Barbara Merlo

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 Whartons 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.

Quality and Fertilizing Ability In Vivo of Sex-Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex-sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14-propidium iodide), mitochondrial function (JC-1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 x 10(6) X-bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non-sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post-thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste.

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P>0.05). Sperm membrane integrity was positively affected (P<0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P>0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6h after thawing (P>0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P>0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.

97 PRELIMINARY DESCRIPTIVE STUDY OF EQUINE PLACENTA GENERATED AFTER TRANSFER OF IN VIVO- AND IN VITRO-PRODUCED EMBRYOS

Placental changes associated with artificial reproductive technologies have been described in several species, but little information is available in horses. Joy et al. (2012) reported that human placentas from intracytoplasmic sperm injection derived embryos were heavier and thicker than those produced after natural conception. Despite the most growing interest and efficiency of artificial reproductive technologies in equine species, only recently, Pozor et al. (2016) described placental abnormalities in pregnancies generated by somatic cell NT, but there are no studies on equine placenta generated by intracytoplasmic sperm injection and traditional embryo transfer. In the present preliminary study, macroscopic differences of placentas generated after transfer of in vitro- or in vivo-produced embryos were registered. Twelve Standardbred recipient mares with pregnancy generated after transfer of in vivo-derived (Group 1) and in vitro-derived (Group 2) embryos were enrolled; 10 Standardbred mares with pregnancy derived by traditional AI were included as control (Group 3). All pregnancies were physiological, and newborn foals were healthy. Mare age, parity, length of pregnancy, gross evaluation and weight of placenta, total length of umbilical cord (UC), length of UC, number of UC coils, foal sex, and weight at birth were registered. Collected data are listed in Table 1 and are expressed as mean±standard deviation. Differences between groups were evaluated by 1-way ANOVA, and the difference in proportion of overweight placentas was evaluated with the Fisher test. The gross evaluation of placenta revealed 8/12 placentas (2/4 Group 1; 6/8 Group 2) were heavier than 11% (Madigan, 1997) due to oedema of the chorioallantois. No overweight placentas were registered in Group 3. In Group 1, 1/4 placentas had villous hypoplasia, and in Group 2, 1/8 placentas had cystic pouches on the UC. There were no significant differences among groups. However, the proportion of overweight placentas between Group 2 (6/8) and Group 3 (0/10) approached significance (P=0.06). Although preliminary, the results of the present study suggest that production of equine embryos in vitro may lead to alterations in placental development. Several studies in cattle and sheep have suggested that alterations in the placentas of pregnancies derived from in vitro-produced embryos are related to effects of culture on epigenetic regulation. Less is known in the horse about the effects of in vitro embryo production on placental development; thus, further research in this area is necessary.

Effects of induced endometritis on uterine blood flow in cows as evaluated by transrectal Doppler sonography

This study was conducted to evaluate the effects of induced endometritis on uterine blood flow in cows. Transrectal Doppler sonography was performed on uterine arteries of six cyclic cows before and for 4 days after inducing acute endometritis by intrauterine infusion of 720 mg of policresulen, and for 4 days of the following estrous cycle. Time-averaged maximum velocity (TAMV) increased (p < 0.001) and pulsatility index (PI) decreased (p < 0.0001) within 1 h of policresulen administration, and did not change (p > 0.05) in the next 4 days of the same cycle. TAMV and PI values in the subsequent cycle did not differ (p > 0.05) from the values measured before infusion and showed no changes (p > 0.05) within the cycle. Blood flow parameters were not related (p > 0.05) to plasma concentrations of progesterone and estrogen. All cows showed an acute endometritis determined by histopathological findings of biopsy samples taken 1 day after infusion and fibrotic endometrial alterations detected in the subsequent cycle. No relationships were observed between fibrotic changes of the endometrium and uterine blood flow during either cycle. In conclusion, acute inflammation is accompanied by a rise in uterine blood flow, but fibrotic alterations do not seem to be related to Doppler sonographic findings.

Could hypoxia influence basic biological properties and ultrastructural features of adult canine mesenchymal stem /stromal cells?

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. 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.

In vitro developmental competence of horse embryos derived from oocytes with a different corona radiata cumulus-oocyte morphology

The 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.

Comparative characterization of human and equine Wharton’s jelly derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) have the capability to differentiate into wide range of specialized cells of mesodermal origin such as osteocytes, chondrocytes, adipocytes, cardiomyocytes, muscle fibers. Due to these properties, MSCs are considered as a new emerging treatment option and therapeutic agent in regenerative medicine. Promising results have been obtained after application of MSCs for treating tendon and joint disease in the equine model, making it favorable for therapeutic application. While the horse is considered a highly suitable model for orthopedic diseases, knowledge is lacking regarding the level of analogy of equine MSCs and their human counterparts. Therefore, the aim of this study was to assess the properties of human and equine Wharton’s jelly derived MSCs in a direct comparison. Obtained MSCs, were characterized for their staminal markers, proliferation and adhesion potential, ultrastructural morphology and their ability in differentiate towards osteogenic, chondrogenic and adipogenic lineages. Results showed a similar pattern in the expression of staminal markers, while a light difference was observed in the proliferation and adhesion potential. Ultramorphological analysis showed nuclear and citoplasmatic features comparable in human and equine MSCs. Finally, both MSCs were able to differentiate towards osteogenic, chondrogenic and adipogenic lineages. In conclusion, although revealing some potentially relevant differences, the study demonstrates a high level of analogy between human and equine MSCs, providing a basis for translational research in the equine model.

186 BOVINE AMNIOTIC FLUID MESENCHYMAL STEM CELLS CHARACTERIZATION AFTER CULTURE IN VITRO

In recent years, fetal adnexa and fluids have been recognised as important sources of mesenchymal stem cells (MSC). The aim of this study was to characterise cell populations of bovine amniotic fluid, studying phenotypic characterisation, RNA expression, and differentiation potential of samples after in vitro culture for different lengths of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at the slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1 : 1) plus 10% fetal bovine serum (FBS) in 5% CO2 at 38.5°C. At passages P3 and P7, a sample for each of the 4 population found was characterised. Immunophenotypic characterisation was performed for MSC (CD90, CD105, CD44) and haematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4, and α-SMA and the ratio between positive cells and total nuclei was evaluated. Gene expression profile was analysed by RT–PCR for pluripotency markers (Oct4, Nanog, Sox2). At the same passages chondrogenic, osteogenic and adipogenic differentiation were induced and evaluated morphologically and cytologically using, respectively, Alcian blue to identify cartilage matrix, Von Kossa for extracellular calcium deposition, and Oil Red O for intracellular lipid droplets. Cell population appeared heterogeneous and we could identify 2 main cell types: round (R) and spindle-shaped (S) cells. Each isolated sample was classified into one of the following 4 types depending on percentages of R or S cells: prevalence of S-cells (S), prevalence of R-cells (R), and samples showing both morphologies with ~10% of S-cells (S10) or 40% S-cells (S40). S-cells percentage decreased with passages in S10 and S40. After FACS, all lines were positive for CD90, CD105, CD44, and CD34 and negative for CD14 both at P3 and at P7. After ICC, Oct4 was negative in all samples analysed, few S cells stained for SSEA4 (8%) at P3 but increased at P7 to 22%; R, S10, and S40 did not express SSEA4 both at P3 and at P7. α-SMA was expressed in all samples at P3 (9.4% S; 0.9% R; 2.5% S10; 27% S40) but not at P7 (27.5% S; 0% R; 0% S10; 0% S40). After RT–PCR analyses, Oct4 was negative in all samples; at P3, Nanog was clearly positive in S-cells, weak in S40, and negative in R and S10, but all samples turned negative at P7. Sox2 was weakly expressed (S) or not expressed (S10, S40, R) at P3 and it was negative in all cells at P7. Only S showed high differentiation potential into all 3 lineages at both P3 and P7, R had the lowest differentiation potential, whereas S10 and S40 were intermediate at both end points. In conclusion, bovine amniotic fluid showed heterogeneous cell populations and S-type had the characteristics of MSCs. S10 and S40 showed more MSC markers at P3, when S cells were still present, and this aspect suggests that S population is the presumptive MSC one. Although prevalent, R-type showed only some MSC characteristics. Further studies are under way to improve S-type isolation, purification, and culture, and to determine the lifespan of these cell types. This work was supported by grant PRIN2009.

319 In vitro maturation of equine oocytes in a completely defined medium supplemented with progesterone

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500 ng mL-1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1 IU mL-1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50 ng mL-1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2 mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytes mL-1): (1) MSOF + FCS 10% (MSOF-FCS), (2) MSOF + progesterone 100 ng mL-1 (MSOF-P4), (3) MSOF. After 24 h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10 μg mL-1 in PBS) for 30 min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P < 0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P < 0.05) increases the nuclear maturation rate at 24 h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4 ± 19.3 ng mL-1, healthy preovulatory follicle 1094.3 ± 170.9 ng mL-1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system. *All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media