In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Neuhauser S, Handler J, Schelling C, Pieńkowska-Schelling A.Chromosomal abnormalities are notable causes of infertility in horses. Mares show various degrees of estrous behavior, and ultrasound examination often reveals an underdeveloped genital tract. This article reports investigations on fertility in a Haflinger sibship with a healthy, normally developed, fertile mare with at least three healthy offspring. Chromosomal analysis performed incidentally and blinded for this mare revealed 63,X/64,XX/65,XXX mosaicism. Two closely related mares were also mosaics (63,X/64,XX), and one of them was a carrier of a marker chromosome. Repeated examinations of th...
Van Cleemput J, Poelaert KCK, Laval K, Van den Broeck W, Nauwynck HJ.The horse's respiratory tract daily encounters a plethora of respirable hazards including air pollutants, mycotoxins and airborne pathogens. To date, the precise effect of air pollution and mycotoxins on respiratory epithelial integrity and subsequent pathogen invasion in the horse has not been studied. Here, diesel exhaust particles (DEP) and three major mycotoxins (deoxynivalenol [DON], aflatoxin B1 [AFB1] and fumonisin B1 [FB1]) were applied to the apical surfaces of both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells (EREC) cultivated at the a...
Martinez de Andino EV, Brom-de-Luna JG, Canesin HS, Rader K, Resende HL, Ripley AM, Love CC, Hinrichs K.To assess meiotic and developmental competence after transfer of immature cumulus-oocyte complexes (COCs) to the preovulatory follicles of mares (intrafollicular oocyte transfer (IFOT)). Methods: In Experiment 1, mares received an ovulatory stimulus at IFOT. Thirty hours later, COCs were recovered from the follicle, and mature oocytes underwent ICSI and embryo culture. In Experiments 2 and 3, autologous vs. allogeneic COCs were used. The mares were inseminated and embryos were recovered. In Experiment 3, the ovulatory stimulus was administered 9 h (autologous) and 15 h (allogeneic) before IF...
Ząbek T, Witarski W, Semik-Gurgul E, Szmatoła T, Kowalska K, Bugno-Poniewierska M.We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-...
Kinoshita Y, Cloutier AK, Rozak DA, Khan MSR, Niwa H, Uchida-Fujii E, Katayama Y, Tuanyok A.Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules). B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but...
Pessôa LVF, Pires PRL, Del Collado M, Pieri NCG, Recchia K, Souza AF, Perecin F, da Silveira JC, de Andrade AFC, Ambrosio CE, Bressan FF....Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objective: We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Metho...
Khatibzadeh SM, Menarim BC, Nichols AE, Werre SR, Dahlgren LA.Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels see...
Polly SS, Nichols AEC, Donnini E, Inman DJ, Scott TJ, Apple SM, Werre SR, Dahlgren LA.Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expressi...
Folmar CN, Cywes-Bentley C, Bordin AI, Rocha JN, Bray JM, Kahn SK, Schuckert AE, Pier GB, Cohen ND.The bacterium Rhodococcus equi can cause severe pneumonia in foals. The absence of a licensed vaccine and limited effectiveness of commercial R. equi hyperimmune plasma (RE-HIP) create a great need for improved prevention of this disease. Objective: Plasma hyperimmune to the capsular polysaccharide poly-N-acetyl glucosamine (PNAG) would be significantly more effective than RE-HIP at mediating complement deposition and opsonophagocytic killing (OPK) of R. equi. Methods: Venipuncture was performed on 9 Quarter Horses. Methods: The ability of the following plasma sources to mediate complement com...
Marzano G, Mastrorocco A, Zianni R, Mangiacotti M, Chiaravalle AE, Lacalandra GM, Minervini F, Cardinali A, Macciocca M, Vicenti R, Fabbri R....Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidativ...
Ghallab ARM, Abou-Ahmed MM, Fad AM, El-Badry DA, Shahat AM, Moawad AR.Cryopreservation of Arabian stallion semen is important in order to improve the function and fertility of frozen/thawed semen in this breed. Objective: To investigate the effects of centrifugation, type of semen extenders, and type of cryoprotectants on the quality of frozen/thawed Arabian stallion spermatozoa. Methods: Semen samples collected from four adult Arabian stallions (one ejaculate per week for 10 consecutive weeks) were either processed directly without centrifugation (no centrifugation; NC) or subjected to centrifugation on the gel-free fraction. Centrifugation protocols were divid...
Desantis S, Accogli G, Albrizio M, Rossi R, Cremonesi F, Lange Consiglio A.Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and...
Ortiz-Rodriguez JM, Martín-Cano FE, Ortega-Ferrusola C, Masot J, Redondo E, Gázquez A, Gil MC, Aparicio IM, Rojo-Domínguez P, Tapia JA....Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact ...
Xia Y, Qin S, Shen Y.In this study, we isolated four Weissella confusa strains from the healthy horse feces to test their potential as equine probiotics. The identification and characteristics of these isolates were determined as per standard methods. Resistance and susceptibility of the isolated strains were tested to low pHs, different heat treatments, commonly used antibiotics and against the pathogenic strains of Salmonella, Pasteurella, Staphylococcus aureus, and Escherichia coli. After 3 h cultural in different pH medium, the 4 strains still had a certain amount of survival above pH 3.0. WH2 and WH4 were s...
Pawlikowska-Warych M, Deptuła W.One of the most common reasons for horse lameness is subchondral bone cysts (SBCs), which are especially evident in young horse athletes. It is believed that SBC development is strongly associated with an individual's bone growth and/or bone microstructure impairment. Current methods of SBC treatment include pharmacological treatment or surgical procedures which may allow the bone within the cyst to rebuild and be restored to properly developed bone tissue. Thus, we propose filling the SBCs with a 3D complex of alginate hydrogel and autologous adipose derived mesenchymal stem cells (ASCs). We ...
Arévalo-Turrubiarte M, Olmeo C, Accornero P, Baratta M, Martignani E.Mesenchymal stem cells (MSCs) have been used in equines as an alternative therapy. A comparative study about the phenotype and in vitro performance of different MSCs tissue sources in adult equines was needed. This study might serve to provide the knowledge to select a valuable harvesting source of MSCs. Bone marrow, synovial and adipose (mesenteric, neck and tail fat) tissues were collected from adult equines. Cell surface markers expression (CD11α/CD18, CD45, CD79α, CD90, CD105 and MHC II) and in vitro differentiation assays were made. In vitro cell migration, cell growth and wound healing...
Ducheyne KD, Rizzo M, Daels PF, Stout TAE, de Ruijter-Villani M.Vitrified-warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus-oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years). COCs were then either directly matured invitro or vitrified and warmed before IVM. Spindle morphology and chromosome alignment within MII stage oocytes were assessed usin...
Cehak A, Krägeloh T, Zuraw A, Kershaw O, Brehm R, Breves G.Fructo-oligosaccharides are commonly administered as prebiotics to horses in order to reduce the risk of disruption of microbial populations in the hindgut. Their microbial degradation to SCFA already begins in the stomach potentially resulting in increased gastric concentrations of SCFA such as butyric acid. The impact of butyric acid on the squamous mucosa is postulated to be detrimental, its effects on the glandular mucosa are yet unknown. Thus, the aim of this study was to determine the effects of butyric acid exposure on the functional integrity and morphology of the equine nonglandular a...
Sales F, Ferreira-Silva J, Vieira J, Basto S, Filho P, Rocha J, Moura MT, Oliveira M.Post-diluents could potentially increase semen cryotolerance, but remain poorly explored in horses. The aim was to evaluate the efficiency of post-diluents on frozen-thawed semen viability of two stallions (S1-S2). The cryopreserved semen was thawed at 50°C for 40 seconds. Semen motility and acrosin activity (AA) were determined during the thermo-resistance test (TRT). Progressive motility of S2 semen decreased after 60 and 90 minutes of TRT (TRT60 and TRT90) on the control compared to both post-diluents. The total motility of both S1 and S2 decreased on TRT60 and TRT90 semen control versus b...
Martins HS, Martins-Filho OA, Araujo MS, Martins NR, Lagares MA.Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. Objective: This work aimed...
Adegbeye MJ, Elghandour MMMY, Barbabosa-Pliego A, Monroy JC, Mellado M, Ravi Kanth Reddy P, Salem AZM.Several concerns exist regarding horse rearing such as environmental pollution, antibiotics resistance, digestive disorders, mycotoxins contamination of animal feed, gut health management, and improvement of feed efficiency. Nanoparticles have the potential to address these issues and thus could be used as feed additive. Citrate reduces and stabilizes gold nanoparticles, alongside biosynthesized silver nanoparticles have the potential to prolong and improve digestive enzyme activity, which would enhance starch digestibility in the stomach. Zinc oxide and selenium nanoparticles could be used to...
Pfeiffenberger M, Bartsch J, Hoff P, Ponomarev I, Barnewitz D, Thöne-Reineke C, Buttgereit F, Gaber T, Lang A.Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tiss...
Saini S, Singha H, Siwach P, Tripathi BN.Interleukin (IL)-4 and IL-10 activate plethora of immune cells and induce the humoral immune response. However, recombinant version of horse IL-4 and IL-10 has not been investigated to understand their immunomodulating activities. This study aimed to produce recombinant horse mature IL-4 and IL-10 in . Immune-modulating activities of recombinant horse IL-4 and IL-10 were investigated in peripheral blood mononuclear cells (PBMCs). Methods: Equine PBMCs were stimulated with recombinant IL-4 and IL-10. A proliferation of PBMCs was measured by XTT assay and cytokines induction was measured by enzy...
Hunt S, Kuo J, Aristizabal FA, Brown M, Patwardhan A, Hedman T.The mechanical properties of the soft palate can be associated with breathing abnormalities. Dorsal displacement of the soft palate (DDSP) is a naturally occurring equine soft palate disorder caused by displacement of the caudal edge of the soft palate. Snoring and a more serious, sometimes life-threatening, condition called obstructive sleep apnea (OSA) are forms of sleep-related breathing disorders in humans which may involve the soft palate. The goal of this study was to investigate the effect of injecting the protein crosslinker genipin into the soft palate to modify its mechanical propert...
Roberts EL, Dang T, Lepage SIM, Alizadeh AH, Walsh T, Koch TG, Kallos MS.Equine mesenchymal stromal cells (MSCs) are increasingly investigated for their clinical therapeutic utility. Such cell-based treatments can require cell numbers in the millions or billions, with conventional expansion methods using static T-flasks typically inefficient in achieving these cell numbers. Equine cord blood-derived MSCs (eCB-MSCs), are promising cell candidates owing to their capacity for chondrogenic differentiation and immunomodulation. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to generate clinically rel...
Lindström H, Mazari AMA, Musdal Y, Mannervik B.Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 μM concentration afforded at ...
Gérard N, Robin E.Terminal follicular differentiation and ovulation are essential steps of reproduction. They are induced by the increase in circulating LH, and lead to the expulsion from the ovary of oocytes ready to be fertilized. This review summarizes our current understanding of cellular and molecular pathways that control ovulation using a broad mammalian literature, with a specific focus to the mare, which is unique in some aspects of ovarian function in some cases. Essential steps and key factors are approached. The first part of this review concerns LH, receptors and signaling, addressing the descripti...
Van Cleemput J, Poelaert KCK, Laval K, Impens F, Van den Broeck W, Gevaert K, Nauwynck HJ.Pollens are well-known triggers of respiratory allergies and asthma. The pollen burden in today's ambient air is constantly increasing due to rising climate change and air pollution. How pollens interact with the respiratory mucosa remains largely unknown due to a lack of representative model systems. We here demonstrate how pollen proteases of Kentucky bluegrass, white birch and hazel selectively destroy integrity and anchorage of columnar respiratory epithelial cells, but not of basal cells, in both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells...
Duchesne R, Castagnet S, Maillard K, Petry S, Cattoir V, Giard JC, Leon A.This study aimed to analyse antimicrobial susceptibility evolution of equine pathogens isolated from clinical samples from 2006-2016. A collection of 25 813 bacterial isolates was studied, clustered according to their origins (respiratory tract, cutaneous, genital and other), and analysed for their antimicrobial susceptibility using the disk diffusion method. The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%), and Rhodococcus equi ...
Longhini ALF, Salazar TE, Vieira C, Trinh T, Duan Y, Pay LM, Li Calzi S, Losh M, Johnston NA, Xie H, Kim M, Hunt RJ, Yoder MC, Santoro D, McCarrel TM....Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improve...
Meier K, Jaguva Vasudevan AA, Zhang Z, Bähr A, Kochs G, Häussinger D, Münk C.Human myxovirus resistance protein B (hMXB) is a restriction factor of HIV-1 that also inhibits a variety of retroviruses. However, hMXB is not antiviral against equine infectious anemia virus (EIAV). We show here that equine MX2 (eMX2) potently restricts EIAV in vitro. Additionally, eMX2 inhibits HIV-1 and other lentiviruses, including murine leukemia virus. Previously, it was reported that hMXB repression is reduced in hMXB Δ1-25, but not in GTP-binding mutant K131A and GTP-hydrolysis mutant T151A. In contrast to this phenomenon, our study indicates that eMX2 restriction is not diminished i...
Birch HL, Rutter GA, Goodship AE.Hypoxia has been suggested as a possible cause of tissue degeneration and subsequent rupture in equine tendons. To determine whether low oxygen tension is likely to be detrimental to tendon cell function, experiments were designed to investigate oxidative energy metabolism in freshly isolated and cultured equine tendon cells. Freshly isolated tenocytes and cultured fibroblasts possessed activities of the mitochondrial enzyme citrate synthase similar to those of other mammalian cells, with well defined oxidative metabolism. D-[6(-14)C]-glucose oxidation was measurable in both freshly isolated a...
Salamon Z, Tollin G.Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, ...
Moe KK, Yano T, Kuwano A, Sasaki S, Misawa N.Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponema...
Briski O, Salamone DF.During the past 2 decades, intracytoplasmic sperm injection (ICSI) has become a routine technique for clinical applications in humans. The widespread use among domestic species, however, has been limited to horses. In horses, ICSI is used to reproduce elite individuals and, as well as in humans, to mitigate or even circumvent reproductive barriers. Failures in superovulation and conventional in vitro fertilization (IVF) have been the main reason for the use of this technology in horses. In pigs, ICSI has been successfully used to produce transgenic animals. A series of factors have resulted in...
Dobrinsky JR.The development of embryo freezing technologies revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Recently, technologies have been developed to cryopreserve pig embryos, notorious for their extreme sensitivity to cooling; horse embryo cryopreservation is in its infancy. While cryopreservation can enhance the utilization of in vitro embryo production technologies, cryosurvival of in vitro-produced ...
Muñoz PM, Ferrusola CO, Lopez LA, Del Petre C, Garcia MA, de Paz Cabello P, Anel L, Peña FJ.Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the prese...
Bagge J, Berg LC, Janes J, MacLeod JN.Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteog...
Payne SL, La Celle K, Pei XF, Qi XM, Shao H, Steagall WK, Perry S, Fuller F.The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapte...
Carneiro JAM, Canisso IF, Bandeira RS, Scheeren VFC, Freitas-Dell'Aqua CP, Alvarenga MA, Papa FO, Dell'Aqua JA.This study aimed to evaluate the antioxidant properties of coenzyme Q10 (CoQ10) during cryopreservation of semen obtained from stallions having good and bad semen freezing ability (GFA vs. BFA, respectively). Forty ejaculates (n = 20 stallions) were split into five centrifugation and five freezing extenders containing different concentrations of CoQ10 (0, 25, 50, 75 and 100 μmols/L). If CoQ10 was added to the centrifugation extender, the freezing extender had no CoQ10 added; similarly, if CoQ10 was added to the freezing extender, the centrifugation extender had no CoQ10. Semen cryoprese...
Pillet E, Labbe C, Batellier F, Duchamp G, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these ...
Spizziri BE, Fox MH, Bruemmer JE, Squires EL, Graham JK.Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acr...
Parker RA, Clegg PD, Taylor SE.To investigate the effects of commonly used antibiotics on cell viability and gene expression of equine bone marrow-derived mesenchymal stromal cells (MSC) in vitro. Methods: Bone marrow-derived MSC were cultured in media containing gentamicin, amikacin, penicillin, enrofloxacin or ceftiofur at concentrations of 50, 100, 200 and 500 µg/ml. The alamarBlue fluorescence assay was used to assess cell viability over 48 h. After 5 days the cells were released and lysed prior to RNA extraction and reverse transcription. RNA levels were assessed using spectrophotometry and quantitative PCR was used...
Love CC, Kenney RM.The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pre...
Tassone S, Renna M, Barbera S, Valle E, Fortina R.Successful studies on in vitro digestibility measurement of feedstuffs with fecal inoculum have been reported for horses, whereas data on donkeys are currently lacking. In this study, we evaluated the use of the Daisy Incubator for in vitro digestibility measurement of feedstuffs using donkey feces as source of microbial inoculum. The method was tested using seven feedstuffs commonly used in donkey diets (alfalfa, bromegrass, ryegrass, and timothy hays; wheat bran and wheat straw; barley grains). Feces were obtained from four female donkeys, and incubations were carried out at 1-week interva...
Yoon JH, Brooks RL, Khan A, Pan H, Bryan J, Zhang J, Budsberg SC, Mueller PO, Halper J.Fluoroquinolone antibiotics have been used widely in humans and domestic animals, including horses, because of their broad-spectrum bactericidal activity, and relative safety. The use of fluoroquinolones, however, is not without risk. Tendonitis and spontaneous tendon rupture have been reported in people during or following therapy with fluoroquinolones. We have studied the effects of enrofloxacin, a fluoroquinolone antibiotic used commonly in domestic animals, on tendon cell cultures established from equine superficial digital flexor tendons. Effects on cell proliferation and morphology were ...
Bordin AI, Liu M, Nerren JR, Buntain SL, Brake CN, Kogut MH, Cohen ND.Rhodococcus equi is an intracellular bacterium that causes pneumonia in foals and immunocompromised adult horses. Evidence exists that foals become infected with R. equi early in life, a period when innate immune responses are critically important for protection against infection. Neutrophils are innate immune cells that play a key role in defense against this bacterium. Enhancing neutrophil function during early life could thus help to protect foals against R. equi infection. The objective of our study was to determine whether in vitro incubation with the TLR9 agonist CpG 2142 would enhance d...
Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the sa...
Bromfield EG, Aitken RJ, Gibb Z, Lambourne SR, Nixon B.While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop a...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Hamada D, Kuroda Y, Kataoka M, Aimoto S, Yoshimura T, Goto Y.One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the i...
Matera MG, Calzetta L, Rogliani P, Bardaro F, Page CP, Cazzola M.Equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO) is a common equine pulmonary disease with some similarities to human asthma and COPD, which represents a major cause of morbidity and loss of lung performance. Salbutamol has been widely used for the treatment of human airway diseases and has usually been prepared as the racemic form of the drug. However, recently the R-enantiomer of salbutamol has been introduced into clinical practice in the treatment of asthma in humans and this has been suggested to be an improvement on the racemic form of the ...
Dorado J, Acha D, Ortiz I, Gálvez MJ, Carrasco JJ, Gómez-Arrones V, Calero-Carretero R, Hidalgo M.The main aim of this study was to evaluate the efficacy of two commercially available liquid stallion semen extenders for the preservation of Andalusian donkey semen at 5°C for up to 72h, and to evaluate the effect of amino acid addition on sperm quality of cooled donkey semen. In addition, this study investigated the effect of seasons on semen characteristics of Andalusian jackasses. Throughout a year, 50 ejaculates were collected from ten adult donkeys and a complete semen evaluation was performed immediately after collection. In Experiment 1, semen samples (n=32) were pooled, divided into ...
Taddei N, Stefani M, Magherini F, Chiti F, Modesti A, Raugei G, Ramponi G.Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were...
Lee MC, Seonwoo H, Jang KJ, Pandey S, Lim J, Park S, Kim JE, Choung YH, Garg P, Chung JH.Hydroxyapatite (HA) is a representative substance that induces bone regeneration. Our research team extracted nanohydroxyapatite (EH) from natural resources, especially equine bones, and developed it as a molecular biological tool. Polyethylenimine (PEI) was used to coat the EH to develop a gene carrier. To verify that PEI is well coated in the EH, we first observed the morphology and dispersity of PEI-coated EH (pEH) by electron microscopy. The pEH particles were well distributed, while only the EH particles were not distributed and aggregated. Then, the existence of nitrogen elements of PEI ...
Baselgia S, Doherr MG, Mellor P, Torsteinsdottir S, Jermann T, Zurbriggen A, Jungi T, Marti E.Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. Objective: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. Methods: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluate...
Venugopal CS, Eades S, Holmes EP, Beadle RE.One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. Objective: Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. Methods: Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrode's solution at...
dos Santos MC, D'Império Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W.Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immu...
Hinrichs K, Choi YH, Varner DD, Hartman DL.We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2....
Honess NA, Gibson JS, Cossins AR.The effects of oxygen tension (PO2) upon the K influx pathways of equine red cells have been studied using 86Rb+ as congener for K. Equilibration of cells in 100% nitrogen led to a low and Cl-independent K flux. Change to an atmosphere of 100% air led to a rapid sixfold increase in K flux. The oxygen-activated flux was entirely Cl dependent and was maintained for up to 3 h. Oxygenation-evoked activation was dependent upon PO2 over the physiological range with little effect up to 70% saturation of haemoglobin with oxygen but significant effects between 70 and 100%. K flux at low PO2 was unaffec...
