Derivation of Canine Induced Pluripotent Stem Cells

Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose‐derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re‐programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c‐myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re‐activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.     

Advances in Holding and Cryopreservation of Equine Oocytes and Embryos

Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or higher holding temperatures, may be detrimental. Small equine embryos (<300 μm), either in vivo derived or in vitro produced, can be slow frozen or vitrified successfully. In the last decade, methods have been developed to allow in vivo–derived expanded blastocysts, up to Day 8, to be vitrified successfully after blastocoele collapse. These methods of shipment and preservation allow mare owners in remote locations to have access to sophisticated assisted reproductive technologies.     

A Botanical-Based Equine Nutraceutical Reduces Gastric Smooth Muscle Contractile Force In Vitro

The objective of this study was to evaluate the effect of a botanical-based equine nutraceutical on contractility of gastric smooth muscle in vitro. Gastric ulcers are prevalent in performance horses and negatively impact horse welfare. Gastric hypermotility has been positively associated with the development of gastric ulceration in nonequine species, and reduction of hypermotility may be protective against their development. Stomachs from 12 pigs processed for food at a provincially inspected abattoir were collected within 1 hour of slaughter. Explants of nonglandular gastric tissue were prepared and suspended in a tissue bath, attached to a force transducer, in the presence or absence of a simulated digest extract of the nutraceutical. Tissue was stimulated to contract using increasing doses of acetylcholine. Peak and mean contractile force over 1 and 2 minutes after exposure to acetylcholine were measured. Exposure of gastric smooth muscle to the nutraceutical significantly reduced contractility of the tissue. These data provide support for the use of this nutraceutical to reduce contractility of nonglandular gastric smooth muscle and may indicate a protective effect of this nutraceutical in horses with mechanically induced gastric ulcers. Future studies are needed to clarify the role of gastric hypermotility on development of equine gastric ulcers and to determine the effect of this nutraceutical on equine gastric contractility and ulcerogenesis in vivo.     

Generation of porcine induced pluripotent stem cells and evaluation of their major histocompatibility complex protein expression in vitro

Induced pluripotent stem cells (iPSCs) are thought to be highly beneficial in the field of regenerative medicine and are believed to overcome immunogenic barriers to cell transplantation. However, issues remain regarding their safety and efficiency for medical use. Furthermore, some recent reports have suggested that iPSCs could be targeted by the autologous immune system. To promote practical applications of iPSCs, in depth research using appropriate animal models is needed and porcine species appear to provide an ideal model. Recent studies have focused on the generation of porcine iPSC cells, but no investigations of their immunological properties have been conducted to date. In the present study, we generated putative iPSCs from porcine somatic cells and measured major histocompatibility complex (MHC) expression on the iPSCs and their derivatives. Compact colonies that expressed pluripotent markers appeared 11 days after viral infection. Embryonic bodies (EB) were produced and differentiated into three germ layers in vitro. Karyotyping and swine leukocyte antigen (SLA) typing showed that the iPSCs were identical to parental somatic cells. Porcine iPSCs expressed only low levels of MHC class I and moderately increased levels on their differentiated derivatives, whereas MHC class II was rarely expressed. In the presence of interferon-gamma (IFN-γ), the expression of MHC class I was elevated on differentiated iPSCs, and gradually decreased after withdrawal of the cytokine. Our data suggest that porcine iPSCs could be useful for preclinical studies of the efficiency and viability of iPSCs, and for devising strategies to rescue transplanted cells from the autologous immune system.     

Molecular signature and colony morphology affect in vitro pluripotency of porcine induced pluripotent stem cells

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.     

Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance

Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC‐like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram both mouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additional studies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC‐driven bovine technologies can be realized.     

In Vitro Expression of the Extracellular Matrix Components Aggrecan,Collagen Types I and II by Articular Cartilage‐Derived Chondrocytes

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan‐based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long‐term results of therapeutic procedures including cell‐based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage‐derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT‐qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells.     

An application of temperature mapping of horse's back for leisure horse‐rider‐matching

Leisure riding is a popular way of using horses however, unlike sport or racing horses, those are mostly not associated with one rider with high skills. Constant overload of equine musculoskeletal system causes pathologies, which are affecting horse mobility and decreases the horse‐rider communication. The aim was to propose the new scoring system of thermograph analysis as an aspect of differences in heat distributions on horseback before and after leisure ridings. The study was conducted on sixteen Polish warmblood horses, scanned with a non‐contact thermographic camera. Heat pattern of the thoracolumbar area was evaluated on thermograms taken before and after exercise. The criteria with point values for horse‐rider‐matching were created: heat points on the dorsal midline of saddle‐back contact area and degree of muscle unit overload. The results of thermograph analysis were compared with the results of a questionnaire on horse‐rider communication during riding in order to estimate the relevance of matching. The maximum score was obtained in 38.3% and 39.8% of combinations based on the thermograph analysis and questionnaire, respectively. Results of both scoring systems were strongly positive correlated (r = .937), demonstrating high sensitivity (61.72%) and specificity (90.23%) of the matching. The horse‐rider matching may improve horse comfort during leisure type of work.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Effect of Mineral Block Supplementation on In Vivo Digestibility and In Vitro Gas Production With Equine Fecal Bacteria

The effects of a mineral block for horses on in vivo digestibility and in vitro fermentability with equine fecal inoculum were evaluated. Fifty healthy horses from three groups (lactating mares n = 19, working horses n = 18, and maintenance horses n = 13) were randomly assigned to two treatment groups (with or without the mineral block; Ca 10.0%, P 12.0%, Zn 12.1 mg/kg, Cu 2,050 mg/kg, Mn 4,050 mg/kg, Se 30 mg/kg, and I 105 mg/kg). Dry matter digestibility was estimated with an internal marker. Samples of diet were incubated with equine fecal bacteria with varying amounts of mineral block (0, 1.1, 3.6, and 6.2 mg/g dry matter [DM]) to record gas production and to estimate in vitro DM digestibility. The results showed that mineral supplementation with the blocks increased in vivo DM digestibility (P < .01) in all groups, but there was an interaction (P < .01) with a greater response in the maintenance horses (55.5% vs. 78.0%) compared to lactating mares (62.8% vs. 79.6%) and working (70.3% vs. 75.1%). Block consumption was lowest in the lactating mares (12.8 g/d), intermediate in the working horses (44.6 g/d), and highest in the maintenance horses (74.2 g/d). The mineral supplementation did not affect the kinetics of gas production but tended (P = .10) to improve the in vitro DM digestibility (37.01% vs. 38.34%). Mineral block supplementation increased dry matter digestibility in horses. The unsupplemented control diet was deficient in several minerals, and block intake was not proportional to the mineral requirements.     

Effects of exercise and glucose administration on content of insulin-sensitive glucose transporter in equine skeletal muscle

OBJECTIVES: To characterize insulin-sensitive glucose-transporter (GLUT-4) protein in equine tissues and determine effects of exercise and glucose administration on content of GLUT-4 protein in equine skeletal muscle. SAMPLE POPULATION: Tissue samples from 9 horses. PROCEDURES: Western blot analyses were performed on crude membrane preparations of equine tissues to characterize GLUT-4. In a crossover, randomized study, horses were strenuously exercised for 3 consecutive days and then administered 13.5% glucose or isotonic saline (0.9% NaCl; control) solution, i.v., at similar infusion rates for 12.1 hours. Samples were collected from the middle gluteal muscle before and after exercise and 10.1 hours after completion of an infusion and used for measurements of glycogen concentration and total content of GLUT-4 protein. RESULTS: Immunoblot analyses detected specifically immunoreactive bands for GLUT-4 in insulin-sensitive tissues. Content of GLUT-4 protein in skeletal muscle increased significantly by 27.3 and 12.3% 22.2 hours after exercise for control and glucose groups, respectively. Intravenous infusion of glucose resulted in a significantly higher rate of glycogenesis, compared with results for the control group (mean +/- SD, 3.98 +/- 0.61 and 1.47 +/- 0.20 mmol/kg/h, respectively). Despite enhanced glycogenesis, we did not detect an increase in content of GLUT-4 protein after glucose infusion, compared with values after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: GLUT-4 protein was expressed in equine skeletal and cardiac muscles. Exercise increased total content of GLUT-4 protein in skeletal muscle, and replenishment of muscle glycogen stores after glucose infusion attenuated the exercise-induced increase in the content of GLUT-4 protein in equine skeletal muscle.     

An In Vitro Investigation into the Effects of a Marine-Derived,Multimineral Supplement in Simulated Equine Stomach and Hindgut Environments

Management of the performance horse often incorporates meal feeding of highly digestible starches and reduced access to high-fiber forage. Such regimens are associated with equine gastric ulceration syndrome (EGUS) and can alter hindgut homeostasis. In-feed buffering of gastric contents and promotion of energy derivation from high-fiber forage in the hindgut are therefore desirable properties of a nutritional supplement. A marine-derived, multimineral supplement with known buffering properties containing calcium, magnesium, and short-chain fructo-oligosaccharides (scFOS) was tested under in vitro simulations of equine stomach and hindgut conditions. Six fiber:concentrate diets were incubated for 4 hours with or without the supplement at 37°C in pepsin HCl solution adjusted to pH 4.1 and 2.6. pH was measured at 1, 2, and 4 hours postincubation. Highest overall pH values were observed with the high cereal feeds; however, the supplement significantly increased (P < .001) the pH across all feeds by 0.17 and 0.19 for feeds incubated at pH 4.1 and 2.6, respectively. A gas production technique was used to measure the fermentation of four fiber:concentrate diets with and without additional supplement, using equine feces as the microbial inoculum. Addition of the supplement decreased (P < .05) the lag time and increased the initial fermentation rate, although as the incubation continued, this effect was reduced. These results demonstrate that the supplement had a significant buffering action for 4-6 hours under simulated in vitro stomach digestion conditions and also stimulated in vitro hindgut fermentation activities.     

Are mule pregnancies really longer than equine pregnancies? Comparison between mule and equine pregnancies

In equine management, it is important to predict the approximate foaling date of mares to monitor parturition and allow early identification and intervention of problems during the perinatal period. There are no studies comparing accurate gestational length (GL) when mares are carrying mule foals and no controlled comparisons between GL of mares pregnant with equine or mule foals. Therefore, the objectives of this study were to compare GL of mares pregnant with equine or mule foals and establish normal reference values for GL of mares pregnant with male and female mules. Gestational length of Mangalarga Paulista breed mares pregnant with equine (n = 54) or with mule (n = 54) foals during the breeding seasons of 2007 to 2016 was evaluated. The mean GL was 347.2 ± 1.4 days (range of 326–368 days) and 341.1 ± 1.6 days (range of 307–360 days) for equine and mule pregnancies, respectively. The normal GL reference for mule pregnancies was 316.9–365.3 days. Therefore, GL of equine pregnancies was longer than of mule pregnancies. Gestational length was not different when pregnancies resulted in females or males within each group. This study established an important reference value for normal GL of mule pregnancies, which can be used by practitioners to estimate and predict foaling dates more accurately.     

The clinical features and short-term treatment outcomes of scirrhous cord: A retrospective study of 32 cases

Scirrhous cord (SC) is an uncommon complication of castration, characterised by chronic infection of the spermatic cord remnant. It is reported that surgical excision of the infected tissue is the most effective means of treatment, but there are few published studies assessing the outcomes of horses treated for SC. The aims of this retrospective study were to describe the clinical features and short-term outcomes in horses treated for SC at two equine hospitals in the UK. The clinical records of horses diagnosed with SC over a 10-year period were reviewed. A diagnosis of SC was made if the gelding presented with typical clinical signs with confirmation at surgery. Thirty-two cases of SC were identified at the two equine hospitals. The mean age at presentation was 6 years (range 2–14 years, n = 22), and the median time from castration to presentation was 29.5 days (range 20–2500 days). Mean age at castration was 4.3 years (range 6 months to 10 years, n = 10). Clinical signs included scrotal swelling, discharging wounds, hindlimb lameness and pyrexia. Five horses demonstrated hyperfibrinogenaemia (n = 8). Microbial culture isolated various bacterial species. All 32 cases were treated with surgical excision of the infected tissue and discharged from the hospitals between 1 and 10 days post-operatively. A limitation of this study is that it was a retrospective study with no long-term follow-up available. It was concluded that the results of this study confirm that SC can present at variable time points following castration, even many years later, and that a variety of bacterial species may be involved. Surgical excision of infected tissue is a successful treatment with a good short-term prognosis for survival.     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

Clinical Experience Using MicroLactin for the Treatment of Equine Inflammatory Disease

MicroLactin is a patented milk protein concentrate whose mode of action is proposed to inhibit neutrophil activation in inflammation and to bolster the immune response in musculoskeletal diseases. MicroLactin was empirically used in the treatment of a series of equine clinical cases. MicroLactin was given in two trials to 166 horses in which neutrophils were associated with an inflammatory response. The primary clinical groups having the greatest positive responders to the use of MicroLactin were: respiratory (92%), joint lameness/foot trauma (90%), muscle injury/myositis (92%), equine protozoal myeloencephalitis (EPM) (81%), skin trauma/hypersensitivity (89%), and toxic enteritis (89%). Positive clinical results were seen within 10 to 14 days when MicroLactin was used as a daily treatment either alone or in combination with other anti-inflammatory agents or as an adjunct to the primary treatment.     

Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4

Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%.     

The role of scintigraphy in the lameness evaluation

Bone scanning to help diagnose orthopedic disease has been used in human patients for over two decades. The value of this diagnostic tool has been well established in helping to identify a variety of musculoskeletal conditions. It has only recently been used by veterinarians for more accurate characterization of equine musculoskeletal disease. The technique offers the major advantage of increased sensitivity over standard radiographic imaging. The case material illustrated here shows that except for consistent identification of bone cysts, most of the pathologic changes to the horse's musculoskeletal system that might cause lameness are detected on bone scans. Many acute bone diseases can be diagnosed by scintigraphy that cannot be discerned by radiographs until the condition has become chronic: Because of their body size, these conditions may not be diagnosed at all in horses. Scintigraphy in horses offers the other major advantage of affording accurate imaging of the upper limbs, pelvis, and vertebral column without general anesthesia. Therefore, it has a final advantage of increased safety over conventional radiography because it eliminates the need to perform general anesthesia to study these areas. In the author's experience, if abnormal uptake of isotope in the upper limbs, pelvis or spine is not observed, general anesthesia to radiograph those areas is not warranted. A second major benefit of scintigraphic imaging is to differentiate mixed lameness conditions in which the component of bone disease must be separated from that of soft tissues to arrive at a rational course of treatment or prognosis. Finally, for athletic horses suspected of having lameness due to localized myositis, scintigraphy not only allows confirmation of muscle inflammation but also identifies the muscle bellies injured reasonably accurately so that specific local treatment may be given. Nuclear imaging of equine skeletal disease is an option that should be employed more frequently by equine practitioners for diagnosis of difficult lameness cases. The technique is safe and comparatively inexpensive when one considers the total expense of multiple examinations or radiographic surveys of patients without conclusively diagnosing the source or sources of skeletal pain. This is particularly true when a horse owner becomes dissatisfied and enlists the services of one or more other veterinarians. The equine specialist will maintain better client rapport if he or she suggests referral of the horse to a veterinary medical teaching center or private clinic where scintigraphic imaging can be done rather than having the client become frustrated and seek another opinion elsewhere.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.