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IntroductionThe increasing prevalence of infertility in male dogs in clinical practice mirrors current trends seen in human medicine. Acquired infertility is notably more common in dogs compared to congenital causes, with conditions such as testicular degeneration leading to irreversible loss of fertility. Current diagnostic methods for testicular degeneration, such as histopathological and cytological examinations, rely on testicular biopsy or fine needle aspiration, making them less feasible for routine use. Anti-Müllerian hormone (AMH), produced by Sertoli cells, has emerged as a potential alternative biomarker for testicular health, which can be measured in serum. This study evaluates AMH as a potential marker for testicular degeneration, using cryptorchid dogs as models for impaired fertility and altered testicular histology.MethodsThe relationship between serum AMH levels and AMH tissue expression with impaired spermatogenesis and altered histology was investigated. Serum AMH levels were determined in intact, cryptorchid, and castrated individuals using an immuno-enzymatic ELISA kit and compared between subgroups based on testicular location. Tissue AMH immuno-expression was differentially quantified in two regions of interest (ROIs), the interstitial space and the seminiferous tubule, in both descended and retained gonads. Furthermore, testicles were analyzed using histomorphometric analysis in seminiferous tubules, while spermatogenesis was evaluated using the Johnsen score.ResultsSerum AMH levels were positively correlated with AMH expression assessed in both interstitial space (ρ = 0.494, p ≤ 0.01) and seminiferous tubules (ρ = 0.610, p ≤ 0.001). Conversely, serum AMH levels showed a negative correlation with the seminiferous tubule area (ρ = −0.435, p ≤ 0.05). Smaller seminiferous tubule areas were linked to increased AMH reactivity in both seminiferous tubules (ρ = −0.774, p ≤ 0.001) and interstitial space (ρ = −0.725, p ≤ 0.001). Additionally, lower Johnsen scores were associated with higher serum AMH levels (ρ = −0.537, p ≤ 0.01) and elevated AMH expression in both seminiferous tubules (ρ = −0.756, p ≤ 0.001) and interstitial space (ρ = −0.679, p ≤ 0.001).DiscussionOur results suggest that higher serum levels and tissue expression of AMH are linked to smaller seminiferous tubules and poorer Johnsen scores, reflecting degenerative changes and Sertoli cell dysfunction in retained testicles. Given the similarities in the mechanisms that increase AMH levels in both cryptorchid and non-cryptorchid testicles affected by testicular degeneration, this study recommends using AMH as a marker for diagnosing testicular degeneration in dogs.

IntroductionIn this proof-of-concept study, the post-mortem feasibility of dual-energy computed tomography (DECT) in the detection of bone edema-like lesions in the equine foot is described in agreement with the gold standard imaging technique, which is magnetic resonance imaging (MRI).MethodsA total of five equine cadaver feet were studied, of which two were pathological and three were within normal limits and served as references. A low-field MRI of each foot was performed, followed by a DECT acquisition. Multiplanar reformations of DECT virtual non-calcium images were compared with MRI for the detection of bone edema-like lesions. A gross post-mortem was performed, and histopathologic samples were obtained of the navicular and/or distal phalanx of the two feet selected based on pathology and one reference foot.ResultsOn DECT virtual non-calcium imaging, the two pathological feet showed diffuse increased attenuation corresponding with bone edema-like lesions, whereas the three reference feet were considered normal. These findings were in agreement with the findings on the MRI. Histopathology of the two pathologic feet showed abnormalities in line with bone edema-like lesions. Histopathology of the reference foot was normal.ConclusionDECT virtual non-calcium imaging can be a valuable diagnostic tool in the diagnosis of bone edema-like lesions in the equine foot. Further examination of DECT in equine diagnostic imaging is warranted in a larger cohort, different locations, and alive animals.

IntroductionThe effect of dietary fiber on pig production has been extensively evaluated. Inspired by observations of the diet of wild, young piglets, this study aimed to examine the possibility of feeding grass hay to suckling piglets besides concentrated creep feed.MethodsThe sow-nursed piglets in this study were divided into two groups based on balanced sow parities. The control group (CON, n = 7 sows) only received a regular, concentrated creep feed, while the treatment piglets (GH, n = 8 sows) were also provided with chopped grass hay from 2 days of age until weaning (28 days). At weaning, one piglet with a median weight was selected from each litter for post-mortem evaluation. Subsequently, six pigs around median weight per sow were grouped into nursery pens and monitored for their feed intake and body weight gain until 9 weeks of age.Results and discussionPiglets in GH consumed, on average, 57 g of grass hay per piglet during the entire lactation period. The emptied weight of the small and large intestine was significantly greater in GH (280 vs. 228 g, 88.8 vs. 79.3 g, respectively, p < 0.05), and the length of the large intestine was stimulated by the grass hay (164 vs. 150 cm, p < 0.05). Morphologically, the villus height in the jejunum was higher in GH (p < 0.05). In the large intestine, the crypt depth of the mid-colon was lower in GH. Moreover, the short-chain fatty acid (SCFA) concentrations in the cecum were increased in GH compared to CON (1,179 vs. 948 µmol/g dry matter, p < 0.05), whereas in the colon, SCFA concentrations were lower in CON (341 vs. 278 µmol/g dry matter, p < 0.05). There was no major impact of grass hay inclusion on the colonic microbiota composition. Only a trend was observed for a lower inverse of the classical Simpson (InvSimpon) index and a higher abundance of Lactobacillus genera in GH. After weaning, no significant differences in feed intake and body weight gain were observed. In conclusion, supplementing the grass hay to suckling piglets led to alterations in intestinal morphology, increased SCFA fermentation in proximal sections of large intestine, stimulation of gastrointestinal tract growth, and subtle modifications in colonic microbiota.

Scientific literature on veterinary dentistry in alpacas has historically focused on the description of tooth root abscesses. However, recent studies have shown a variety of other, sometimes preceding dental conditions to be widespread in this species. To allow the development and finetuning of treatment strategies in this species, a more thorough understanding of the underlying etiopathogenesis of dental disease is required. Histological studies focusing on normal dental and surrounding tissues might serve as a basis for this purpose. Nine teeth, extracted from seven alpacas were collected. All samples were retrieved from animals that died or were euthanized for non-dental reasons. Histological sections were prepared at three different levels in each tooth and examined using light microscopy focusing on the assessment of pulp tissue, dentin, cementum, periodontal tissues and the apical region. The histological appearance of the investigated dental tissues in alpacas showed great similarities with other hypsodont species. However, a rather rare type of dentin called “vasodentin” could be identified in all examined cheek teeth. Another species-specific finding was the extremely close proximity of varying neighboring tooth roots that seemed to be responsible for inducing massive resorptive lesions. The results of this study might contribute to a better understanding of the etiopathogenesis of some dental diseases in the alpaca.

Mesenchymal stem cells (MSCs) are a promising candidate for both human and veterinary regenerative medicine applications because of their abundance and ability to differentiate into several lineages. Mesenchymal stem cells are however a heterogeneous cell population and as such, it is imperative that they are unequivocally characterized to acquire reproducible results in clinical trials. Although the tri-lineage differentiation potential of MSCs is reported in most veterinary studies, a qualitative evaluation of representative histological images does not always unambiguously confirm tri-lineage differentiation. Moreover, potential differences in differentiation capacity are not identified. Therefore, quantification of tri-lineage differentiation would greatly enhance proper characterization of MSCs. In this study, a method to quantify the tri-lineage differentiation potential of MSCs is described using digital image analysis, based on the color deconvolution plug-in (ImageJ). Mesenchymal stem cells from three species, i.e., bovine, equine, and porcine, were differentiated toward adipocytes, chondrocytes, and osteocytes. Subsequently, differentiated MSCs were stained with Oil Red O, Alcian Blue, and Alizarin Red S, respectively. Next, a differentiation ratio (DR) was obtained by dividing the area % of the differentiation signal by the area % of the nuclear signal. Although MSCs isolated from all donors in all species were capable of tri-lineage differentiation, differences were demonstrated between donors using this quantitative DR. Our straightforward, simple but robust method represents an elegant approach to determine the degree of MSC tri-lineage differentiation across species. As such, differences in differentiation potential within the heterogeneous MSC population and between different MSC sources can easily be identified, which will support further optimization of regenerative therapies.

The use of mesenchymal stem cells (MSCs) for the treatment of equine tendon disease is widely investigated because of their regenerative and immunomodulatory potential. However, questions have been raised concerning the immunogenic properties of allogeneic MSCs. Therefore, two studies were conducted to assess the safety of equine allogeneic peripheral blood-derived tenogenic primed MSCs (tpMSCs). The objective was to evaluate if a single and repeated tpMSC administration induced a cellular and humoral immune response in horses suffering from tendon injuries. Horses enrolled in the first study (n = 8) had a surgically induced superficial digital flexor tendon core lesion and were treated intralesionally with tpMSCs. Before and after treatment the cellular immunogenicity was assessed by modified mixed lymphocyte reactions. The humoral immune response was investigated using a crossmatch assay. Presence of anti-bovine serum albumin (BSA) antibodies was detected via ELISA. Horses enrolled in the second study (n = 6) suffered from a naturally occurring tendon injury and were treated twice with tpMSCs. Blood was collected after the second treatment for the same immunological assays. No cellular immune response was found in any of the horses. One out of eight horses in the first study and none of the horses in the second study had anti-tpMSC antibodies. This particular horse had an equine sarcoid and further investigation revealed presence of antibodies against sarcoid cells and epithelial-like stem cells before treatment, which increased after treatment. Additionally, formation of antibodies against BSA was observed. These findings might indicate a non-specific immune response generated after treatment. Serum from the other horses revealed no such antibody formation. These two studies showed that the administration of tpMSCs did not induce a cellular or humoral immune response following an intralesional single or repeated (two consecutive) allogeneic tpMSC treatment in horses with tendon injury, except for one horse. Therefore, a larger field study should confirm these findings and support the safe use of tpMSCs as a therapeutic for horses suffering from tendon injuries.

Background: Tendon injuries are very common in horses and jeopardize the athletic performance, and due to the high risk of reinjury may lead to early retirement. The use of mesenchymal stem cells for the treatment of equine tendon disease is widely investigated because of their regenerative potential. The objective of this study is to investigate the safety and efficacy of equine allogeneic tenogenic primed mesenchymal stem cells (tpMSCs) for the management of tendinitis in horses.Methods: A core lesion was surgically induced in the superficial digital flexor tendon of both forelimbs of eight horses. After 7 days, one forelimb was treated with tpMSCs, while the contralateral forelimb served as an intra-individual control and was treated with saline. A prescribed exercise program was started. All horses underwent a daily clinical evaluation throughout the entire study period of 112 days. Blood samples were taken at different time points for hematological and biochemical analysis. Tendon assessment, lameness examination, ultrasound assessment and ultrasound tissue characterization (UTC) were performed at regular time intervals. At the end of the study period, the superficial digital flexor tendons were evaluated macroscopically and histologically.Results: No suspected or serious adverse events occurred during the entire study period. There was no difference in local effects including heat and pain to pressure between a single intralesional injection of allogeneic tpMSCs and a single intralesional injection with saline. A transient moderate local swelling was noted in the tpMSC treated limbs, which dissipated by day 11. Starting at a different time point depending on the parameter, a significant improvement was observed in the tpMSC treated limbs compared to the placebo for echogenicity score, fiber alignment score, anterior-posterior thickness of the tendon and echo type by UTC assessment. Immunohistochemistry 112 days post-injection revealed that the amount of collagen type I and Von Willebrand factor were significantly higher in the tendon tissue of the tpMSC group, while the amount of collagen type III and smooth muscle actin was significantly lower.Conclusion: Equine allogeneic tenogenic primed mesenchymal stem cells were shown to be well-tolerated and may be effective for the management of tendon injuries.

The current study was designed to evaluate the pathogenesis, pathology and immune response of female genital tract infection with Chlamydia trachomatis L2c, the most recently discovered lymphogranuloma venereum strain, using a porcine model of sexually transmitted infections. Pigs were mock infected, infected once or infected and re-infected intravaginally, and samples were obtained for chlamydial culture, gross and microscopic pathology, and humoral and cell-mediated immunity. Intravaginal inoculation of pigs with this bacterium resulted in an infection that was confined to the urogenital tract, where inflammation and pathology were caused that resembled what is seen in human infection. Re-infection resulted in more severe gross pathology than primary infection, and chlamydial colonization of the urogenital tract was similar for primary infected and re-infected pigs. This indicates that primary infection failed to induce protective immune responses against re-infection. Indeed, the proliferative responses of mononuclear cells from blood and lymphoid tissues to C. trachomatis strain L2c were never statistically different among groups, suggesting that C. trachomatis-specific lymphocytes were not generated following infection or re-infection. Nevertheless, anti-chlamydial antibodies were elicited in sera and vaginal secretions after primary infection and re-infection, clearly resulting in a secondary systemic and mucosal antibody response. While primary infection did not protect against reinfection, the porcine model is relevant for evaluating immune and pathogenic responses for emerging and known C. trachomatis strains to advance drug and/or vaccine development in humans.

Pediatric drug development, especially in disease areas that only affect children, can be stimulated by using juvenile animal models not only for general safety studies, but also to gain knowledge on the pharmacokinetic and pharmacodynamic properties of the drug. Recently, the conventional growing piglet has been suggested as juvenile animal model. However, more studies with different classes of drugs are warranted to make a thorough evaluation whether the juvenile pig might be a suitable preclinical animal model. Ibuprofen is one of the most widely used non-steroidal anti-inflammatory drugs in human. The present study determined the PK parameters, gastro-intestinal and renal safety of 5 mg/kg BW ibuprofen after single intravenous, single oral and multiple oral administration to each time eight pigs (four males, four females) aging 1, 4, 8 weeks and 6–7 months. Oral administration was performed via a gastrostomy button. A jugular catheter was used for intravenous administration and blood sampling. To assess NSAID induced renal toxicity, renal function was evaluated using iohexol and p-aminohippuric acid as markers for glomerular filtration rate and renal plasma flow, respectively. After the trial, necropsy and histology was performed to evaluate macroscopic and microscopic gastro-intestinal as well as renal lesions. Both enantiomers, R-ibuprofen and S-ibuprofen, were determined in plasma using an in-house developed and validated UHPLC-MS/MS method. Pharmacokinetic parameters were estimated using compartmental analysis. Clearance and volume of distribution of total ibuprofen and both enantiomers increased with age as was observed in human. The rate of stereochemical conversion decreased with age. Multiple oral dosing decreased the absolute oral bioavailability and maximum plasma concentration of R-ibuprofen and food consumption did not influence drug absorption. Based on the limited available pediatric literature, the current study might suggest the conventional pig as suitable animal model to evaluate NSAIDs for pediatric use.