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In vitro developmental competence of horse embryos derived from oocytes with a different corona radiata cumulus-oocyte morphology
Full text linkThe increase in demand for in vitro produced horse embryos is fostering the development of commercial laboratories for this purpose. Nevertheless, blastocyst production after intracytoplasmic sperm injection (ICSI) is still not as great as desired in most of these laboratories. In relation to horse oocyte classification, both expanded and compact cumulus-oocyte-complexes (COCs) are used for in vitro embryo production. The aim of this study was to compare in vitro embryo developmental capacity of COCs from horses including those with only the corona radiata, frequently collected after aspiration procedures. Horse oocytes were collected by follicular aspiration of abattoir-derived ovaries. After classification as expanded, compact or corona radiata COCs, these were in vitro matured, fertilized by ICSI and in vitro cultured for 7.5 days. Maturation rate, cleavage rate and morula/blastocyst rates were recorded. No significant differences (P > 0.05) were detected among groups in maturation rate. Cleavage rate was less (P < 0.05) for embryos derived from oocytes with a corona radiata as compared to compact-derived embryos, but embryo development after 7.5 days of culture was similar among groups (P > 0.05). In conclusion, even if embryos derived from oocytes with corona radiata had a lesser cleavage rate after ICSI, the developmental capacity was similar to embryos derived from oocytes with a compact and expanded cumulus morphology, indicating these can be an useful source of embryos in horses
Could hypoxia influence basic biological properties and ultrastructural features of adult canine mesenchymal stem /stromal cells?
Full text linkThe aim of the present study was to compare canine adipose tissue mesenchymal stem cells cultured under normoxic (20% O2) and not severe hypoxic (7% O2) conditions in terms of marker expression, proliferation rate, differentiation potential and cell morphology. Intra-abdominal fat tissue samples were recovered from 4 dogs and cells isolated from each sample were cultured under hypoxic and normoxic conditions. Proliferation rate and adhesion ability were determined, differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced; the expression of CD44, CD34, DLA-DQA1, DLA-DRA1 was determined by PCR, while flow cytometry analysis for CD90, CD105, CD45 and CD14 was carried out. The morphological study was performed by transmission electron microscopy. Canine AT-MSCs, cultured under different oxygen tensions, maintained their basic biological features. However, under hypoxia, cells were not able to form spheroid aggregates revealing a reduction of their adhesivness. In both conditions, MSCs mainly displayed the same ultrastructural morphology and retained the ability to produce membrane vesicles. Noteworthy, MSCs cultivated under hypoxya revealed a huge shedding of large complex vesicles, containing smaller round-shaped vesicles. In our study, hypoxia partially influences the basic biological properties and the ultrastructural features of canine mesenchymal stem /stromal cells. Further studies are needed to clarify how hypoxia affects EVs production in term of amount and content in order to understand its contribution in tissue regenerative mechanisms and the possible employment in clinical applications. The findings of the present work could be noteworthy for canine as well as for other mammalian species
Evaluation of Some Physical, Haemathological and Clinical Chemistry Parameters in Healthy Newborn Italian Holstein Calves
Full text linkAbstract: The aim of the present study was to investigate some physical,
haematological and clinical chemistry parameters in the newborn Italian
Holstein calf at birth and at 24 h of life, to evaluate changes during the
immediate post-partum period. Forty-six Italian Holstein Friesian calves
were included in this study. Heart rate, respiratory rate and body
temperature were recorded at birth and at 24 h of life. The time needed to
raise the head, acquire sternal recumbency, stand up were also recorded.
Blood samples were collected before first feeding and at 24 h of age and
CBC count, L-lactate, glucose and total protein concentrations were
evaluated. The head was raised immediately in 46/46 calves, suckling
reflex was acquired within 12±9 min, sternal position in 5±2 min and
newborn stood up in 38±30 min. Some of the physical data,
haematological and biochemical values showed statistical differences
between birth and 24 h of age. The results from this study provide some
information about physical and laboratory data of Italian Holstein
Friesian calves, at birth and at 24 h of life. Our results confirm that
several clinical and laboratory values in newborn calves differ from adult
reference intervals and from calves of different breeds
Overnight holding aids in selection of developmentally competent equine oocytes
Full text linkThe demand for equine in vitro produced embryos has increased over the last decade. The aim of this study was to compare the effects of an extended IVM or a prolonged period before fertilization, including holding time, on equine immature oocyte developmental competence. Oocytes, collected from abattoir-derived ovaries, were divided into 4 groups: H0/24 (n = 165) 0 h holding + standard 24-26 h IVM; H8/36 (n = 160) 8 h holding + 36 h IVM; H20/24 (n = 187) 20 h holding + 24 h IVM; H0/44 (n = 164) 0 h holding + 44 h IVM. Oocytes matured to MII were fertilized by intracytoplasmic sperm injection (ICSI) and cultured for 10 days. The oocyte degeneration rate was higher (P 0.05). Timing of blastocyst development was not different among groups. Overnight holding of equine immature oocytes followed by a standard IVM interval may induce a pre-selection of the most competent oocytes thereby improving cleavage and embryo development rates after ICSI
Equine Colostrum-Derived Mesenchymal Stromal Cells: A Potential Resource for Veterinary Regenerative Medicine
Full text linkBeyond its immunological role, colostrum has emerged as a promising, non-invasive source of bioactive factors, including mesenchymal stem/stromal cells (MSCs). This study represents the first attempt to isolate and characterize MSCs from equine colostrum (C-MSCs) to assess their potential use in veterinary regenerative medicine. Colostrum (n = 6) was collected from mares immediately after their delivery and centrifuged, and the recovered cells were cultured under standard conditions. The C-MSCs displayed plastic adherence and a heterogeneous morphology, including spindle-shaped and epithelial-like cells. The population doubling time (PDT) values varied among the samples, and four out of six showed rapid proliferation (< 0.05). Spheroid formation assays revealed differences in cell–cell adhesion: four out of six samples formed stable spheroids within four days. A migration assay showed significant variability (p < 0.05): one out of six achieved complete wound closure within 72 h, whereas five out of six reached ~30% at 96 h. All samples were positive for adipogenic, chondrogenic, and osteogenic differentiation as shown via staining. RT-PCR confirmed MSC marker expression, while hematopoietic markers were absent. MHC-I expression was weak in five out of six samples, whereas MHC-II was consistently negative. These findings support equine colostrum as a viable MSC source, though its variability requires further validation with larger samples. Additional research is needed to investigate C-MSCs’ immunomodulatory properties and therapeutic potential
Effects of holding and the addition of naloxone on vitrification of equine immature oocytes
Full text linkThis study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification
outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment
combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10 8 M) (VIT and VIT-
Nx), oocytes vitrified after overnight holding with or without Nx (10 8 M) (H-VIT and H-VIT-Nx). They were
assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution,
apoptosis, and apoptotic gene expression. At survival rate determination, the degeneration rate was higher in VIT
and VIT-Nx compared to F (P < 0.05). The highest maturation rate was observed in VIT-Nx. A significant
reduction in ROS levels was observed in H compared to F (P < 0.05). ROS levels were similar between F and VIT,
while the Nx supplementation tended to increase them (VIT-Nx vs F: P = 0.053; VIT-Nx vs VIT: P = 0.069).
Conversely, in oocytes vitrified after overnight holding, vitrification induced an increase in ROS levels (H vs VIT:
P < 0.05), which was not observed in H-VIT-Nx. GSH intracellular levels showed significant differences only in
held oocytes, with higher GH levels in H compared to H-VIT and H-VIT-Nx (P < 0.05). All treatments induced an
increase in HMMP levels compared to F (P < 0.05). In H oocytes, mitochondria were distributed throughout the
entire oolemma (TOMM20) and active mitochondria (D-LAT) were detected in the outermost region. Incontrast,
in H-VIT-Nx, potentially active mitochondria were spread throughout the cytoplasm. AnnexinV/PI staining
revealed that the percentage of viable oocytes was higher (P < 0.05) in F and H than in all vitrified/warmed
oocytes, and H-VIT-Nx had the highest degeneration rate (P < 0.05). RT-PCR analysis confirmed the detection for
both reference genes, and target genes BCL2 and Survivin in all samples. In contrast, BAX and p53 transcripts
were consistently undetectable. No significant differences were observed in the expression of BCL2 and Survivin
between groups. In conclusion, overnight holding at uncontrolled room temperature can alter oocyte charac-
teristics and lead to variable results after vitrification. Nx demonstrated contrasting antioxidant effects
depending on the vitrification timing, but it appeared to improve IVM outcomes in oocytes vitrified immediately
after collection
Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality
Full text linkThis study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8°C), the other in a styrofoam box with ice (+3°C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4°C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 ± 0.6°C and 8.6 ± 2.5°C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3°C) or higher (+8°C) temperature than +5°C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions
Equine Bone Marrow and Adipose Tissue Mesenchymal Stem Cells: Cytofluorimetric Characterization, In Vitro Differentiation, and Clinical Application
Full text linkThe aim of the present work was to isolate, cultivate, differentiate, and conduct cellular
characterization of mesenchymal stem cells (MSCs) derived from equine adipose tissue
(eAT) and bone marrow (eBM). Isolated and characterized cells were used in racehorses
suffering from a superficial flexor tendon injury. Equine adipose tissue collection was
performed at the base of the horse tail, whereas eBM was aspirated from iliac crest.
Mononuclear cell fraction was isolated and cultured. In vitro differentiation and molecular
characterization at P3 of culture were performed. No statistically significant differences in
the number of cell doublings were found among different culture passages (P > .05).
Doubling time was greater for eBM than eAT (3.2 1.5 vs. 1.3 0.7; P < .05). Positive von
Kossa and Alizarin Red staining confirmed osteogenesis. Alcian Blue and Oil Red O staining
illustrated chondrogenesis and adipogenesis, respectively. Isolated cells resulted positive
for CD90, CD44, and CD105, whereas negative for hematopoietic markers, CD14, CD45, and
CD34. Using isolated cells for injured tendon therapy, no adverse reactions were observed,
and all inoculated horses returned to race competitions. In vitro results revealed the
immunophenotypic characterization of isolated cells similar to that observed in human
MSCs from the same sources; furthermore, in the present study, their clinical use proves
the safety of eBM-derived and eAT-derived MSCs and a successful outcome for the treated
animals that returned to their previous level of sport activity
Observational Study on Cryptosporidiosis in an Equine Perinatology Unit
Full text linkThe present study aimed to describe clinical signs of cryptosporidiosis in neonatal foals hospitalized in an Equine Perinatology Unit and to compare the clinical signs between Cryptosporidium parvum and Cryptosporidium horse genotype infection. The study was divided into two parts. In the retrospective study, nine foals infected by C. parvum were considered. In the prospective study, 70 foals, less than 15 days old, were prospectively included. Historical and clinical data were recorded, and in the prospective study, multiple fecal samples were collected. C. parvum (n = 13) and Cryptosporidium horse genotype (n = 7) were isolated. In four foals, there was a mixed infection with both the Cryptosporidium. Diarrhea, when present, showed similar duration and characteristics. Sixteen foals showed decreased abdominal sounds and colic pain before evidence of diarrhea. Nineteen foals had hyperthermia at least once. Although survival rates were similar between C. parvum (77%), C. horse genotype (100%), and cryptosporidial mixed infection (100%), foals affected by C. parvum presented anorexia (P <.0031) and received specific therapy (P <.014) more frequently than the others. Recorded data strengthen the thought that C. parvum infection is more severe in foals, suggesting that they would have developed host adaptations in response to the C. horse genotype or that C. parvum is a more pathogenic strain. Because healthy and asymptomatic foals can shed oocysts of Cryptosporidium spp., students and staff should always wear the personal protective equipment to avoid zoonotic infection
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