Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes.

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10-9 M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10-9 M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2',7'-dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10-9 M melatonin during in vitro maturation had no effect on these parameters.

Superovulation protocols for dairy cows bred with SexedULTRA™ sex-sorted semen.

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 1,000 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-hr interval with frozen-thawed SexedULTRA™ sex-sorted semen (total of 10 × 106 sex-sorted sperm), starting 18 hr after onset of oestrus, with embryos/ova recovered 7 d after oestrus. 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 1,000 IU, with SexedULTRA™ sex-sorted semen yielding satisfactory fertilization rates and embryo quality.

Luteal Blood Flow and progesterone concentration during first and second postpartum estrous cycle in lactating dairy cows.

The aim of the present study was to determine the differences in corpus luteum (CL) functionality between the first postpartum estrous cycle and the following cycle in lactating dairy cows. Luteal blood flow (LBF), luteal size and blood progesterone (P4) concentration were monitored during the first and second postpartum estrous cycle. During the first and second postpartum estrous cycle, the mean LBF value increased (p < .05) from early to late dioestrus, while it decreased rapidly in proestrus, resulting statistically lower (p < .05) than those registered in all previous phases. Statistically significant differences were not observed between overall LBF during first and second postpartum estrous cycle (p > .05). During the first postpartum estrous cycle, P4 blood concentrations showed a significant reduction (p < .05) from dioestrus to proestrus. A different trend of P4 concentrations was observed during the second postpartum estrous cycle, where mean P4 value registered in proestrus resulted statistically lower than those registered in the previous cycle phases (p < .05). The mean P4 concentration registered over the first postpartum estrous cycle resulted statistically lower (p < .05) than that registered during the second one. A significant correlation between P4 concentrations and LBF was registered only during the second postpartum estrous cycle. Results indicate that during the first postpartum estrous cycle, P4 concentration was independent of luteal blood flow and luteal size.

Ultrastructural characteristics and immune profile of equine MSCs from fetal adnexa.

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-ß1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equine WJ-MSCs constitutively contain a very impressive number of large vesicular structures, scattered throughout the cytoplasm. Moreover, an abundant extracellular fibrillar matrix was located in the intercellular spaces among WJ-MSCs. Data recorded in this study reveal that MSCs from different fetal tissues have different characteristics that may drive their therapeutic use. These finding could be noteworthy for horses as well as for other mammalian species, including humans.

Therapeutic Application of Extracellular Vesicles Derived from Mesenchymal Stem Cells in Domestic Animals.

Recently, the therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) has been extensively studied in both human and veterinary medicine. EVs are nano-sized particles containing biological components commonly found in other biological materials. For that reason, EV isolation and characterization are critical to draw precise conclusions during their investigation. Research on EVs within veterinary medicine is still considered in its early phases, yet numerous papers were published in recent years. The conventional adult tissues for deriving MSCs include adipose tissue and bone marrow. Nonetheless, alternative sources such as synovial fluid, endometrium, gingiva, and milk have also been intermittently used. Fetal adnexa are amniotic membrane/fluid, umbilical cord and Wharton's jelly. Cells derived from fetal adnexa exhibit an intermediate state between embryonic and adult cells, demonstrating higher proliferative and differentiative potential and longer telomeres compared to cells from adult tissues. Summarized here are the principal and recent preclinical and clinical studies performed in domestic animals such as horse, cattle, dog and cat. To minimize the use of antibiotics and address the serious issue of antibiotic resistance as a public health concern, they will undoubtedly also be utilized in the future to treat infections in domestic animals. A number of concerns, including large-scale production with standardization of EV separation and characterization techniques, must be resolved for clinical application.

Could assisted reproductive techniques affect equine fetal membranes and neonatal outcome?

Embryo transfer (ET) and intracytoplasmic sperm injection (ICSI) are widely used in equine species, but their effects on fetal adnexa and neonates have not been investigated yet. The aim of this study was to retrospectively evaluate whether pregnancies obtained by ET or ICSI could be associated with the presence of macroscopic alterations of fetal membranes (FM) and umbilical cord (UC) and if the use of these techniques could influence neonatal outcome. Sixty-six light breed mares hospitalized at the Veterinary Teaching Hospital, University of Bologna, for attending delivery were included in the study. Mares were divided into Artificial Insemination (AI; 32/66 mares, 48 %), Embryo Transfer (ET; 12/66 mares, 18.2 %) and Intracytoplasmic Sperm Injection (ICSI; 22/66 mares, 33 %) groups. All the medical reports of mares and their foals were reviewed and data about mare, pregnancy, foaling, fetal membranes, umbilical cord and foal were recorded. The occurrence of dystocia resulted statistically different between AI group and ICSI group (p = 0.0066), and between AI group and ET group (p = 0.044). Macroscopic examination of FM revealed alterations in 30/66 mares (46 %): 8/32 in AI (25 %), 7/12 in ET (58 %) and 15/22 in ICSI (68 %) with significant lower incidence in AI compared to ET (p = 0.04) and ICSI (p = 0.002) groups. Alterations reported were chorionic villi hypoplasia, chorioallantois edema, allantois cysts, necrotic areas and greenish-grey concretions. Total length of UC resulted significantly shorter in ICSI group (49 ± 9 cm; p < 0.03) compared to AI (60 ± 17 cm) and ET (59 ± 15 cm). However, there were no differences in the incidence of foals' diseases at birth and in foals' survival among groups (p > 0.05). The results demonstrate that transfer of in vivo or in vitro produced embryos may lead to alterations of placental development, as observed in other species, without being associated with a higher incidence of neonatal morbidity and mortality. Further studies about trophoblast development, FM histological evaluation, and placental gene expression should be carried out to clarify the mechanisms underlying the placental alterations.

Effect of Maca aqueous extract addition to a freezing extender for canine semen.

This study investigated the effect of the addition of Lepidium meyenii (Maca) to the freezing extender on the post-thaw quality of dog semen. Ten canine ejaculates were frozen following a two-step protocol using a tris-glucose-citrate egg yolk extender with or without the addition of 10 µl/mL of aqueous extract of Maca (Maca and ctrl groups, respectively). Prior to (fresh semen) and after freezing (T0) sperm motility, kinetic parameters, viability and mitochondrial membrane potential (MMP), as well as the levels of malondialdehyde (MDA) were evaluated. In addition, sperm motility, kinetic parameters, viability and MMP were examined up to 2 h of incubation of 37 °C after thawing (T1 and T2) to evaluate thermo-resistance. The addition of Maca reduced MDA concentration at T0 (p < 0.05) and increased total motility, the percentage of sperm with medium velocity and WOB at T1. Progressive motility decreased (p < 0.05) at T1 in the ctrl group, whereas it was not affected in Maca group at any time point. In addition, the percentage of hyperactivated spermatozoa remained constant at T1 in the ctrl, while in the Maca group an increase (p < 0.05) of this parameter was recorded. Although no differences were found for MMP between groups at any time points, a decrease of viable sperm with low MMP was observed in ctrl group between T0 and T1 and in Maca group between T1 and T2. The addition of Maca prior freezing reduced the extent of lipid peroxidation and activated canine sperm motility and hyperactivation after thawing.

Beyond Canine Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells Transplantation: An Update on Their Secretome Characterization and Applications.

A dog is a valuable animal model and concomitantly a pet for which advanced therapies are increasingly in demand. The characteristics of mesenchymal stem/stromal cells (MSCs) have made cell therapy more clinically attractive. During the last decade, research on the MSC therapeutic effectiveness has demonstrated that tissue regeneration is primarily mediated by paracrine factors, which are included under the name of secretome. Secretome is a mixture of soluble factors and a variety of extracellular vesicles. The use of secretome for therapeutic purposes could have some advantages compared to cell-based therapies, such as lower immunogenicity and easy manufacturing, manipulation, and storage. The conditioned medium and extracellular vesicles derived from MSCs have the potential to be employed as new treatments in veterinary medicine. This review provides an update on the state-of-the-art characterization and applications of canine adipose tissue-derived MSC secretome.

Gross and histological examination of Wharton's Jelly in the equine umbilical cord.

Wharton's jelly (WJ) is fundamental for the well-being of the fetus, binding to the umbilical vessels and protecting them from twisting and compression. Gross and microscopic studies have been undertaken on the umbilical cord (UC) of human placentae in both normal and high-risk pregnancies, however there is limited research on equine UC. The aim of this study was to describe microscopically and immunohistochemically the equine UC in normal pregnancies, with particular attention to WJ. Forty-seven healthy mares, with no complications during pregnancy, admitted to the hospital for attending delivery were enrolled. Clinical data was collected at foaling on foal health and placental characteristics. UC samples were collected from three sites (amniotic, allantoic and in the region of vein anastomosis) for histology. The thickness of different layers of arteries and veins and WJ in different UC portions were measured (µm). Wharton's Jelly was weighted (g) and its sections were stained with Masson's trichrome, orcein technique and silver impregnation. Immunohistochemistry was undertaken using antibodies raised-against collagen type I, V, VI and fibrillin. Forty-seven UCs, from 19 colt and 28 filly foals, were analyzed for WJ weight and 8/47 UCs were examined histologically. Warton's jelly was only found in the amniotic portion of the UC closest to the foal's abdomen. The weight of WJ (4.0 ± 3.3 g) did not vary between colts and fillies and it was not correlated with any of the clinical or UC parameters measured. The tunica media of arteries and veins was thicker in the amniotic portion of the UC, as described in human UCs in late pregnancy. This finding could be an adaptation to aid in resisting compression because of fetal movements and UC twisting. The umbilical vein was thicker than the umbilical arteries in the tunica media and tunica adventitia in the sections examined throughout the length of the cord. This preliminary study describes gross and histological WJ's structure in the equine species. However, further studies are required to better characterize UC's changes throughout pregnancy and in the presence of mare's or fetal disease.

Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse.

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 staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

Stem Cells in Domestic Animals: Applications in Health and Production.

In the last decade, researchers described Mesenchymal Stem/stromal cells (MSCs) as a possible population of cells for cell-based therapies in regenerative medicine, both for humans and animals [...].

An Update on Applications of Cattle Mesenchymal Stromal Cells.

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.

Shipping Temperature, Time and Media Effects on Equine Wharton's Jelly and Adipose Tissue Derived Mesenchymal Stromal Cells Characteristics.

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. Overall, this study found that, despite equine adipose tissue MSCs being usable after 24 h of storage, cells derived from Wharton's jelly need to be used within 6 h. Moreover, while for adipose cells the best conservation solutions seems to be plasma, the cell viability of Wharton's jelly MSCs declined in both saline and plasma solution, confirming their reduced resistance to conservation.

Overnight holding aids in selection of developmentally competent equine oocytes.

The 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) for H20/24 than the other groups (H0/24 38.2 %, H8/36 43.1 %, H20/24 54.5 %, H0/44 32.9 %). Cleavage was higher (P < 0.05) in H20/24 (70 %) compared to H0/24 (45 %) and H8/36 (54 %) but not to H0/44 (63 %). No differences among groups were observed in the number of blastocysts per oocyte. Injected oocytes that reached the blastocysts stage were higher (P < 0.05) for H20/24 (20 %) than H0/24 (7 %) and H0/44 (7 %) but not H8/36 (12 %). For cleaved oocytes, a higher blastocyst rate (P < 0.05) was observed for H20/24 (28 %) than H0/44 (11 %), while H0/24 (15 %) and H8/36 (21 %) were not different from any group (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.

Effect of platelet lysate on uterine response of mares susceptible to persistent mating-induced endometritis.

Many mares are susceptible to persistent mating-induced endometritis (PMIE), an important cause of reduced fertility. Platelet lysate (PL) derives from freeze-thawing platelets after concentration, so that growth factors are released from the platelets. Among the advantages of PL compared to platelet-rich plasma (PRP), it can be frozen stored and allogenic use for PL might also be conceivable. Platelet-rich plasma beneficially reduced inflammatory response in PMIE mares when administered 24 h pre- or 4 h post-AI. The aim of this study was to test the effect of PL on inflammatory uterine response in mares susceptible to PMIE. A total of 14 mares susceptible to PMIE (based on presence of fluid or inflammatory cells 24 h after AI) underwent an untreated (Ctr) cycle followed by a treated (PL) cycle. From each mare, 100 mL of citrated whole blood was obtained for PRP production by centrifugation. The resultant PRP was brought to a final volume of 10 mL with platelet poor plasma and frozen at -80 °C to obtain PL. On untreated cycles, mares were inseminated with frozen-thawed semen 36 h after ovulation induction. On treated cycles, PL was thawed, infused into the uterus 12 h after ovulation induction, and AIs were performed 24 h later. The number of neutrophils in uterine cytology (score 1(normal)-3(severe inflammation)) evaluated by optical microscopy, uterine fluid accumulation (height x width) and uterine edema (score 0-3) observed in ultrasonography, were analysed. Pregnancy was evaluated by ultrasonography 14 days after ovulation. A significant decrease (P < 0.05) was observed on cytology score (PL 1.3 ± 0.1 vs Ctr 2.0 ± 0.1), fluid accumulation (PL 79.5 ± 30.1 mm2 vs Ctr 342.7 ± 52.9 mm2) and edema score (PL 1.8 ± 0.2 vs Ctr 2.3 ± 0.2) in treated mares. Pregnancy rate in PL-treated cycles (3/12) and control cycles (2/14), were not significantly different (P > 0.05). According to the results, we conclude that treatment with PL in mares classified as susceptible to PMIE appears to reduce the inflammatory response after breeding, based on clinical signs of uterine edema, IUF accumulation and PMNs migration.

Assessment of an open-access CASA software for bovine and buffalo sperm motility analysis.

The aim of this study was to verify the reliability of an open access CASA software (BGM) to evaluate the sperm motility of cattle and buffalo, comparing motility and kinematic parameters to those of a commercial one (HTM). Thirty frozen-thawed samples for each species were analyzed with both HTM and BGM, after 1 h of incubation at 37 °C. Sperm viability and mitochondrial membrane potential (MMP) were evaluated through flow cytometric analysis. Agreement of all motility variables between the two systems was assessed. Correlation analysis was performed to identify relationships between motion parameters and sperm viability and MMP. Bland Altman analysis showed good agreement between methods for all motility parameters except for curvilinear velocity (VCL) in cattle, and for average path (VAP), VCL and (amplitude of lateral head displacement) ALH in buffalo, that showed a proportional bias (P > 0.05). In both systems, positive correlation between both viability and high MMP and total and progressive motility of cattle spermatozoa were found; viability and the sperm with high MMP were positive correlated only with VAP, straight-line (VSL), VCL and ALH evaluated with HTM system. Different results were found for buffalo sperm motility parameters, since viability had positive correlations and mitochondrial activity negative ones. Results suggested that motility assessment performed by these two systems are comparable. The discrepancy of VCL, VAP, and ALH could be due to the difference in the algorithms between software. The open-access CASA plug-in is a reliable alternative to the expensive commercial CASA system for sperm motility assessment in cattle and buffalo.

Peptide Mediated Adhesion to Beta-Lactam Ring of Equine Mesenchymal Stem Cells: A Pilot Study.

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 α4ß1 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 α4ß1 integrin agonist GM18 affects equine AT- and WJ-MSCs adhesion ability with a donor-related variability. These preliminary results represent a first step in the study of equine MSCs adhesion to PLLA scaffolds containing GM18, suggesting that WJ-MSCs might be more suitable than AT-MSCs. However, the results need to be confirmed by increasing the number of samples before drawing definite conclusions.

Effect of Aqueous Extract of Maca Addition to an Extender for Chilled Canine Semen.

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 24 h of refrigerated storage with the higher concentration tested was observed. Although Maca was not able to protect canine semen with extended refrigeration storage time, it increased hyperactivation and preserved DNA integrity in short-term storage.

Current Status on Canine Foetal Fluid and Adnexa Derived Mesenchymal Stem Cells.

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 present review is to offer an update on the state of the art on canine MSCs derived from foetal adnexa and fluid focusing on the findings in their clinical setting.

Biological characteristics and metabolic profile of canine mesenchymal stem cells isolated from adipose tissue and umbilical cord matrix.

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 of CD34, mostly by AT-MSCs. DLA-DRA1 and DLA-DQA1 were weakly expressed only at passage 0 by UC-MSCs, while they were expressed at different passages for AT-MSCs. There was no difference (P>0.05) in total ATP production between cell cultures, but the ratio between the "mitochondrial ATP Production Rate" and the "glycolytic ATP Production Rate" was higher (P<0.05) in AT- than in UC-MSCs. However, in both MSCs types the mitochondrial respiration was the main pathway of ATP production. Mitochondrial respiration and ATP turnover in UC-MSCs were higher (P<0.05) than in AT-MSCs, but both had a 100% coupling efficiency. These features and the possibility of increasing the oxygen consumption by a spare respiratory capacity of four (AT-MSCSs) and two (UC-MSCs) order of magnitude greater than basal respiration, can be taken as indicative of the cell propensity to differentiate. The findings may efficiently contribute to select the most appropriate MSCs, culture and experimental conditions for transplantation experiments in mesenchymal stem cell therapy for companion animals.