Alessia Gloria

Stemness characteristics and osteogenic potential of sheep amniotic epithelial cells.

We set out to characterize stemness properties and osteogenic potential of sheep AEC (amniotic epithelial cells). AEC were isolated from 3‐month‐old fetuses and expanded in vitro for 12 passages. The morphology, surface markers, stemness markers and osteogenic differentiation were inspected after 1, 6 and 12 passages of expansion, with an average doubling time of 24 h. AEC clearly expressed the stemness markers Oct‐3/4 (octamer‐binding protein‐3/4), Nanog, Sox2 and TERT (telomerase reverse transcriptase) and displayed low levels of global DNA methylation. Culture had moderate effects on cell conditions; some adhesion molecules progressively disappeared from the cell surface, and the expression of Sox2 and TERT was slightly reduced while Nanog increased. No changes occurred in the levels of DNA methylation. Cells organized in 3D spheroids were used for IVD (in vitro differentiation). Within these structures the cells developed a complex intercellular organization that involved extensive intercellular coupling despite continuous cell migration. Marked deposition of calcein in the ECM (extracellular matrix), increased ALP (alkaline phosphatase) activity, expression of bone‐related genes (osteocalcin) and the matrix mineralization shown by Alizarin Red staining demonstrate that AEC can undergo rapid and extensive osteogenic differentiation. AEC introduced in experimental bone lesions survived in the site of implantation for 45 days and supported consistent bone neoformation, thus showing promising potential applications in osteogenic regenerative medicine.

Blood analysis in newborn donkeys: hematology, biochemistry, and blood gases analysis

The knowledge of reference ranges for hematologic, biochemical, and blood gas parameters in the different species and the influence of breed and age on them is a fundamental tool for the clinician. For this reason, the aim of this study was to evaluate the age-related changes of hematologic and biochemical parameters in Martina Franca donkey foals during the first 3 weeks of life and of blood gases during the first 24 hours of age. Fifteen healthy donkey foals were enrolled; blood samples were collected from each foal at 10 minutes after birth, 1 hour after the first and second suckles, 12 and 24 hours after birth, daily from Day 2 to 7, and at Days 10, 14, and 21 of life. Erythrocytes, leukocytes, and platelets counts were assessed; also metabolic (alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, creatinphospokinase, lactate dehydrogenase, alkaline phosphatase, glucose, blood urea nitrogen, creatinine, total proteins, albumins, cholesterol, and total bilirubin) and electrolytic parameters (Ca, P, Mg, Na, K, and Cl) were evaluated. Finally, blood gases and metabolic parameters (pH, pCO2, pO2, sO2, TCO2, HCO3, lactate, and base excess) on venous blood were assessed with a portable analyzer. A statistical analysis to evaluate the influence of age and sex was performed. Several differences were found between sampling times, demonstrating that age influences these parameters. Moreover differences were found compared with data reported in literature for donkey foals of another species, horse foals, and adult donkeys. Although a great interindividual variation for some parameters exists, this study demonstrated that interval references should be addressed not only to different species, but also to specific breeds and to the neonatal period.

Kinematic study on the effect of pH on bull sperm function

Since the mammalian spermatozoa became capable of motion, during the epididymal transit, the spermatozoon swims in a liquid medium and it is completely dependent on the environmental conditions. Some reports have suggested an influence of pH on sperm kinetic characteristics, but no study has objectively described how motility changes in a different environmental pH. In this study, we evaluated the effect of different environmental pHs (5.5, 6, 6.5, 7, 7.5, 8, and 8.5) on kinetic parameters, sperm viability, mitochondrial activity, and sperm morphology of bull semen immediately and 1h after dilution. The results showed higher values for sperm motility characteristics, viability, and mitochondrial activity at pH 7 and 7.5. Values of pH lower than 6.5 and higher than 8 resulted in suboptimal motility, with a decrease in most parameters. At pH 8 and 8.5, a discrepancy between viability and total and progressive motility was found, with a significant amount of spermatozoa that were live but immotile. This reduction seemed related to a decrease in mitochondrial activity, possibly due to the increase in pH. The flow cytometric evaluation of sperm viability assessed by calcein AM was very consistent with the amount of spermatozoa with membrane integrity, evaluated in fluorescence by propidium iodide/SYBR-14 stain. Thus, the calcein AM stain could be used as viability stain instead the classic propidium iodide/SYBR-14 stain because this could allow the addiction of other functional stains without a overlapping of the fluorescent signal in the flow cytometer.

Characteristics of donkey spermatozoa along the length of the epididymis

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated.

Effect of sperm concentration on characteristics of frozen-thawed semen in donkeys

In this study, the effect of donkey sperm concentration in the straw during cryopreservation on the quality of thawed semen was evaluated. Samples from seven adult Martina Franca jackasses were collected three times using a Missouri artificial vagina. After estimation of volume and concentration, raw semen was evaluated for motility using a computer-assisted sperm analyzer (CASA); viability and acrosome integrity were also determined. Fresh semen was then centrifuged and re-suspended at five different concentrations (100, 250, 500, 750, and 1000×10(6)sperm/ml) with a commercial extender, packaged in 0.5ml straws, and frozen. After thawing, motility parameters, viability, and acrosome integrity were analyzed. The analysis of the data showed similar parameters of fresh semen compared with those of centrifuged and cooled samples. The sperm concentration in the straw affected the semen parameters analyzed after thawing, as suggested by evidence that when the concentration increased, the quality of the post-thawed semen decreased. Furthermore, the differences in total and progressive motility among samples at different concentrations are due to the immobilization of spermatozoa, as suggested by the finding that the percentage of static spermatozoa increased when the concentration increased. The reason for the impairment of semen quality when the sperm concentration increased was discussed. A great variability in cryo-resistance was found between jackasses but not within the same male, suggesting the presence of donkey males with semen that has acceptable and unacceptable freezing qualities.

The effect of the chamber on kinetic results in cryopreserved bull spermatozoa

In this study, the effect of the chamber used for the automated analysis of sperm motility by a computer‐assisted semen analysis system on sperm kinematics was evaluated, and the cause of this effect was also verified. Twenty‐three bull semen batches were thawed, and semen was diluted, aliquoted and analysed with six different chambers, (three capillary‐loaded chambers and three droplet (DR)‐loaded chambers). For each chamber type, each sample was analysed in quadruplicate, and the reliability of the analysis was tested using an intraclass correlation coefficient (ICC). Furthermore, sperm membrane integrity (MI) was evaluated, for each sample and chamber, in 12 randomly selected central and 12 edge fields. The ICC analysis showed that some parameters could have a significant variability related to the chamber. High stability of results was detected in Leja 4‐chamber slide. Furthermore, as previously reported in other studies, capillary‐loaded chambers seemed to affect the total and progressive motility and sperm velocities (average path velocity, straight line velocity and curvilinear velocity). These findings were corroborated by the evaluation of sperm MI that was significantly higher in the DR‐loaded chambers. This study confirms that the chamber used for the objective kinetic evaluation of bull‐thawed spermatozoa significantly affects the result. These differences could be present also in other species, even if the specific effect on the sperm kinematics should be verified. The Makler chamber seemed to give reliable results with negligible effects on sperm kinematics.

Effect of the season on some aspects of the estrous cycle in Martina Franca donkey

The Martina Franca (MF) donkey breed, with 48 jackasses and 515 jennies, is considered an endangered breed according to the data from the Monitoring Institute for Rare Breeds and Seeds in Europe. The knowledge of the estrous cycle characteristics has a great impact for assisted reproduction, especially in endangered species. In this study, the estrous cycle characteristics were investigated in 12 MF jennies throughout the year. Estrous cycle, estrous and diestrous lengths, follicular growth and ovulation, and estradiol-17β (E2) and progesterone (P4) plasma concentrations were monitored in MF jennies and compared in different seasons. In all jennies (100%) estrous cycle was detected during the whole year, with no differences in the estrous cycle length among seasons. However, a significant increase of estrous length in spring and summer compared with autumn and winter was found. Diestrus was shorter in summer than in the other seasons. Estrous behavior was always shown and characterized by rhythmic eversion of the vulvar labia (winking) with exhibition of the clitoris, urination, male receptivity and clapping, with sialorrhoea, neck and head extension, and back ears. Estrus was characterized by the ovulation of a larger follicle in spring and summer than in autumn and winter. The pattern of E2 and P4 plasma concentrations during the estrous cycle were similar to that reported for the mare, but without differences among the four seasons, so that a negligible effect of environmental conditions on ovarian E2 and P4 secretion was hypothesized, despite the larger diameter of the ovulating follicle in spring and summer.

Osteo-regenerative potential of ovarian granulosa cells: An in vitro and in vivo study

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates.

BackgroundDensity gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation, was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars, but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.ResultsSingle layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample.ConclusionsThese data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.

Neospora caninum seropositivity and reproductive risk factors in dogs

Despite the importance of Neospora caninum in veterinary medicine, knowledge of distribution of neosporosis in dog populations in some countries is still poor. The aims of the present study were to determine the occurrence of anti-N. caninum antibodies in one-hundred dogs living in cattle farms or dog breedings in central Italy and to evaluate the risk factors associated with seropositivity. The incidence of reproductive system disorders (e.g. infertility after first pregnancy) was also evaluated. Serum from breeding and farm dogs was tested to an indirect immunofluorescent antibody test (IFAT) to assess the occurrence of seropositivity. Management and individual data were collected and analysed both by linear and logistic multiple-regression models to find reliable predictors of seroprevalence and anti-N. caninum antibody level. The seropositivity for N. caninum was 32%. Dogs reared for breeding and presence of cattle on the farm were associated with seropositivity for N. caninum. Dogs living in the cattle farms showed a higher seropositivity for N. caninum (46%) compared with those living in dogs breeding (18%) (P < 0.05). The high presence of seropositive dogs in cattle farms of the study region demonstrates the potential risk of horizontal transmission of N. caninum between dogs and cattle, regardless the occurrence of reproductive system disorders or with infectious bovine tissues contact. Although the Neospora seropositivity in dog breedings may appear relatively low if compared with that found in dogs living with livestock, this infection, apparently underestimated, should be considered as a potential serious problem in canine medicine.