Effect of rearing systems on immune status, stress parameters, intestinal morphology, and mortality in conventional and local chicken breeds.

The majority of poultry meat used to be sourced from intensively housed birds. However, consumer preference has since demanded poultry producers develop more sustainable farming systems. Although free-range farming is considered beneficial for animal welfare, it is not as easy to standardize as an intensive system, which makes the choice of bird genotype appear crucial for alternative systems. In this study, we aimed to evaluate the effect of conventional and free-range rearing systems on the immune status, stress parameters, intestinal morphology and mortality in commercial hybrids (Ross 308) and local poultry strains, Bionda Piemontese (BP), Robusta Maculata (RM), BP x Sasso (BPxS), and RM x Sasso (RMxS). RNA was extracted from the jejunum and spleen to assess the mRNA expression of IL-2, IL-6, IL-10, IL-18, IL-1ß, inducible nitric oxide synthase (iNOS), toll-like receptor (TLR)-4, and interferon gamma (IFN-γ). The heterophil:lymphocyte (H/L) ratio and intestinal histomorphometric evaluation were also calculated. We found that compared to the conventional system, the rearing system significantly affected the jejunum expression of IL-10, iNOS, IL-2, and IL-6, where these genes were upregulated in free-range system. A significant interaction between the rearing system and the genotype was also shown. More specifically, local breeds showed a significantly higher expression (P < 0.001) of IL-6 in the free-range system compared to the same genotypes in the conventional system. Moreover, IL-6 is constantly upregulated in local breeds within the free-range system compared to Ross hybrids. We also found significantly increased H/L and mortality rates in the latter, compared to the local breeds in the free-range reared system. The jejunum morphology also demonstrated a significantly higher villus height in BP and BPxS compared to the Ross hybrids. Overall, the results of our study confirm that the intense selection for growth in broiler chickens may have reduced their ability to react to the environmental stimuli related to free-range systems, resulting in a lower adaptability to a free-range environment, thus making them inappropriate for any farming system other than the conventional one. On the contrary, local chicken breeds are able to adapt and survive in the free-range system of rearing, and represent a genetic resource especially when adaptability to free-range conditions is required.

Sedation quality of alfaxalone associated with butorphanol, methadone or pethidine in cats injected into the supraspinatus or the quadriceps muscle.

OBJECTIVES: The aim of this study was to compare the quality of sedation with three different anaesthetic protocols (alfaxalone combined with butorphanol, methadone or pethidine) administered intramuscularly in cats, and to evaluate the influence of the injection site (between supraspinatus and quadriceps muscles) on the onset and quality of sedation. METHODS: A total of 151 cats were selected for this study. Cats were sedated with alfaxalone (3 mg/kg) combined with either butorphanol (0.3 mg/kg; n = 50), methadone (0.3 mg/kg; n = 53) or pethidine (5 mg/kg; n = 48). The combination was injected intramuscularly into the supraspinatus (n = 79) or quadriceps muscle (n = 72). The data included a scoring system for the quality of sedation and physiological parameters, such as heart rate (HR), respiratory rate, body temperature and occurrence of mydriasis, monitored during the first 30 mins of anaesthesia. RESULTS: The opioid associated with alfaxalone influenced the overall sedation score, the degree of myorelaxation, the occurrence of mydriasis and HR. The overall sedation score was poorer with butorphanol than with methadone (P = 0.008), and butorphanol induced a lower degree of myorelaxation than methadone (P = 0.013). The injection into the supraspinatus showed better qualitative results for sedation and a faster onset time (in about 3 mins) than that into the quadriceps (P <0.001). HR decreased from baseline (P <0.001) and over time (P <0.001), mainly in cats of the butorphanol-supraspinatus and pethidine-quadriceps groups (P = 0.004). The occurrence of mydriasis was lower after butorphanol than after methadone and pethidine (P = 0.025), while the incidence of side effects did not differ among groups. CONCLUSIONS AND RELEVANCE: All three protocols provided a good quality of sedation and allowed performing the scheduled procedure. Moreover, the injection into the supraspinatus muscle showed superior results in all the qualitative scores of sedation and quicker onset time than that into the quadriceps muscle.

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Goji Berries Supplementation in the Diet of Rabbits and Other Livestock Animals: A Mini-Review of the Current Knowledge.

In the last decades, several nutraceutical substances have received great attention for their potential role in the prevention and treatment of different diseases as well as for their beneficial effects in promoting the health of humans and animals. Goji berries (GBs) are the fruit of Lycium barbarum and other species of Lycium, used in traditional Chinese medicine, and they have recently become very popular in the Occidental world because of their properties, such as anti-aging, antioxidant, anticancer, neuroprotective, cytoprotective, antidiabetic, and anti-inflammatory activities. These effects are essentially evaluated in clinical trials in humans; in experimental animal models, such as mice and rats; and in cell lines in in vitro studies. Only recently has scientific research evaluated the effects of GBs diet supplementation in livestock animals, including rabbits. Although studies in the zootechnical field are still limited and the investigation of the GB mechanisms of action is in an early stage, the results are encouraging. This review includes a survey of the experimental trials that evaluated the effects of the GBs supplementation on reproductive and productive performances, immune system, metabolic homeostasis, and meat quality principally in the rabbit with also some references to other livestock animal species. Evidence supports the idea that GB supplementation could be used in rabbit breeding, although future studies should be conducted to establish the optimal dose to be administered and to assess the sustainability of the use of GBs in the diet of the rabbit.

FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors.

Despite promising immunotherapy strategies in human melanoma, there are few studies on the immune environment of canine melanocytic tumors. In humans, the activation of immunosuppressive cell subpopulations, such as regulatory T cells (Tregs) that express forkhead box protein P3 (FoxP3), the engagement of immunosuppressive surface receptors like cytotoxic T lymphocyte antigen (CTLA-4), and the secretion of molecules inhibiting lymphocyte activation, such as indoleamine-pyrrole 2,3-dioxygenase (IDO), are recognized as immunoescape mechanisms that allow tumor growth and progression. The aim of our study was to investigate the expression of these immunosuppression markers in canine melanocytic tumors and to postulate their possible role in melanoma biology and progression. Fifty-five formalin-fixed, paraffin-embedded canine melanocytic tumors (25 oral melanomas; 20 cutaneous melanomas; 10 cutaneous melanocytomas) were selected to investigate the expression of FoxP3, CTLA-4, and IDO by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). All of the tested markers showed high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanomas when compared to their benign counterpart. IDO expression was associated with an increased hazard of death both in univariable and multivariable analyses (P < .05). FoxP3 protein expression >6.9 cells/HPF (high-power field) was an independent predictor of death (P < .05). CTLA-4 gene and protein expressions were associated with a worse prognosis, but only in the univariable analysis (P < .05). FoxP3, CTLA-4, and IDO likely play a role in canine melanoma immunoescape. Their expression, if supported by future studies, could represent a prognostic tool in canine melanoma and pave the way to future immunotherapeutic approaches in dogs.

Gonadectomy in Raccoons: Anesthetic and Cardiorespiratory Effects of Two Ketamine-Based Pre-Anesthetic Protocols before Sevoflurane-Sufentanil.

Nineteen raccoons were enrolled in this study. The aim was to evaluate and compare the quality of anesthesia and the cardiorespiratory effects following treatment with a ketamine-based combination with either dexmedetomidine (KD group) or midazolam (KM group) in raccoons undergoing ovariohysterectomy/orchiectomy. General anesthesia was maintained with sevoflurane in oxygen and sufentanil infusion. The time required to approach the animals was similar among groups resulting in a median of 5 min after IM injection. Animals in group KD were scored with greater myorelaxation (p < 0.01) and easier intubation (p < 0.05). Moreover 70% of them did not require other drugs for tracheal intubation unlike animals in group KM, which required propofol in 100% of cases. After intubation and connection to the breathing circuit, physiological parameters were monitored continuously and recorded every 5 min. Sevoflurane requirements were lower in group KD than KM (p = 0.005). Blood pressure was maintained within physiological ranges in both groups but with higher values in group KM (p < 0.05). Mild respiratory depression occurred during surgery and animals in group KM showed greater respiratory acidosis (p < 0.05). Recovery was smooth and uneventful in all animals. Both anesthetic protocols can be recommended for safe anesthesia in wild raccoons.

Effect of aglepristone (RU534) administration during follicular phase on progesterone, estradiol-17ß and LH serum concentrations in bitches.

Aglepristone was administered in bitches during the follicular phase to evaluate its effects on progesterone, estradiol-17ß and LH serum concentrations. Ten German Shepherds were divided into two groups (treated n = 5; control n = 5). Treated bitches received 10 mg/kg BW of aglepristone subcutaneously during the early follicular phase, 24 hr after and then 7 days later. The control group was injected, at the same time periods, with saline solution (0.3 ml/kg BW). For the steroid evaluations, blood was collected daily from the onset of proestrus until the first day of cytological dioestrus. For LH base-line serum determination, blood was also collected every 20 min for 2 hr at the onset of proestrus. For LH surge identification, blood was collected daily (every 6 hr) starting from the day of the first administration of aglepristone or saline solution until the first day of dioestrus. All animals ovulated but the treated group presented longer ovulation-dioestrus intervals than the control group (5.2 ± 2.2 days p < .05). Serum concentrations of the evaluated hormones were similar between experimental animals except for serum LH. Indeed, no LH peaks were detected in the treated group while LH surges were clearly observed in the control group (9 ± 1 days after the beginning of proestrus. In particular, the area under the curve for LH was significantly lower in treated than control animals (12 ± 4 ng/ml x Day; p = .01). In conclusion, administrations of aglepristone during the follicular phase of the bitch does not affect the steroid hormone patterns but does prevent the occurrence of a LH surge. This work raises significant questions and opens perspectives concerning the mechanisms of ovulation in bitches.

E-Cadherin Expression in Canine Melanocytic Tumors: Histological, Immunohistochemical, and Survival Analysis.

E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

Effects of Diets Enriched in Linseed and Fish Oil on the Expression Pattern of Toll-Like Receptors 4 and Proinflammatory Cytokines on Gonadal Axis and Reproductive Organs in Rabbit Buck.

Infections of the genital tract can perturb the fertility in humans and animals. Pathogen recognition and activation of innate immunity onset through the pattern recognition receptor activation, such as Toll-like receptor 4 (TLR4), leading to the production of proinflammatory cytokines and mediators. TLR4 is expressed both on leukocytes and nonimmune cells. Rabbit TLR4 shows great similarity to its human counterpart. Moreover, the TLR4 signalling pathway could be modulated by long-chain polyunsaturated fatty acids (LC-PUFA). The objectives of this study were (i) to determine the expression levels of TLR4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit and (ii) to evaluate if the n-3 PUFA-enriched diets can modify their expression levels in the tissues and LC-PUFA profiles in seminal plasma. Fifteen rabbit bucks (n = 5/experimental group) were fed with different diets: commercial standard (group C), rich in extruded linseed (10%, group L), and in fish oil (3%, group FO) for 110 days. TLR4, TNF-α, and IL-1ß mRNA were ubiquitously expressed throughout the hypothalamic-gonadal axis. However, TLR4 mRNA expression was lower in the hypothalamus than the epididymis (P < 0.01), seminal vesicles (P < 0.01), and pituitary gland (P < 0.05). Dietary enrichment in PUFA did not modify the gene expression profile nor the histological characteristics of the tissues. Conversely in seminal plasma, rabbits fed with L and FO had lower n-6 (P < 0.05), LC-PUFA n-6 (P < 0.05), and n-6/n-3 ratio (P < 0.05) but higher n-3 (P < 0.001) and LC-PUFA n-3 (P < 0.01) compared to the control group. Our study builds a map of the gene expression of TRL4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit, fundamental step for understanding the immune defence mechanisms. Diets enriched in LC-PUFA did not affect basal gene expression but modulated sperm fatty acid composition. Finally, rabbit may be an excellent animal model to study the relationship between inflammation and infertility, and the nutritional modulation of immune functions.

Tumour-infiltrating lymphocytes in canine melanocytic tumours: An investigation on the prognostic role of CD3+ and CD20+ lymphocytic populations.

The study of the immune response in several types of tumours has been rapidly increasing in recent years with the dual aim of understanding the interactions between neoplastic and immune cells and their importance in cancer pathogenesis and progression, as well as identifying targets for cancer immunotherapy. Despite being considered one of the most immunogenic tumour types, melanoma can progress in the presence of abundant lymphocytic infiltration, therefore suggesting that the immune response is not able to efficiently control tumour growth. The purpose of this study was to investigate whether the density, distribution and grade of tumour-infiltrating lymphocytes (TILs) in 97 canine melanocytic tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in the dog. As a further step in the characterization of the immune response in melanocytic tumours, an immunohistochemical investigation was performed to evaluate the two main populations of TILs, T-lymphocytes (CD3+ ) and B-lymphocytes (CD20+ ). The results of our study show that TILs are present in a large proportion of canine melanocytic tumours, especially in oral melanomas, and that the infiltrate is usually mild. The quantity of CD20+ TILs was significantly associated with some histologic prognostic factors, such as the mitotic count, the cellular pleomorphism and the percentage of pigmented cells. Remarkably, a high infiltration of CD20+ TILs was associated with tumour-related death, presence of metastasis/recurrence, shorter overall and disease-free survival, increased hazard of death and of developing recurrence/metastasis, hence representing a potential new negative prognostic factor in canine melanocytic tumours.

Tumor Thickness and Modified Clark Level in Canine Cutaneous Melanocytic Tumors.

Breslow thickness and Clark level are prognostic factors for human cutaneous melanomas. Breslow thickness is measured with an ocular micrometer from the top of the granular layer of the epidermis to the deepest invasive cell across the broad base of the tumor, while Clark level is based on the anatomical level of invasion through the layers of the dermis. Because of the anatomical differences between humans and dogs, we evaluated the tumor thickness and a modified Clark level in 77 canine primary cutaneous melanocytic tumors. Tumor thickness (using both a traditional and a more convenient system) and modified Clark level were measured and associated with histological diagnosis and clinical outcome. Tumor thickness was a prognostic factor, being greater in animals with shorter overall survival and disease-free time. Cutoffs of 0.95 cm and 0.75 cm defined a higher hazard for an unfavorable outcome and to develop recurrence/metastasis, respectively. Because of an excellent agreement between the 2 methods, it was concluded that tumor thickness could be measured with a ruler when an ocular micrometer is not available. Modified Clark level was not found to be relevant for prognosis. However, we suggest that both tumor thickness and a modified Clark level can be valid additional parameters when histological diagnosis is uncertain. Further studies, including a wider sample population, would be worthwhile to confirm the prognostic significance of these 2 parameters.

FoxP3 and IDO in Canine Melanocytic Tumors.

Human melanoma is one of the deadliest forms of cancer, with poor prognosis and high resistance to chemotherapy and radiotherapy. The discovery of immunosuppressive mechanisms in the human melanoma microenvironment led to the use of new prognostic markers and to the development of immunotherapies targeting immune checkpoint molecules. Immunoescape mechanisms in canine melanoma have not yet been investigated, and no such immunotherapy has been tested. The aim of this study was to provide preliminary data on the expression of transcription factor forkhead box protein P3 (FoxP3) and indoleamine 2,3-dioxygenase (IDO) in primary canine melanocytic tumors and to investigate their prognostic role. Formalin-fixed, paraffin-embedded samples from 74 canine melanocytic tumors (26 oral melanomas, 23 cutaneous melanomas, and 25 cutaneous melanocytomas) were retrospectively evaluated by immunohistochemistry to explore the expression of FoxP3 and IDO. An increased risk of death due to melanoma was associated with a higher number of FoxP3+ cells per high-power field (FoxP3+/HPF), a higher percentage of CD3+ cells that were also FoxP3+ infiltrating and surrounding the tumor (%FoxP3), and a higher number of IDO+ cells/HPF (IDO+/HPF). A prognostic value for FoxP3 and IDO is suggested by our study, with optimal cutoffs of 14.7 FoxP3+ cells/HPF, 6.1 IDO+ cells/HPF, and 12.5% FoxP3+ cells. Both markers were also associated with tumor type. Multivariable analysis identified IDO+/HPF ( P < .001) as an independent prognostic marker. Even though stratification by diagnosis caused a loss of significance, results from the present study suggest a prognostic role for IDO and FoxP3, possibly related to the establishment of an immunosuppressive microenvironment.

Effects of local lipopolysaccharide administration on the expression of Toll-like receptor 4 and pro-inflammatory cytokines in uterus and oviduct of rabbit does.

Inflammation of the uterus and oviduct is associated with reduced reproductive performance in humans and domestic animals. Toll-like receptors are expressed in various immune and non-immune cells and play a crucial role in innate immunity. Toll-like receptor - 4 (TLR4) can detect lipopolysaccharide (LPS) from Gram-negative bacteria leading to the secretion of pro-inflammatory cytokines, chemokines, antimicrobial peptides and other inflammatory mediators. To investigate the effects of a local inflammation on the expression levels of TLR4 and pro-inflammatory cytokines, 12 female rabbits received an intracervical infusion with either saline solution endotoxin-free (carrier, 2 mL; n = 6) or LPS (500 µg diluted in 2 mL of saline solution; n = 6). Blood samples were performed at 0, 30, 60 and 90 min and 2,4,6 and 24 h after treatment to evaluate interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) plasma concentrations. Animals were sacrificed 24 h post-treatment. The uterus and oviducts were immediately collected. The gene expression and protein levels of TLR4 and pro-inflammatory cytokines were detected by quantitative real-time PCR (qRT-PCR) and immuno-histochemical assay, respectively. Our study showed that the intracervical administration of LPS induced local inflammation given that the animals showed no clinical signs, the histological samples revealed signs of inflammation and plasma levels of pro-inflammatory cytokines were unchanged compared to the control. LPS produced an increase in the TLR4 mRNA expression levels in the uterus with respect to the control (P < 0.05). In LPS-treated rabbits the gene expression of IL-1ß was higher in the uterus and oviducts and TNF-α only in the oviduct (P < 0.05) as compared to the control. The immuno-histochemical assay showed that TLR4, IL-1ß and TNF-α were expressed in the reproductive tissues of the rabbit. Moreover, after the LPS stimulation the stromal cells of the uterus exhibited a higher staining for TLR4 (P < 0.05) and the epithelial cells of the oviduct for TNF-α and IL-1ß (P < 0.05) with respect to the control. These results suggest that (1) TRL4, IL-1ß and TNF-α are expressed in uterus and oviducts of the doe, and (2) LPS up-regulates the gene and protein expression of TLR4 and pro-inflammatory cytokines in uterus and oviducts. Therefore, the rabbit could be a useful animal model for studying the local mechanisms involved in reproductive dysfunctions caused by subclinical infections.

Contrast-enhanced ultrasonographic characteristics of the diseased canine prostate gland.

The work was carried out on a total of 26 male dogs that on the basis of clinical examination, prostate ultrasound and prostate biopsy, were divided prospectively into four groups: (1) normal dogs (control group; n = 8); (2) dogs with benign prostatic hyperplasia (group BPH; n = 8); (3) dogs suffering from prostatitis (group prostatitis; n = 4); (4) dogs with prostatic tumors (group tumors; n = 6). The examination of the prostate by means of contrast medium and dedicated ultrasound system allowed a detailed qualitative and quantitative analysis of prostatic vessels in normal and diseased conditions, enabling the detection and characterization of different disease states, and quantification of parameters such as peak intensity of perfusion (%), arrival time of the contrast medium to its maximum value of video intensity (time to peak [TTP; seconds]), regional blood volume, regional blood flow, and mean transit time (MTT [seconds]). The hemodynamic indices TTP (P < 0.01) and MTT (P < 0.001) of diseased prostate groups were significantly lower than those in the normal prostate group although there were no differences among diseases. Optimal cutoff values were 31 seconds (Sensitivity: 72%; Specificity: 88%) and 47 seconds (Sensitivity: 100%; Specificity: 88%) while area under receiver operating characteristic curves were 0.86 (P < 0.01) and 0.97 (P < 0.01) for TTP and MTT, respectively. The qualitative evaluation of vascular patterns showed differences between normal and diseased prostate glands. The latter were characterized by an alteration of the normal vascular appearance consisting of loss of the subcapsular arterioles and lack of a centripetal vascular pattern. The qualitative aspect of the study highlighted the different vascular architecture between BPH, prostatitis, adenocarcinoma, and lymphoma. This study shows how contrast-enhanced ultrasound represents a valid and noninvasive method for highlighting and characterizing prostatic vasculature. Furthermore, it allows the operator to obtain qualitative and quantitative data that are useful for the diagnosis of selected prostatic diseases. In conclusion, contrast-enhanced ultrasound can be a valuable noninvasive diagnostic tool to improve the diagnostic accuracy of prostate diseases in the dog.