, speeding the rate of genetic improvement. Although in the past decades several studies implemented novel protocols to enhance embryo production in sheep, the conditions of every single stage of IVEP can significantly affect embryo yield and successful transfer into the recipients. Moreover, the recent progresses on embryo production and freezing technologies might allow wider propagation of valuable genes in small ruminants populations and may be used for constitution of flocks without risks of disease. In addition, they can give a substantial contribution in preserving endangered breeds. The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.

Intrafollicular oocyte transfer (IFOT): Potential feasibility in the ovine species.

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 µL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

The Design of a New Catheter for Transcervical Artificial Insemination in Ewes.

In ovine species, transcervical artificial insemination (TCAI) is limited by the poor quality of frozen-thawed semen and by the convoluted cervical lumen hampering the passage of inseminating devices. The aim of the study was to test the efficiency of three newly designed catheters with bent tips of 3.5 mm, 5.0 mm or 8.0 mm in terms of reproductive performances (experiment 1) and to compare the results of TCAI with the best performing catheter of experiment 1 to those obtained in ewes submitted to surgical incision of cervical folds (SICF) prior to insemination (experiment 2). The following parameters were assessed: time to pass the cervix; depth of cervical penetration; site of deposition of semen; pregnancy (PR); and lambing rates (LR). The results of experiment 1 indicated that the 5.0 mm tip catheter resulted in deeper and faster TCAI and higher PR and LR compared to 3.5 mm and 8.0 mm tip catheters (p < 0.05). In experiment 2, TCAI with the 5.0 mm catheter did not differ from TCAI after SICF in terms of depth of semen deposition, time to pass the cervix, PR and LR (p < 0.05). In conclusion, the use of a catheter that allowed transcervical uterine deposition of semen without excessive manipulation led to satisfactory pregnancy rates.

Investigation of ß-hydroxybutyrate in early lactation of Simmental cows: Genetic parameters and genomic predictions.

Genomic information allows for a more accurate calculation of relationships among animals than the pedigree information, leading to an increase in accuracy of breeding values. Here, we used pedigree-based and single-step genomic approaches to estimate variance components and breeding values for ß-hydroxybutyrate milk content (BHB). Additionally, we performed a genome-wide association study (GWAS) to depict its genetic architecture. BHB concentrations within the first 90 days of lactation, estimated from milk medium infrared spectra, were available for 30,461 cows (70,984 records). Genotypes at 42,152 loci were available for 9,123 animals. Low heritabilities were found for BHB using pedigree-based (0.09 ± 0.01) and genomic (0.10 ± 0.01) approaches. Genetic correlation between BHB and milk traits ranged from -0.27 ± 0.06 (BHB and protein percentage) to 0.13 ± 0.07 (BHB and fat-to-protein ratio) using pedigree and from -0.26 ± 0.05 (BHB and protein percentage) to 0.13 ± 0.06 (BHB and fat-to-protein ratio) using genomics. Breeding values were validated for 344 genotyped cows using linear regression method. The genomic EBV (GEBV) had greater accuracy (0.51 vs. 0.45) and regression coefficient (0.98 vs. 0.95) compared to EBV. The correlation between two subsequent evaluations, without and with phenotypes for validation cows, was 0.85 for GEBV and 0.82 for EBV. Predictive ability (correlation between (G)EBV and adjusted phenotypes) was greater when genomic information was used (0.38) than in the pedigree-based approach (0.31). Validation statistics in the pairwise two-trait models (milk yield, fat and protein percentage, urea, fat/protein ratio, lactose and logarithmic transformation of somatic cells count) were very similar to the ones highlighted for the single-trait model. The GWAS allowed discovering four significant markers located on BTA20 (57.5-58.2 Mb), where the ANKH gene is mapped. This gene has been associated with lactose, alpha-lactalbumin and BHB. Results of this study confirmed the usefulness of genomic information to provide more accurate variance components and breeding values, and important insights about the genomic determination of BHB milk content.

Histological and Immunohistochemical Features of Trichoblastoma in a Sarda Breed Sheep.

Skin tumors with adnexal differentiation are commonly reported in dogs and cats, while only anecdotal evidence is available in sheep. Here we illustrate the macroscopic, histologic, and immunohistochemical features of a cutaneous lesion with adnexal differentiation in a 6-year-old female Sarda breed sheep, surgically treated for a horn-like mass located in the left pinna. Additionally, we investigate a possible contribution of Ovine Papillomaviruses (OaPVs). Histologically, the dermis was expanded by an expansive and unencapsulated multilobulated nodule composed of cuboidal to spindle basaloid cells arranged in variably-sized cytokeratins (CK) AE1-AE3, CK 5/6 and CK 34 beta E12, p63-positive winding cords with a characteristic palisade arrangement of neoplastic cells in the periphery of the tumor. Based on these results, the cutaneous neoplasm was diagnosed as a trabecular trichoblastoma with spindle cells and rare structures resembling papillary mesenchymal bodies. Additionally, multiple enlarged sebaceous lobules clustered around dilated ducts suggestive of sebaceous gland hyperplasia were detected near the trichoblastoma. No PV DNA was found in the examined tissues, suggesting that ovine PVs are not involved in the pathogenesis of the present skin tumors with adnexal differentiation. Further investigations and efforts are required to elucidate the prevalence of skin tumors with adnexal differentiation in this species.

Resveratrol supplementation and cryopreservation of buck semen.

Resveratrol (Resv) was tested to assess its effects on buck semen freezability. Ejaculates of 4 bucks were collected, washed and diluted in a commercial extender at 30 °C. Extended semen was divided into 4 aliquots supplemented with increasing concentrations of Resv: 0 µM (control); 10 µM; 25 µM and 50 µM. Aliquots were cooled to 4 °C in 5h and frozen in LN2. Thawing was performed at 37 °C for 30 s. At the 3 stages of the experiment (30 °C, 4 °C, thawing), motility (CASA), osmotic resistance (Hos test) and integrity of cytoplasm and acrosome membranes (PI/PSA staining) were assessed. Moreover, in thawed samples, the oxidative status (MDA assay) and early apoptosis (DNA fragmentation by TUNEL assay) were evaluated. Resveratrol supplementation did not affect most of the motility parameters analysed, except for total motility, ALH (lateral head displacement) and velocity distribution (P < 0.05). Functional and morphological integrity of membranes was not affected at any stage of the experiment (P > 0.05). In thawed spermatozoa, the oxidative status was not preserved by Resv (P > 0.05) while early apoptosis, was significantly decreased in the 50 µM Resv group (P < 0.05). Resveratrol did not improve buck semen freezability; the observed effects on motility and DNA were not dose dependent and not mediated by a potential anti-oxidant activity.

Reproductive Performance Following Transcervical Insemination with Frozen Thawed Semen in Ewes Submitted to Surgical Incision of Cervical Folds (SICF): Comparison with Laparoscopic Artificial Insemination.

Transcervical artificial insemination (AI) after the surgical incision of cervical folds (SICF) could represent a valid alternative to laparoscopic AI when frozen thawed semen is used. The aim of this experiment was to compare pregnancy (PR) and lambing rates (LR) of ewes submitted either to transcervical AI after SICF or to laparoscopic AI using frozen thawed semen. Pregnant at term ewes (n = 80) were allocated in two experimental groups. After lambing, one group (n = 39) was submitted to SICF. The remaining ewes that were regularly lambed were allocated to the group of laparoscopic AI (n = 40). Six months later, oestrous cycle of both experimental groups was synchronised and all ewes were artificially inseminated with frozen thawed semen. Ewes submitted to SICF underwent transcervical insemination and intrauterine deposition of semen was recorded. The remaining animals were submitted to laparoscopic AI. Pregnancy and LR were recorded. Intrauterine deposition of semen was possible in 89.7% pf ewes submitted to SICF. This group showed similar PR and LR compared to the laparoscopic group (respectively: PR, 71.8% vs. 70% and LR, 64.1% vs. 65%; p > 0.05). Transcervical AI after SICF may represent a valid alternative to laparoscopy in AI protocols requiring the use of frozen thawed semen.

Resveratrol treatment during maturation enhances developmental competence of oocytes after prolonged ovary storage at 4 °C in the domestic cat model.

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus-oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (-) 5 µM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P < 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P < 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.

Interaction between Copper Oxide Nanoparticles and Amino Acids: Influence on the Antibacterial Activity.

The increasing concern about antibiotic-resistance has led to the search for alternative antimicrobial agents. In this effort, different metal oxide nanomaterials are currently under investigation, in order to assess their effectiveness, safety and mode of action. This study focused on CuO nanoparticles (CuO NPs) and was aimed at evaluating how the properties and the antimicrobial activity of these nanomaterials may be affected by the interaction with ligands present in biological and environmental media. Ligands can attach to the surface of particles and/or contribute to their dissolution through ligand-assisted ion release and the formation of complexes with copper ions. Eight natural amino acids (L-Arg, L-Asp, L-Glu, L-Cys, L-Val, L-Leu, L-Phe, L-Tyr) were chosen as model molecules to investigate these interactions and the toxicity of the obtained materials against the Gram-positive bacterium Staphylococcus epidermidis ATCC 35984. A different behavior from pristine CuO NPs was observed, depending on the aminoacidic side chain. These results were supported by physico-chemical and colloidal characterization carried out by means of Fourier-Transform Infrared spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and Thermo-Gravimetric Analysis (TGA), Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and light scattering techniques (Dynamic Light Scattering (DLS), Electrophoretic Light Scattering (ELS) and Centrifugal Separation Analysis (CSA).

Surgery on cervical folds for transcervical intrauterine artificial insemination with frozen-thawed semen enhances pregnancy rates in the sheep.

In sheep industry, genetic progress rate achieved by artificial insemination (AI) is limited by the convoluted anatomy of the cervix, which does not allow the passage of an insemination catheter for uterine semen deposition. The aim of this study was to test, in 98 pregnant at term Sarda ewes, the effects of: Experiment 1) total or partial ablation of cervical folds and Experiment 2) 4 or 2 incisions of cervical folds, on the passage of an insemination catheter, deposition of frozen-thawed semen and pregnancy rates. Surgical procedures were performed within 24 h from parturition providing deep sedation and epidural anaesthesia. Duration of surgeries and post-operatory recovery were carefully monitored. For both experiments, 5 months since surgery, independently of the stage of oestrus cycle, cervical patency was tested through the transcervical passage of a palpation probe. Six months since surgery, in Experiment 1, ewes were naturally mated with fertile rams. In Experiment 2, ewes submitted to incisions of the cervical folds and a control group underwent synchronisation of oestrus and transcervical AI with frozen-thawed semen. Thirty days later, for both experiments, pregnancy rates were assessed by ultrasonography and lambing rates were recorded. Five months after surgery, in Experiment 1, transcervical passage of a palpation probe to reach the uterine lumen was possible in all ewes submitted to total and partial ablation of folds. In Experiment 2, this was achievable in 90.5% ewes with 4 incisions of the folds and in 89.6% ewes with 2 incisions with no significant differences among groups (P = 0.44). In Experiment 1, pregnancy rates in ewes mated to rams after total or partial ablation of the cervical folds was 100%. In Experiment 2, following transcervical AI, pregnancy rates were higher in groups submitted to 4 (63.7%) or 2 (41.4%) incisions of the cervical folds compared to the control group (8%; P<0.05). These data were confirmed at lambing with rates of 56.8% and 41.4% in ewes submitted to 4 or 2 incisions respectively, significantly higher than the control group (4%; P<0.05). Surgical ablation or incision of the cervical folds in post-partum ewes represent valid procedures for transcervical intrauterine deposition of semen for AI, obtaining satisfactory pregnancy rates. These procedures might be useful in programs of genetic selection and MOET.

Perspectives of nanotechnology in male fertility and sperm function.

Recent advances in nanotechnology have tremendously expanded its possible applications in biomedicine. Although, the effects of nanoparticles (NPs) at cellular and tissue levels have not been fully understood, some of these biological effects might be employed in assisted reproduction to improve male fertility particularly by enhancing sperm cell quality either in vivo or in vitro. This review summarises the available literature regarding the potential applications of nanomaterials in farm animal reproduction, with a specific focus on the male gamete and on different strategies to improve breeding performances, transgenesis and targeted delivery of substances to a sperm cell. Antioxidant, antimicrobial properties and special surface binding ligand functionalization and their applications for sperm processing and cryopreservation have been reviewed. In addition, nanotoxicity and detrimental effects of NPs on sperm cells are also discussed due to the increasing concerns regarding the environmental impact of the expanding use of nanotechnologies on reproduction.

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model.

BACKGROUND: Storage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model. METHODS: Ovaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/ß tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes. RESULTS: The results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage. CONCLUSIONS: Storage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours.

BACKGROUND: Cerium oxide nanoparticles (CeO2 NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO2 NPs for 96 h on the main structural and kinematic parameters of spermatozoa. METHODS: The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO2 NPs (0, 44 and 220 µg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H2DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni's as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. RESULTS: Exposure of ram spermatozoa to increasing doses of CeO2 NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO2 NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. CONCLUSIONS: We reported beneficial effects of CeO2 NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO2 NPs. These effects could not be related to an antioxidant activity of CeO2 NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

Hypoluteoidism in a dog associated with recurrent mammary fibroadenoma stimulated by progestin therapy.

BACKGROUND: Hypoluteoidism in the bitch is characterized by insufficient production and secretion of progesterone by the corpora lutea. It is a rare pathologic condition and during pregnancy, it leads to embryonic resorption or fetal abortion. Supplementary therapy with progestins is indicated during pregnancy to obtain delivery of vital puppies but unwarranted side effects of such treatment are poorly documented. CASE PRESENTATION: A 4-year-old, nulliparous, female Istrian Shorthaired Hound dog had been mated repeatedly in six heats with different dogs of proven fertility but signs of pregnancy did not develop. Estrous cycles, mating and pregnancies were monitored as hypoluteoidism or genital disease was suspected. During the first monitored estrus, the bitch was mated and on day 18 [day 0, day of estimated peak of luteinizing hormone (LH)], ultrasound examination showed three amniotic vesicles that were however found to be resorbed between day 20 and 23. Progesterone concentrations, measured by ELISA, were >8 ng/mL until day 12 and 1-2.5 ng/mL on days 20, 23 and 26. Primary hypoluteoidism was therefore suspected. In the second monitored estrus, the bitch was mated and during pregnancy, progesterone concentrations were >8 ng/mL until day 17 and 1-2.5 ng/mL on day 19. On days 20 and 22, two out of three embryonic vesicles had been resorbed. The bitch was treated with progesterone in oil from day 19 to day 58. Increase in the size of 2nd left thoracic mammary gland (T2-L) was observed and on day 46, ultrasound evaluation and biopsy were performed revealing a low-cellularity fibroadenoma. Parturition started spontaneously at day 65 but due to dystocia caused by fetal macrosomia, a Caesarean section was performed. During the next (third) monitored estrus, the bitch was bred again and during pregnancy, early decrease in progesterone concentration confirmed the diagnosis of primary hypoluteoidism. The bitch was treated with synthetic progestin (altrenogest) from day 8 to day 57. Five amniotic vesicles were detected by ultrasonography. Recurrence of swelling of T2-L was observed. On day 60, the bitch whelped five pups, two males and three females. As reported later by the owner, the latter did not show any sign of heat over the past 3 years. In one of them, clitoral hypertrophy and a blind ending vagina were diagnosed. CONCLUSIONS: This is the first description of early hypoluteoidism in a pregnant bitch developing a mammary fibroadenoma under progestin treatment.

Vaginal fold prolapse in a dog with pyometra and ovarian papillary cystadenocarcinoma.

CASE DESCRIPTION: A 7-year-old 42-kg (92.4-lb) sexually intact nulliparous female Italian Mastiff was examined because of a history of vaginal prolapse during diestrus. CLINICAL FINDINGS: A physical examination revealed vaginal fold prolapse. Abdominal ultrasonography revealed an enlarged uterus with hypoechogenic content, corpora lutea in the ovaries, and a cyst in the right ovary. Hematologic abnormalities included leukocytosis, neutrophilia, mild anemia, and low Hct. Progesterone and estradiol concentrations were 9.36 ng/mL and 30.42 pg/mL, respectively, in serum and 72.72 ng/mL and 792 pg/mL, respectively, in the ovarian cystic fluid. TREATMENT AND OUTCOME: Ovariohysterectomy was performed; the prolapsed tissue was repositioned by external manipulation and maintained in situ by temporary apposition of the vulvar lips with a retention suture. Anatomic and histologic examinations of the excised tissues revealed pyometra and papillary cystadenocarcinoma in the right ovary. The vaginal hyperplasia completely regressed at 35 days after surgery; 5 months after surgery, the dog's general condition was considered good. CLINICAL RELEVANCE: Findings in this case were indicative of a hormonally active ovarian papillary cystadenocarcinoma in a female dog in diestrus. Hormone production by the cystadenocarcinoma was the predisposing factor that induced pyometra, mucosal hyperplasia, and vaginal fold prolapse in the dog. On the basis of these concurrent disorders, ovariohysterectomy was an appropriate treatment.

Cerium dioxide nanoparticles did not alter the functional and morphologic characteristics of ram sperm during short-term exposure.

The aim of the study was to investigate the interaction and the short-term effects of increasing doses of cerium dioxide nanoparticles (CeO2 NPs) on ram spermatozoa, stored at 4 °C for up to 24 hours, on the main functional and kinematic parameters. Spermatozoa were incubated with 0, 22, 44, and 220 µg/mL of CeO2 NPs at 4 °C and submitted at 0, 2, and 24 hours to the following analyses: (1) intracellular uptake of CeO2 NPs by the spermatozoa; (2) kinematic parameters; (3) acrosome and membrane integrity; (4) integrity of DNA; (5) mitochondrial activity; (6) ROS production. The results indicated that the exposure of spermatozoa to increasing doses of nanoceria was well tolerated. No intracellular uptake of NPs by the cells was observed and both kinematic parameters and status of the membranes were not affected by the incubation with NPs (P > 0.05). Moreover, no influence on the redox status of spermatozoa and on the levels of fragmentation of DNA was reported among groups at any time (P > 0.05). The data collected provide new information about the impact of CeO2 NPs on the male gamete in large animal model and could open future perspectives about their biomedical use in the assisted reproductive techniques.