Barbara Merlo - Academia.edu (original) (raw)

Papers by Barbara Merlo

Animals

To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed... more To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed in a laboratory. Cells are then sent back to the veterinary clinic. The main goal of storage conditions during cell transport is to preserve their biological properties and viability. The aim of this study was to evaluate the effects of storage solutions, temperature and time on the characteristics of equine adipose tissue and Wharton’s jelly-derived MSCs. We compared two different storage solutions (plasma and 0.9% NaCl), two different temperatures (4 °C and room temperature) and three time frames (6, 24, 48 h). Cell viability, colony-forming units, trilineage differentiation, the expression of CD45 and CD90 antigens and adhesion potentials were evaluated. Despite the molecular characterization and differentiation potential were not influenced by storage conditions, viability, colony-forming units and adhesion potential are influenced in different way, depending on MSCs sources. Overal...

Animals

Attention on mesenchymal stromal cells (MSCs) research has increased in the last decade mainly du... more Attention on mesenchymal stromal cells (MSCs) research has increased in the last decade mainly due to the promising results about their plasticity, self-renewal, differentiation potential, immune modulatory and anti-inflammatory properties that have made stem cell therapy more clinically attractive. Furthermore, MSCs can be easily isolated and expanded to be used for autologous or allogenic therapy following the administration of either freshly isolated or previously cryopreserved cells. The scientific literature on the use of stromal cells in the treatment of several animal health conditions is currently available. Although MSCs are not as widely used for clinical treatments in cows as for companion and sport animals, they have the potential to be employed to improve productivity in the cattle industry. This review provides an update on state-of-the-art applications of bovine MSCs to clinical treatments and reproductive biotechnologies.

PLOS ONE

Despite the increasing demand of cellular therapies for dogs, little is known on the differences ... more Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production. Frozen-thawed samples were used for this study. No differences in mean cell proliferation were found (P>0.05). However, while AT-MSCs showed a progressive increase in doubling time over passages, UC-MSCs showed an initial post freezing-thawing latency. No differences in migration, spheroid formation ability, and differentiation potential were found (P>0.05). RT-PCR analysis confirmed the expression of CD90 and CD44, the lack of CD14 and weak expression ...

Animals

Antioxidant supplementation has been proposed as a new strategy to improve the long-term preserva... more Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 2...

11th Annual Conference of the European Society of Domestic Animal Reproduction, 2007

VII Congresso Nazionale SIRA, 2009

Animals, 2022

Regenerative medicine applied to skin lesions is a field in constant improvement. The use of biom... more Regenerative medicine applied to skin lesions is a field in constant improvement. The use of biomaterials with integrin agonists could promote cell adhesion increasing tissue repair processes. The aim of this pilot study was to analyze the effect of an α4β1 integrin agonist on cell adhesion of equine adipose tissue (AT) and Wharton’s jelly (WJ) derived MSCs and to investigate their adhesion ability to GM18 incorporated poly L-lactic acid (PLLA) scaffolds. Adhesion assays were performed after culturing AT- and WJ-MSCs with GM18 coating or soluble GM18. Cell adhesion on GM18 containing PLLA scaffolds after 20 min co-incubation was assessed by HCS. Soluble GM18 affects the adhesion of equine AT- and WJ-MSCs, even if its effect is variable between donors. Adhesion to PLLA scaffolds containing GM18 is not significantly influenced by GM18 for AT-MSCs after 20 min or 24 h of culture and for WJ-MSCs after 20 min, but increased cell adhesion by 15% GM18 after 24 h. In conclusion, the α4β1 in...

Animals, 2021

Effective standards of care treatment guidelines have been developed for many canine diseases. Ho... more Effective standards of care treatment guidelines have been developed for many canine diseases. However, a subpopulation of patients is partially or completely refractory to these protocols, so their owners seek novel therapies such as treatments with MSCs. Although in dogs, as with human medicine, the most studied MSCs sources have been bone marrow and adipose tissue, in recent years, many researchers have drawn attention towards alternative sources, such as foetal adnexa and fluid, since they possess many advantages over bone marrow and adipose tissue. Foetal adnexa and fluid could be considered as discarded material; therefore, sampling is non-invasive, inexpensive and free from ethical considerations. Furthermore, MSCs derived from foetal adnexa and fluid preserve some of the characteristics of the primitive embryonic layers from which they originate and seem to present immune-modulatory properties that make them a good candidate for allo- and xenotransplantation. The aim of the ...

The aim of this study was to verify the effects of vitrification on in vitro produced bovine blas... more The aim of this study was to verify the effects of vitrification on in vitro produced bovine blastocysts. Day 7 blastocysts and expanded blastocysts were divided in two groups: vitrified embryos (V); control group (C). Cryopreserved embryos were thawed on the same day and cultured for 48 h to asses the re-expansion and hatching. Expanded blastocysts were stained with Propidium Iodide and Hoechts 33258. The number of live and total cells was similar in both groups (58.65\ub113.64 vs 63.92\ub126.66; 79.50\ub121.37 vs 74.60\ub128.36); the number of dead cells and the proportion of expanded blastocysts were significantly different (20.46\ub111.58 vs 10.68\ub110.24; 50.94% vs 68.25%)

Reproduction, 2012

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbi... more Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets s...

International Journal of Dairy Science, 2020

Reproduction in Domestic Animals, 2019

Reproduction in Domestic Animals, 2019

The objective was to compare embryo yield and quality in lactating dairy cows superovulated (SO) ... more The objective was to compare embryo yield and quality in lactating dairy cows superovulated (SO) with varying amounts of gonadotropins and FSH:LH ratios and inseminated with SexedULTRA™ sex-sorted semen. The SO treatments (n=77) involved 3 protocols: groups F700 and F1000 were given total doses of 700 and 1000 IU of Folltropin (FSH:LH ratio 49:1), respectively, whereas group F700P300 was given 700 IU of Folltropin + 300 IU of Pluset (FSH:LH ratio 1:1). Cows were artificially inseminated 3 times over a 10-h interval with frozen-thawed SexedULTRA™ sex-sorted semen (total of 10 x 10 6 sex-sorted sperm), starting 18 h after onset of estrus, with embryos/ova recovered 7 d after estrus. Total number of recovered structures and transferable embryos were lower (P<0.05) in F700 (4.7 ± 3.0 and 1.9 ± 1.7, respectively; mean ± SD) compared to F1000 (8.1 ± 3.8 and 4.4 ± 2.6) and F700P300 (8.5 ± 6.4 and 4.5 ± 3.3). Percentage of cows ovulating >50% of follicles ≥0.8 cm in diameter was lower (P<0.05) in F700 (35.5%) than in F1000 (82.4%) and F700P300 (73.1%). Percentage of unfertilized oocytes was higher (P<0.05) in F700 (45.0 vs 27.7% for F1000 and 29.0% for F700P300) whereas percentage of morulae was higher (P<0.05) in F1000 (19.3 vs 8.7% for F700 and 12.2% for F700P300). Embryo quality was similar among groups (P>0.05). In conclusion, embryo production in lactating dairy cows was improved by increasing total dose of gonadotropins from 700 to 1000 IU, with SexedULTRA™ sex-sorted semen yielding satisfactory fertilization rates and embryo quality.

Veterinary Research Communications, 2018

The aim of the present study was to compare canine adipose tissue mesenchymal stem cells cultured... more The 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. Intraabdominal 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 roundshaped 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.

Reproduction, 2017

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and W... more Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton’s jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P < 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P < 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-β1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equ...

Theriogenology, 2017

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional... more Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that sperm-oocyte interaction in equine species has been demonstrated to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrode's medium at pH 7.4, 8.0 and 9.0 for 4 h at 38°C, 5% CO2 with 2.5 IU AP (AP group) or without AP (CAP group); viability with mitochondrial activity, motility and acrosome integrity were

Reproduction, Fertility and Development, 2009

Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in sev... more Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in several species by artificial insemination, although the success rate is often lower than with non-sexed semen. In horses, the problem was partially overcome with hysteroscopic insemination using sex-sorted fresh sperm. However, when sex-sorted frozen–thawed sperm were used the pregnancy rate was heavily reduced in comparison with non-sexed frozen–thawed semen. Because it has been demonstrated that in vitro assisted reproductive techniques, namely intracytoplasmic sperm injection (ICSI), has permitted live foals to be obtained using sperm with low fertility in the field, in this study we investigated the possibility of using ICSI with sexed-sorted frozen–thawed sperm for equine embryo production in vitro. Briefly, semen was collected from two Standardbred stallions of proven fertility (Stallions A, B), sorted using a MoFlo SX flow cytometer and frozen (Johnson LA and Welch GR 1999 Theriogen...

Veterinary Research Communications, 2009

Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injur... more Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injuries in the horse. MSC populate different tissues but the most commonly used for clinical purposes are isolated from bone marrow or adipose tissue. The first objective of this study was to investigate if the donor animal, the tissue of origin and the technique of isolation could influence the number of MSC available for transplantation after a short-term expansion. The second aim was to devise a culture system capable of increasing MSC lifespan and we tested the effect of basic fibroblast growth factor (bFGF). Results indicate that MSC can be efficiently isolated from both sources and supplementation of bFGF enhances proliferation rate maintaining differentiation potential. In addition, this study shows that collection, expansion and storage of frozen MSC can be performed for later therapeutic use.

Theriogenology, 2007

The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomi... more The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species were evaluated performing three experiments. This is the first study about these topics in the domestic cat. In Experiment 1, ketamine or medetomidine effects on cat sperm quality after collection by electroejaculation (E.E.) have been assessed in nine animals. Results showed that mean sperm concentration was significantly higher (p < 0.01) after medetomidine than after ketamine administration. In Experiment 2, ketamine or medetomidine effects on sperm flow in 12 electroejaculated cats were studied. Mean sperm concentration and mean total number of spermatozoa resulted significantly higher (p < 0.01) in medetomidine than in ketamine treated animals. The number of spermatozoa displaced in urethra was significantly higher (p < 0.01) using medetomidine. No significant differences were observed in percentages of retrograde flow. In Experiment 3, ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection, were evaluated. Data obtained showed a significantly higher (p < 0.05) number of spermatozoa displaced in urethra after medetomidine than after ketamine injection. In conclusion, E.E. in the cat after medetomidine administration determined a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction and without increasing sperm retrograde flow.

Animals

To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed... more To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed in a laboratory. Cells are then sent back to the veterinary clinic. The main goal of storage conditions during cell transport is to preserve their biological properties and viability. The aim of this study was to evaluate the effects of storage solutions, temperature and time on the characteristics of equine adipose tissue and Wharton’s jelly-derived MSCs. We compared two different storage solutions (plasma and 0.9% NaCl), two different temperatures (4 °C and room temperature) and three time frames (6, 24, 48 h). Cell viability, colony-forming units, trilineage differentiation, the expression of CD45 and CD90 antigens and adhesion potentials were evaluated. Despite the molecular characterization and differentiation potential were not influenced by storage conditions, viability, colony-forming units and adhesion potential are influenced in different way, depending on MSCs sources. Overal...

Animals

Attention on mesenchymal stromal cells (MSCs) research has increased in the last decade mainly du... more Attention on mesenchymal stromal cells (MSCs) research has increased in the last decade mainly due to the promising results about their plasticity, self-renewal, differentiation potential, immune modulatory and anti-inflammatory properties that have made stem cell therapy more clinically attractive. Furthermore, MSCs can be easily isolated and expanded to be used for autologous or allogenic therapy following the administration of either freshly isolated or previously cryopreserved cells. The scientific literature on the use of stromal cells in the treatment of several animal health conditions is currently available. Although MSCs are not as widely used for clinical treatments in cows as for companion and sport animals, they have the potential to be employed to improve productivity in the cattle industry. This review provides an update on state-of-the-art applications of bovine MSCs to clinical treatments and reproductive biotechnologies.

PLOS ONE

Despite the increasing demand of cellular therapies for dogs, little is known on the differences ... more Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production. Frozen-thawed samples were used for this study. No differences in mean cell proliferation were found (P>0.05). However, while AT-MSCs showed a progressive increase in doubling time over passages, UC-MSCs showed an initial post freezing-thawing latency. No differences in migration, spheroid formation ability, and differentiation potential were found (P>0.05). RT-PCR analysis confirmed the expression of CD90 and CD44, the lack of CD14 and weak expression ...

Animals

Antioxidant supplementation has been proposed as a new strategy to improve the long-term preserva... more Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 2...

11th Annual Conference of the European Society of Domestic Animal Reproduction, 2007

VII Congresso Nazionale SIRA, 2009

Animals, 2022

Regenerative medicine applied to skin lesions is a field in constant improvement. The use of biom... more Regenerative medicine applied to skin lesions is a field in constant improvement. The use of biomaterials with integrin agonists could promote cell adhesion increasing tissue repair processes. The aim of this pilot study was to analyze the effect of an α4β1 integrin agonist on cell adhesion of equine adipose tissue (AT) and Wharton’s jelly (WJ) derived MSCs and to investigate their adhesion ability to GM18 incorporated poly L-lactic acid (PLLA) scaffolds. Adhesion assays were performed after culturing AT- and WJ-MSCs with GM18 coating or soluble GM18. Cell adhesion on GM18 containing PLLA scaffolds after 20 min co-incubation was assessed by HCS. Soluble GM18 affects the adhesion of equine AT- and WJ-MSCs, even if its effect is variable between donors. Adhesion to PLLA scaffolds containing GM18 is not significantly influenced by GM18 for AT-MSCs after 20 min or 24 h of culture and for WJ-MSCs after 20 min, but increased cell adhesion by 15% GM18 after 24 h. In conclusion, the α4β1 in...

Animals, 2021

Effective standards of care treatment guidelines have been developed for many canine diseases. Ho... more Effective standards of care treatment guidelines have been developed for many canine diseases. However, a subpopulation of patients is partially or completely refractory to these protocols, so their owners seek novel therapies such as treatments with MSCs. Although in dogs, as with human medicine, the most studied MSCs sources have been bone marrow and adipose tissue, in recent years, many researchers have drawn attention towards alternative sources, such as foetal adnexa and fluid, since they possess many advantages over bone marrow and adipose tissue. Foetal adnexa and fluid could be considered as discarded material; therefore, sampling is non-invasive, inexpensive and free from ethical considerations. Furthermore, MSCs derived from foetal adnexa and fluid preserve some of the characteristics of the primitive embryonic layers from which they originate and seem to present immune-modulatory properties that make them a good candidate for allo- and xenotransplantation. The aim of the ...

The aim of this study was to verify the effects of vitrification on in vitro produced bovine blas... more The aim of this study was to verify the effects of vitrification on in vitro produced bovine blastocysts. Day 7 blastocysts and expanded blastocysts were divided in two groups: vitrified embryos (V); control group (C). Cryopreserved embryos were thawed on the same day and cultured for 48 h to asses the re-expansion and hatching. Expanded blastocysts were stained with Propidium Iodide and Hoechts 33258. The number of live and total cells was similar in both groups (58.65\ub113.64 vs 63.92\ub126.66; 79.50\ub121.37 vs 74.60\ub128.36); the number of dead cells and the proportion of expanded blastocysts were significantly different (20.46\ub111.58 vs 10.68\ub110.24; 50.94% vs 68.25%)

Reproduction, 2012

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbi... more Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets s...

International Journal of Dairy Science, 2020

Reproduction in Domestic Animals, 2019

Reproduction in Domestic Animals, 2019

The objective was to compare embryo yield and quality in lactating dairy cows superovulated (SO) ... more The objective was to compare embryo yield and quality in lactating dairy cows superovulated (SO) with varying amounts of gonadotropins and FSH:LH ratios and inseminated with SexedULTRA™ sex-sorted semen. The SO treatments (n=77) involved 3 protocols: groups F700 and F1000 were given total doses of 700 and 1000 IU of Folltropin (FSH:LH ratio 49:1), respectively, whereas group F700P300 was given 700 IU of Folltropin + 300 IU of Pluset (FSH:LH ratio 1:1). Cows were artificially inseminated 3 times over a 10-h interval with frozen-thawed SexedULTRA™ sex-sorted semen (total of 10 x 10 6 sex-sorted sperm), starting 18 h after onset of estrus, with embryos/ova recovered 7 d after estrus. Total number of recovered structures and transferable embryos were lower (P<0.05) in F700 (4.7 ± 3.0 and 1.9 ± 1.7, respectively; mean ± SD) compared to F1000 (8.1 ± 3.8 and 4.4 ± 2.6) and F700P300 (8.5 ± 6.4 and 4.5 ± 3.3). Percentage of cows ovulating >50% of follicles ≥0.8 cm in diameter was lower (P<0.05) in F700 (35.5%) than in F1000 (82.4%) and F700P300 (73.1%). Percentage of unfertilized oocytes was higher (P<0.05) in F700 (45.0 vs 27.7% for F1000 and 29.0% for F700P300) whereas percentage of morulae was higher (P<0.05) in F1000 (19.3 vs 8.7% for F700 and 12.2% for F700P300). Embryo quality was similar among groups (P>0.05). In conclusion, embryo production in lactating dairy cows was improved by increasing total dose of gonadotropins from 700 to 1000 IU, with SexedULTRA™ sex-sorted semen yielding satisfactory fertilization rates and embryo quality.

Veterinary Research Communications, 2018

The aim of the present study was to compare canine adipose tissue mesenchymal stem cells cultured... more The 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. Intraabdominal 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 roundshaped 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.

Reproduction, 2017

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and W... more Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton’s jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P < 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P < 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-β1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equ...

Theriogenology, 2017

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional... more Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that sperm-oocyte interaction in equine species has been demonstrated to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrode's medium at pH 7.4, 8.0 and 9.0 for 4 h at 38°C, 5% CO2 with 2.5 IU AP (AP group) or without AP (CAP group); viability with mitochondrial activity, motility and acrosome integrity were

Reproduction, Fertility and Development, 2009

Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in sev... more Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in several species by artificial insemination, although the success rate is often lower than with non-sexed semen. In horses, the problem was partially overcome with hysteroscopic insemination using sex-sorted fresh sperm. However, when sex-sorted frozen–thawed sperm were used the pregnancy rate was heavily reduced in comparison with non-sexed frozen–thawed semen. Because it has been demonstrated that in vitro assisted reproductive techniques, namely intracytoplasmic sperm injection (ICSI), has permitted live foals to be obtained using sperm with low fertility in the field, in this study we investigated the possibility of using ICSI with sexed-sorted frozen–thawed sperm for equine embryo production in vitro. Briefly, semen was collected from two Standardbred stallions of proven fertility (Stallions A, B), sorted using a MoFlo SX flow cytometer and frozen (Johnson LA and Welch GR 1999 Theriogen...

Veterinary Research Communications, 2009

Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injur... more Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injuries in the horse. MSC populate different tissues but the most commonly used for clinical purposes are isolated from bone marrow or adipose tissue. The first objective of this study was to investigate if the donor animal, the tissue of origin and the technique of isolation could influence the number of MSC available for transplantation after a short-term expansion. The second aim was to devise a culture system capable of increasing MSC lifespan and we tested the effect of basic fibroblast growth factor (bFGF). Results indicate that MSC can be efficiently isolated from both sources and supplementation of bFGF enhances proliferation rate maintaining differentiation potential. In addition, this study shows that collection, expansion and storage of frozen MSC can be performed for later therapeutic use.

Theriogenology, 2007

The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomi... more The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species were evaluated performing three experiments. This is the first study about these topics in the domestic cat. In Experiment 1, ketamine or medetomidine effects on cat sperm quality after collection by electroejaculation (E.E.) have been assessed in nine animals. Results showed that mean sperm concentration was significantly higher (p < 0.01) after medetomidine than after ketamine administration. In Experiment 2, ketamine or medetomidine effects on sperm flow in 12 electroejaculated cats were studied. Mean sperm concentration and mean total number of spermatozoa resulted significantly higher (p < 0.01) in medetomidine than in ketamine treated animals. The number of spermatozoa displaced in urethra was significantly higher (p < 0.01) using medetomidine. No significant differences were observed in percentages of retrograde flow. In Experiment 3, ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection, were evaluated. Data obtained showed a significantly higher (p < 0.05) number of spermatozoa displaced in urethra after medetomidine than after ketamine injection. In conclusion, E.E. in the cat after medetomidine administration determined a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction and without increasing sperm retrograde flow.