Morphometry of primary and secondary epidermal laminae in equine hoof

We studied the length of primary and secondary epidermal laminae of the toe and the lateral and medial quarters of horses, distributed into proximal, middle and distal thirds of the hooves. Eight limbs from adult crossbred horses, four females and four males, used to pull carts without pedal conditions. Fragments were taken from different regions of the hooves and subjected to conventional histological techniques. The samples were stained with hematoxylin-eosin and analyzed by light microscopy. The primary epidermal laminae were higher in the hooves of forelimbs compared to hindlimbs in the proximal and middle thirds and the regions of the medial quarter and toe. The secondary laminae were higher in forelimb of the middle third and medial quarter. Comparing the length of the epidermal laminae between hoof parts, it was seen that the primary laminae are lower in the proximal third and higher in the toe, while the secondary laminae are lower in the proximal third and medial quarter. The results suggested that the morphology of the laminae in the different regions of the hooves is influenced through the work performed by the animal, as well as through the different distribution of forces.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

Four cases of equine polysaccharide storage myopathy in the United Kingdom

This paper describes four cases of equine polysaccharide storage myopathy which were confirmed by histological examination of muscle biopsy specimens. The horses were of mixed breeding, with warmblood and thoroughbred dominating. They all had recurrent episodes of rhabdomyolysis, indicated by clinical signs and increased plasma levels of muscle enzymes. They were managed conservatively and have continued athletic careers despite their disease.

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

Reasons for performing study: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. Objectives: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. Methods: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. Results: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. Conclusions: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. Potential relevance: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our under-standing of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field. (c) 2006 Elsevier B.V. All rights reserved.

Equine laminitis: Bites by Bothrops spp cause hoof lamellar pathology in the contralateral as well as in the bitten limb

The envenoming caused by Bothrops snakebite includes local symptoms, such as pronounced edema, hemorrhage, intense pain, vesicles, blisters and myonecrosis. The principal systemic symptom consists in the alteration of blood clotting, due to fibrinogen consumption and platelet abnormalities. The horses involved in this study had this symptomatology and one of them exhibited symptoms consistent with laminitis in the bitten and in the contralateral limbs. Laminitis lesions were characterized by separation of the hoof lamellar basement membrane (BM) from basal cells of the epidermis. These results demonstrated that Bothrops snake venom can induce acute laminitis. We conclude that components of the venom, probably metalloproteinases, cause severe lesions in the hoof early in the envenoming process. Antivenom therapy must be initiated as soon as possible in order to prevent complications, not only to save the life of an envenomed horse, but also to avoid the dysfunctional sequels of laminitis. (c) 2006 Elsevier Ltd. All rights reserved.

Conjugated equine estrogen, raloxifene and arterial stiffness in postmenopausal women

Methods We analyzed the influence of conjugated equine estrogen (CEE) and raloxifene on arterial stiffness. Sixty-seven healthy, normotensive women 1-10 years into menopause were assigned to receive oral placebo, conjugated equine estrogen 0.625mg, or raloxifene 60mg. Arterial stiffness was evaluated by measuring the carotid-femoral and femoral-dorsalis pedis pulse wave velocity (CF PWV, FP PWV). Systolic pressure augmentation index (AI) at the carotid artery was obtained with applanation tonometry. Results Arterial stiffness was not affected by any treatment regimen: placebo (CF PWV before vs. after: 644 vs. 626 cm/s, p = 0.09; FP PWV before vs. after: 1006 vs. 1012 cm/s, p = 0.77; AI before vs. after = 30 vs. 29%, p = 0.55), CEE (CF PWV before vs. after: 642 vs. 600 cm/s, p = 0.11; FP PWV before vs. after: 952 vs. 971 cm/s, p = 0.66; AI before vs. after: 25 vs. 32%, p = 0.82), and raloxifene (CF PWV before vs. after: 636 vs. 601 cm/s, p = 0.12; FP PWV before vs. after: 964 vs. 941 cm/s, p = 0.62; AI before vs. after: 25 vs. 25%, p = 0.65). A correlation occurred between basal stiffness and the degree of reduction in indexes measured, indicating that the higher the basal stiffness, the greater the degree of reduction, particularly in the CEE group: CF PWV (r = -0.602, p = 0.001); FP PWV (r = -0.455, p = 0.022); AI (r = -0.410, p = 0.042). Conclusions Conjugated equine estrogen and raloxifene do not seem to affect arterial stiffness of healthy normotensive women less than 10 years since menopause. Reduction in arterial stiffness seems related to its basal level.

Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.

Arthrodesis of the equine proximal interphalangeal joint: a biomechanical comparison of 3-Hole 4.5 mm locking compression plate and 3-hole 4.5 mm narrow dynamic compression plate, with two transarticular 5.5 mm cortex screws

Objectives To compare the biomechanical characteristics of 2 arthrodesis techniques for the equine proximal interphalangeal joint (PIP) using either a 3-hole 4.5 mm locking compression plate (LCP) or 3-hole 4.5 mm narrow dynamic compression plate (DCP), both with 2 transarticular 5.5 mm cortex screws. Study Design Experimental. Sample Population Cadaveric adult equine forelimbs (*n=6 pairs). Methods For each forelimb pair, 1 limb was randomly assigned to 1 of 2 treatment groups and the contralateral limb by default to the other treatment group. Construct stiffness, gap formation across the PIP joint, and rotation about the PIP joint were determined for each construct before cyclic axial loading and after each of four, 5000 cycle loading regimens. After the 20,000 cycle axial loading regimen, each construct was loaded to failure. Results There were no significant differences in construct stiffness, gap formation, or sagittal plane rotation between the LCP and DCP treatment groups at any of the measured time points. Conclusion Biomechanically, fixation of the equine PIP joint with a 3-hole 4.5 mm LCP is equivalent to fixation with a 3-hole 4.5 mm narrow DCP under the test conditions used.

Prevalence of equine Piroplasmosis and its association with tick infestation in the State of Sao Paulo, Brazil

Serum samples were collected from 582 horses from 40 stud farms in the State of Sao Paulo and tick (Acari: Ixodidae) infestations were evaluated on them. Serum samples were subjected to the complement fixation test (CFT) and a competitive inhibition ELISA (cELISA) for Babesia caballi and Theileria equi. Logistic regression analyses were performed to construct multivariate models that could explain the dependent variable (horses positive for B. caballi or T equi) as a function of the independent variables (presence or abundance of each one of the rick species found on the farms). A higher overall prevalence of B. caballi (54.1%) than of T equi (21.6%) was found by the two tests. The ticks Dermacentor nitens Neumann, 1897, Amblyomma cajennense (Fabricius, 1787) and Rbipicephalus (Boopbilus) microplus (Canestrini, 1887) were present on horses on 38 (95%), 20 (50%), and 4 (10%) farms, respectively. Infestations by D. nitens were statistically associated with B. caballi-positive horses on the farms by either the CFT or cELISA. Infestations by A. cajennense were statistically associated with T equi-positive horses on the farms by either CFT or cELISA.

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TA!) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2 x 2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5 +/- 2.3 d postpartum, received a 3 mg norgestomet ear implant se, plus 3 mg norgestomet and 5 mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 mu g gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means +/- SEM, 1.53 +/- 0.1 vs. 0.48 +/- 0.1 mm/d; P < 0.0001), its diameter on Day 11(11.4 +/- 0.6 vs. 9.3 +/- 0.7 mm; P = 0.03), as well as ovulation rate (80.8% vs. 50.0%, P = 0.02), whereas GnRH improved the synchrony of ovulation (72.0 +/- 1.1 VS. 71.1 +/- 2.0 h). In Experiment 2 (n = 599 cows, 40 to 120 d postpartum), pregnancy rates differed (P = 0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control. GnRH, eCG, and eCG + GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 318%, P = 0.002; and 48.0% vs 37.6%, P = 0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol. (C) 2010 Elsevier Inc. All rights reserved.

Effects of equine chorionic gonadotropin and type of ovulatory stimulus in a timed-AI protocol on reproductive responses in dairy cows

The objectives were to evaluate the effects of equine chorionic gonadotropin (eCG) supplementation (with or without eCG) and type of ovulatory stimulus (GnRH or ECP) on ovarian follicular dynamics, luteal function, and pregnancies per AI (P/AI) in Holstein cows receiving timed artificial insemination (TAI). On Day 0, 742 cows in a total of 782 breedings, received 2 mg of estradiol benzoate (EB) and one intravaginal progesterone (P4) insert (CIDR). On Day 8, the CIDR was removed, and all cows were given PGF2 alpha and assigned to one of four treatments in a 2 x 2 factorial arrangement: (1) CG: GnRH 48 h later; (2) CE: ECP; (3) EG: eCG + GnRH 48 It later; (4) EE: eCG + ECP. There were significant interactions for eCG x ovulatory stimulus and eCG x BCS. Cows in the CG group were less likely (28.9% vs. 33.8%; P < 0.05) to become pregnant compared with those in the EG group (odds ratio [OR] = 0.28). There were no differences in P/AI between CE and EE cows (30.9% vs. 29.1%; OR = 0.85; P = 0.56), respectively. Thinner cows not receiving eCG had lower P/AI than thinner cows receiving eCG (15.2% vs. 38.0%; OR = 0.20; P < 0.01). Treatment with eCG tended to increase serum progestesterone concentrations during the diestrus following synchronized ovulation (P < 0.10). However, the treatment used to induce ovulation did not affect CL volume or serum progesterone concentrations. In conclusion, both ECP and GnRH yielded comparable P/AI. However, eCG treatment at CIDR removal increased pregnancy rate in cows induced to ovulate with GnRH and in cows with lower BCS. (C) 2009 Elsevier Inc. All rights reserved.

Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa

The advantages of using cryopreserved semen in equine reproduction are well known. During cryopreservationl spermatozoa undergo many changes that lead to a decrease in fertility. There is no agreement on the ideal sperm dose and concentration to maximize fertility rates. Thus, the objectives of this experiment were to evaluate sperm motion by computer-assisted analysis (CASA), sperm membrane integrity and function with fluorescence probes of cryopreserved sperm at three concentrations: 100 (C100), 200 (C200) and 400 x 10(6) sperm/mL (C400), and two straw volumes (0.50 and 0.25 mL). There was no interaction between sperm concentration and storage volume (P > .05). Sperm motion characteristics were influenced by concentration (C100 > C200 > C400; P < .05). Curvilinear velocity (VCL) in 0.25-mL straws had higher average values (P < .05). Membrane integrity and function were not changed by straw volume (P > .05). However, sperm concentration changed the percentage of cells with intact plasma membrane (C100 > C200 > C400; P < .05) and the percentage of cells with high mitochondrial membrane potential (C100 = C200; P > .05 and C400 < C100 and C200; P < .05). According to this experiment, the best freeing method was that involving 100 x 10(6) sperm/mL, regardless of straw volume.