Western Blot Analysis of Proacrosin/Acrosin in Frozen Dog Sperm During In Vitro Capacitation

Acrosin is an acrosomal protease synthesized as an inactive precursor, proacrosin, which is processed via autoproteolysis into active forms alpha- and beta-acrosin. In this paper, a comparative study on the immunoreactivity of acrosin during in vitro capacitation of frozen and fresh (control) canine sperm using Western blot analysis is reported. Semen samples were obtained by digital stimulation and ejaculates processed as fresh and frozen samples and then capacitated for 0, 30, 60 and 90 min. At each time period, samples were analyzed with monoclonal antibody C5F10 by Western blot. The antibody specifically recognized, in fresh and frozen/thawed spermatozoa, a 40-, 32- and 27-kDa bands corresponding to proacrosin, alpha- and beta-acrosin, respectively, during capacitation. Western immunoblots showed that the beta-acrosin reactivity in fresh sperm was directly proportional to the time of capacitation, whereas a decreased reactivity of active form of acrosin was observed with frozen-thawed sperm (p   < 0.05). These results suggest that proacrosin is activated to beta-acrosin earlier in frozen/thawed dog spermatozoa than in fresh dog spermatozoa.     

An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E₂ (PGE₂) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE₂ at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.     

Biomechanics of Cranial Cruciate Ligament Reconstruction in the Dog I. In Vitro Laxity Testing

Following an "over-the-top" reconstruction of the cranial cruciate ligament using fascia lata and the lateral one-third of the patellar ligament, joint instability was measured using an in vitro laxity testing device. Cranlocaudal drawer increased between zero and four weeks, then returned to initial values at 26 weeks.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

An In Vitro Biomechanical Comparison of Two Fixation Methods for Transverse Osteotomies of the Medial Proximal Forelimb Sesamoid Bones in Horses

OBJECTIVE: This study compared the mechanical properties of the normal intact suspensory apparatus and two methods of fixation for repair of transverse, midbody fractures of the proximal sesamoid bones of adult horses: transfixation wiring (TW) and screws placed in lag fashion (LS). STUDY DESIGN: An in vitro, paired study using equine cadaver limbs mounted in a loading apparatus was used to test the mechanical properties of TW and LS. ANIMAL OR SAMPLE POPULATION: Seventeen paired (13 repaired, 4 normal) equine cadaver limbs consisting of the suspensory apparatus third metacarpal bone, and first and second phalanges. METHOD: The two methods of repair and normal intact specimens were evaluated in single cycle-to-failure loading. Yield failure was defined to occur at the first notable discontinuity (>50 N) in the load-displacement curve, the first visible failure as evident on the videotape, or a change in the slope of the moment-fetlock angle curve. Ultimate failure was defined to occur at the highest load resisted by the specimen. Corresponding resultant force and force per kg of body weight on the suspensory apparatus, fetlock joint moment, and angle of fetlock dorsiflexion were calculated by use of specimen dimensions and applied load. These were compared along with specimen stiffness, and ram displacement. RESULTS: Load on the suspensory apparatus, load on the suspensory apparatus per kg of body weight, moment, applied load, and angle of fetlock dorsiflexion at yield failure were significantly greater for the TW-repaired than for the LS-repaired specimens. A 3 to 5 mm gap was observed before yield failure in most TW-repaired osteotomies. CONCLUSIONS: Transfixation wiring provided greater strength to yield failure than screws placed in lag fashion in single cycle load-to-failure mechanical testing of repaired transverse osteotomized specimens of the medial proximal forelimb sesamoid bone.     

An In Vitro Biomechanical Investigation of an Interlocking Nail for Fixation of Diaphyseal Tibial Fractures in Adult Horses

The compressive, bending and torsional mechanical properties of osteotomized adult equine tibiae stabilized with an interlocking intramedullary nail (nail-tibia composite) were compared with those of intact tibiae to determine the clinical applicability of the nail for repair of tibial fractures in adult horses. The mean yield load, failure load, and stiffness for the nail-tibia composites were significantly less ( P < .05) than those for the intact tibiae in all loading configurations. The mean compressive yield load for the nail-tibia composites was greater than the compressive load calculated from previously reported in vivo data for walking and trotting, and was equal to the load calculated for recovery from anesthesia. The mean yield bending moment for the nail-tibia composites was greater than the bending moment previously calculated for standing, walking, and recovery from anesthesia. The mean torsional yield load for the nail-tibia composites was less than the torsional load determined for the walk from another in vivo study. The design of the interlocking nail evaluated in the present study should be modified to increase torsional and compressive yield strengths and torsional stiffness before reasonable success could be expected for the treatment of adult equine tibial fractures.     

Species Differences in the Susceptibility of Erythrocytes Exposed to Free Radicals In Vitro

The susceptibility of human, cow, pig, sheep and rabbit erythrocytes to free radicals (peroxyl radicals) generated in vitro by 2,2-azo-bis(2-amidinopropane) hydrochloride (AAPH) was evaluated by means of a haemolysis test and expressed as the time to 50% of maximal haemolysis (HT₅₀). The most sensitive to damage by free radicals appeared to be the erythrocytes of pigs and sheep, their HT₅₀ values being (mean±SEM) 85.1±1.28 min and 89.0±1.31 min, respectively. Human erythrocytes and those of cows and rabbits were about twice as resistant, their HT₅₀ values being (mean±SEM) 174.3±1.53 min, 181.2±1.22 min and 183.4±2.54 min, respectively. Pig and sheep erythrocytes used in the haemolysis test provided an indication of the antioxidant status in a shorter time (2.5 h versus 4.5 h) than those of the other species studied. The results indicate that the HT₅₀ test may be a convenient alternative to the osmotic resistance test for defining the antioxidant resistance of erythrocytes.     

The Brain of the Dog in Section: a Comprehensive View for Veterinary Students

Transversal, horizontal and sagittal sections of the brain were stained by the ancient but efficient Mulligan method, a procedure that establishes a clear macroscopic difference between the white and grey substances. Different structures of each section were studied and most of the details were identified and named according to the NAV. All sections were projected onto the whole brain. By means of this easy and basic procedure the students increase their understanding of (1) the size and/or the form and/or the topography of several prominent structures of the brain, (2) the general distribution of the substancia alba and grisea, and they begin to understand the complexity of the brain.     

GABA Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study examines the involvement of GABA_{A or B} receptors in gonadotrophin‐releasing hormone (GnRH) release in vitro and determines whether oestradiol modulates γ‐aminobutyric acid (GABA)–GnRH interaction. Within 10 min after ewe killing, hypothalamic slices were dissected and placed in oxygenated Minimum Essential Media (MEM)‐α at 4°C; within 2 h, slices were singly perifused at 37°C with oxygenated MEM‐α (0.15 ml/min), with or without oestradiol (24 pg/ml). After 4 h equilibration, fractions were collected for 4 h interposed with a 10 min exposure to specific GABA_{A or B} receptor ligands (0.1–10 mm ). The GABA_{A or B} agonists (muscimol or baclofen) did not greatly influence GnRH release. However, GnRH increased (p < 0.05) after exposure to 10 mm GABA_{A or B} antagonists (bicuculline or CGP52432, respectively). The GABA_A antagonist stimulated greater sustained GnRH release (p < 0.05) in the absence of oestradiol than in its presence. The bioactivity of the released GnRH was studied using a hypothalamus‐pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to the GABA_A antagonist, did the hypothalamic eluate stimulate luteinizing hormone release from pituitary fragments (p < 0.05) confirming that the GABA_A antagonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is predominantly under GABA_A receptor inhibitory control and this is attenuated in the presence of oestradiol.     

Noradrenergic Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study investigates the influence of α₁‐adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates α₁‐adrenoreceptor‐GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media‐α (MEM‐α) at 4°C and within 2 h were singly perifused at 37°C with oxygenated MEM‐α (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4‐h equilibration, 10‐min fractions were collected for 4 h interposed with a 10‐min exposure at 60 min to specific α₁‐adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1–10 mm ). The α₁‐adrenoreceptor agonist (10 mm ) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, α₁‐adrenoreceptor agonist (10 mm )‐induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus–pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to α₁‐adrenoreceptor agonist (10 mm ), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 ± 12.3–36.2 ± 21.6 ng/ml) confirming that the α₁‐adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.     

In Vitro and In Vivo Evaluation of Ferric-Hyaluronate Implants for Delivery of Amikacin Sulfate to the Tarsocrural Joint of Horses

Objective— To assess the antimicrobial elution characteristics, toxicity, and antimicrobial activity of amikacin‐impregnated ferric‐hyaluronate implants (AI‐FeHAI) for amikacin delivery to the tarsocrural joint of horses. Study Design— Experimental study. Sample Population— AI‐FeHAI implants, equine cartilage, and synovium, and horses (n=6). Methods— In vitro study: Five AI‐FeHAI were placed in saline solution with daily replacement until implant degradation. Eluent was tested for amikacin concentration and bioactivity. Synovial and cartilage explants were incubated in the presence or absence of AI‐FeHAI for 72 hours and subsequently assessed for morphology, viability, and composition. Synovial explants were incubated with Staphylococcus aureus in the presence or absence of AI‐FeHAI. Spent medium was cultured daily and explants were assessed for morphology and viability after 96 hours. In vivo study: AI‐FeHAI were placed in 6 tarsocrural joints. Standard cytologic analysis and amikacin concentration (SFAC) were determined in synovia obtained regularly for 28 days thereafter. Similar analyses were conducted after a single intra‐articular injection of amikacin 6 months later. Results— In vitro study: Amikacin concentrations exceeded 16 μg/mL and inhibited S. aureus growth for 8 days. AI‐FeHAI had no effect on cartilage explants. AI‐FeHAI eliminated bacteria from synovial explants. In vitro study: After AI‐FeHAI placement, SFAC was highest (140.78+63.81 μg/mL) at first sampling time. By 24 hours SFAC was <16 μg/mL. After intra‐articular injection, SFAC was the highest (377.91 ± 40.15 μg/mL) at first sampling time. By 48 hours SFAC was <16 μg/mL. Conclusions— A single intra‐articular amikacin injection demonstrated superior pharmacokinetics than AI‐FeHAI prepared as described. Clinical Relevance— AI‐FeHAI cannot be recommended for clinical use.     

Complete Heart Block in a Dog Seropositive for Borrelia burgdorferi: Similarity to Human Lyme Carditis

Lyme disease has been recognized in humans since 1975 when it was associated with an outbreak of oligoarthritis in children in Lyme, Connecticut. Erythema chronicum migrans (ECM) is a clinical marker for the human disease, which usually appears within 3 to 32 days after an infected tick bite. Lyme disease is caused by spirochete, Borrelia burgdorferi, which is vectored by the hard ticks Ixodes dammini or Ixodes pacificus in the United States. In humans, Lyme disease has been found to cause a variety of clinical syndromes including cardiopathy, neuropathy, dermatopathy, and arthropathy. Human Lyme carditis is characterized by varying degrees of atrioventricular (AV) heart block that usually resolve regardless of therapy. Lyme disease has been reported in the dog as an arthropathy. This article reports a case of complete heart block and myocarditis in a dog with a positive titer for B burgdorferi, in which clinical and pathologic findings were similar to those seen in human Lyme myocarditis.     

Noradrenergic Control of Arginine Vasopressin Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study aims at ascertaining the influence of α₁‐adrenoreceptors on arginine vasopressin (AVP) release in vitro and determine whether E₂ modulates the α₁‐adrenoreceptor and AVP interaction. Ten minutes after ewe killing, sagittal midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus with the median eminence, 2 mm thick, 2 per sheep) were dissected, placed in oxygenated minimum essential media‐α (MEM‐α) at 4°C and within 2 h were singly perifused at 37°C with oxygenated MEM‐α (pH 7.4; flow rate 0.15 ml/min), either with or without E₂ (24 pg/ml). After 4 h equilibration, 10 min fractions were collected for 4 h interposed with 10 min exposure at 60 min to a specific α₁‐adrenoreceptor agonist or antagonist at various doses (0.1–10 mm ). At the end of all perifusions, slices responded to KCl (100 mm ) with AVP efflux (p < 0.05). Release of AVP was enhanced (p < 0.05) by the α₁‐adrenoreceptor agonist (methoxamine 10 mm ; no E₂, n = 7 perifusion chambers: from 14.3 ± 2.7 to 20.9 ± 3.9, with E₂, n = 10: from 10.7 ± 1.2 to 18.4 ± 3.4 pg/ml) or the antagonist (thymoxamine 10 mm ; no E₂, n = 5: from 9.5 ± 3.1 to 30.4 ± 6.0, with E₂, n = 10: from 10.8 ± 0.9 to 39.1 ± 6.3 pg/ml). With the agonist, the response occurred only at 80 min (p < 0.05) both in the presence and absence of E₂. Whereas, after the antagonist, values were higher (p < 0.05) throughout the post‐treatment period (80–170 min) without E₂, but declined by 150 min in the presence of E₂. Furthermore, the response to the α₁‐adrenoreceptor antagonist was greater (p < 0.05; 90–140 min) than the agonist only in the presence of E₂. In conclusion, these results reveal direct α₁‐adrenoreceptor‐mediated control of the hypothalamic AVP neuronal system which is modulated by E₂.     

Time Course of In Vitro Maturation of Compact Cumulus Horse Oocytes after Roscovitine‐Induced Meiotic Inhibition: Effects on the Coordination Between Nuclear and Cytoplasmic Maturation

This study was carried out to evaluate the usefulness of a pre‐maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre‐cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM‐ROSC) or in a simple medium (M199‐ROSC). After pre‐maturation, oocytes from both the groups were in part denuded and fixed‐stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre‐cultured in M199‐ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM‐ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199‐ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM‐ROSC group (34/90) compared with M199‐ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre‐culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.     

In Vivo and In Vitro Evidence of the Involvement of CXCL1, a Keratinocyte-Derived Chemokine, in Equine Laminitis

Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans.
Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis.
Animals: Twenty adult horses.
Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression.
Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [ P = .0001]) and DTP groups (21-fold [ P = .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases ( P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H₂O₂ (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA ( P = .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes.
Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.     

Splenocaval Shunting for Alleviation of Portal Hypertension in a Dog: A Case Report

Objective —To describe the construction and use of a splenocaval shunt to prevent portal hypertension in a dog with iatrogenic rupture and subsequent complete occlusion of an intrahepatic portosystemic shunt (IPSS).
Study Design —Case report describing a single, client-owned animal.
Results —During dissection, the back wall of an IPSS was torn. Complete shunt occlusion was required to control the hemorrhage. This resulted in the development of life-threatening portal hypertension. Emergency splenocaval shunt construction reduced the portal pressure from 47 to 20 cm H₂0. The dog experienced minimal postoperative complications. A second surgical procedure was performed a month later to completely ligate the splenocaval shunt.
Conclusions —A splenocaval shunt can be used to divert blood from the portal to the systemic circulation to control portal hypertension. In this dog, it resulted in a successful outcome with few complications.
Clinical Relevance —The splenocaval shunt could be constructed before the dissection of a difficult IPSS if problems arise as occurred in the dog described in this report. Complete IPSS occlusion can be performed without development of portal hypertension.     

Assessment of Suitable Porcine Semen for Freezing,According to the Ejaculate Characteristics in the Iberico × Landrace Breed

A total of 35 ejaculates were studied in order to assess the suitability of porcine semen for freezing according to the ejaculate characteristics. The effects of the freezing procedure were identified; a decrease in motility and acrosome quality was found after thawing. The best results on motility were linked to the ejaculates with a volume of less than 100 ml of the sperm‐rich fraction, a concentration lower than 450 × 10⁶ spermatozoa/ml and an agglutination score below 2. However, the best normal apical ridge (NAR) was found when the volume of the sperm‐rich fraction was greater than 150 ml. For this reason, an intermediate volume of the sperm‐rich fraction of the ejaculate for the best motility and the best NAR, a concentration lower than 450 × 10⁶ spermatozoa/ml and a rate of agglutination below 2 should provide the best quality after freezing. This study also attempted to determine whether a positive effect of ejaculate selection on the overall freezing performance might be expected.