Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Platelet rich plasma in tendinopathies: how to explain the failure.

Tendinopathies are very common in athletes and in people practicing sport activities. The experimental evidence that growth factors (GFs), present in platelets, enhance the recruitment, proliferation and differentiation of cells involved in tissue regeneration, has prompted the use of platelet rich plasma (PRP) preparations in the treatment of these diseases. However, at present, a sound demonstration of the clinical efficacy of PRP is still lacking. Several theoretical and practical reasons can explain the failure of the treatment: a) animal experiments have been carried out on normal tendons submitted to surgical lesions, and it is questionable whether these models may best mimic human pathology; b) the pathway of chronic tendinopathies is very complex, involving, besides GFs, many other pathogenetic factors, which operate at different stages of the disease; c) several methods have been used to produce PRP, which can result in a large variation in GF content, and in kinetics of release. Therefore, further research is desirable. As a preliminary step, it is necessary to standardize PRP preparation, and to establish the modalities of its activation and administration. Secondly, prospective, randomized, double-blind studies are needed, selecting subjects with homogenous forms of tendinopathies: load-bearing and non-load-bearing tendons, midportion and insertional tendinopathies, with or without neovascularization. Finally, new strategies in PRP use should be exploited: among them, the association of PRP with autologous stem cells or the administration of selective GFs (fibroblast growth factor, vascular endothelial growth factor, or anti-angiogenic factors), which could be better options in specific situations.

Oxytocin, vasopressin, prostaglandin F(2alpha), luteinizing hormone, testosterone, estrone sulfate, and cortisol plasma concentrations after sexual stimulation in stallions.

This experiment was designed to determine the effects of sexual stimulation on plasma concentrations of oxytocin (OT), vasopressin (VP), 15-ketodihydro-PGF(2alpha) (PG-metabolite), luteinizing hormone (LH), testosterone (T), estrone sulfate (ES), and cortisol (C) in stallions. Semen samples were collected from 14 light horse stallions (Equus caballus) of proven fertility using a Missouri model artificial vagina. Blood samples were collected at 15, 12, 9, 6, and 3 min before estrous mare exposure, at erection, at ejaculation, and at 3, 6, and 9 min after ejaculation. Afterwards, blood sampling was performed every 10 min for the following 60 min. Sexual activity determined an increase in plasma concentrations of OT, VP, C, PG-metabolite, and ES and caused no changes in LH and T concentrations. The finding of a negative correlation between C and VP at erection, and between C and T before erection and at the time of erection, could be explained by a possible inhibitory role exerted by C in the mechanism of sexual arousal described for men.

Luteal function in jennies following PGF(2alpha) treatment 3 days after ovulation.

Native PGF(2alpha) and its analogs have been used in the horse mare to manipulate ovarian activity, primarily as luteolytic agents to induce estrus. Despite numerous studies on the effects of these luteolysins in the mare, to date only a single investigation has been conducted in the jenny. The aim of this study was to evaluate the response of the corpus luteum (CL) to a single dose of PGF(2alpha) given 3 days (72h) after ovulation and to establish the plasma progesterone (P4) profile from pre-treatment to post-treatment ovulation in the Martina Franca donkey. Twenty-two jennies were ultrasonographically monitored and treated 72h after the detection of ovulation with 0.075 mg i.m. of R-cloprostenol. From the day of ovulation until ovulation post-treatment, blood was collected daily for P4 determination by enhanced luminescence immunoassay. All the jennies except one, exhibited behavioral signs of PGF(2alpha)-induced estrus within 4 days of treatment lasting 5.4+/-1.16 days. Post-treatment ovulation was also hastened, reducing the interovulatory interval (9.6 days). In response to treatment, plasma P4 concentrations fell to estrus levels and then remained constant until the next ovulation in all but the non-responding animal. Our findings indicate that PGF(2alpha) treatment on Day 3 post-ovulation causes the functional regression of the CL in the jenny, reflected both by the rapid induction of estrus and ovulation and by an abrupt drop in circulating P4 concentrations.

Morphological features of the placenta at term in the Martina Franca donkey.

This study was designed to establish the morphological features of the placenta of the Martina Franca jenny. Ten placentas were harvested at the time of foal delivery and examined both for gross and histological characteristics. The following factors were determined: the total weight and volume of the placenta and its components, the surface area of the allantochorion, umbilical cord length and site of insertion, and the diameter of the umbilical cord vessels and vascular pattern type. The weight of the placenta was similar to previously reported for ponies, and represented 12% of foal birth weight. Umbilical cord length was similar to that in the horse but longer than in the pony, while cord weight was intermediate between the two. In a histological examination, numerous strong villi were observed at sites corresponding to the non-pregnant and pregnant horn and uterine body. No villi were detected in the area overlying the cervical star. Despite obvious similarities between the donkey and horse placenta, specific morphological features do exist, and are possibly related to the differences in length of gestation.

Electrolytes changes in mammary secretions before foaling in jennies.

No knowledge regarding the peripartum changes in mammary secretions in the jenny are presently available in literature. In the mare, instead, several studies report the role of these changes as indicators of foetal readiness for birth and impending parturition. This experiment was designed to determine calcium, sodium, potassium concentrations, and the value of sodium/potassium ratio in mammary fluids during prepartum in the jenny. Samples were daily collected by hand milking, after mammary gland size increased noticeably, from 17 Martina Franca jennies. Prepartum mammary secretions were analysed every other day between day 10 and day 2 antepartum, and then once a day from the day before to the day of parturition. Calcium concentration showed a significant increase between day 10 and day 6 antepartum and then between day 6 and days 4 and 2. Afterwards, another statistical significant increase was observed at parturition. Sodium concentration significantly decreased from day 10 to day 2 prepartum. Potassium concentration significantly increased between day 10 and day 8 before parturition, then showed a further increase at day 4, followed by none significant changes until foaling. All jennies showed a reversal in sodium/potassium ratio between 2 days antepartum and the day before. In conclusion, the evaluation of mammary fluid calcium concentrations and the reversal of sodium/potassium ratio could be used as good indicators of foetal maturity in the jenny. As far as the prediction of parturition is concerned, the reversal of sodium/potassium ratio is the best parameter, since it was detected 48-24 h before parturition in all considered animals.

Efficacy of hCG and GnRH for inducing ovulation in the jenny.

Knowledge about management of ovulation in the donkey is limited compared to that in the horse. This experiment was designed to evaluate the efficacy of injecting single doses of lecirelin (a GnRH-analogue) or of hCG to induce ovulation in the jenny and to determine whether effects are dependent upon follicular diameter at time of injection. Ovarian activity and follicular growth were monitored by rectal ultrasonography. Jennies were randomly allotted to the following groups: Group GnRH, treated with 100 microg lecirelin; Group hCG, treated with 2500 IU hCG; Group C, untreated and monitored for spontaneous ovulation. Animals were also categorized into subgroups depending upon follicular diameter: 30-35 mm (GnRH-1, hCG-1 and C-1) or 36-40 mm (GnRH-2, hCG-2 and C-2). Jennies in the two hormone treatment groups did not differ significantly for time from treatment to ovulation, but there was a significant reduction in time to ovulation as follicle size at treatment increased. Jennies treated with either lecirelin or hCG had significantly smaller follicle size at ovulation than jennies in the Control groups that underwent spontaneous ovulation. Treatment groups did not differ significantly in the proportion of jennies that ovulated within 48 h of injection or between 25 and 48 h following injection. These results highlight the usefulness of lecirelin for induction and synchronization of ovulation in the jenny, particularly since it would avoid the risk of reduced hCG response in reproductive management programs in which that hormone was repeatedly used.