Transfer of a single fresh in vitro-produced embryo may prevent twin pregnancy without compromising the fertility of the cow.

This study examines whether the transfer of a fresh in vitro-produced (IVP) embryo can avoid the risk of twin pregnancy without reducing the fertility of a cow. The study population was comprised of 416 lactating dairy cows synchronized for oestrus: 294 were fixed-time inseminated (AI cows), and 122 were given GnRH treatment at the time of embryo transfer (ET) an IVP embryo (ET cows). Of the 416 cows, 167 (40.1%) became pregnant. Twin pregnancy was recorded in 20.8% of the AI pregnant cows (21/101), whereas no ET cows had twins (0/66). Significant interaction (p < .01) was observed between breeding technique and the period of the year for the likelihood of pregnancy. This meant that using AI cows during the warm period (May-September) as reference, the odds ratio for pregnancy in ET cows during the warm period was 3.4 (p = .001). In conclusion, transfer of a single fresh IVP embryo proved useful to prevent the risk of twin pregnancy without affecting fertility.

Use and adequacy of non-pregnancy diagnosis in cow. Which future?

In cattle, early detection of gestation is very important from an economic and management point of view in all types of farming. However, due to the poor efficiency of oestrous detection, it is essential to determine non-pregnant cows as early as possible, in order to minimize the inter-insemination interval, thus de facto, reducing herd open days. Direct and indirect gestation diagnostic methods have been developed with the aim of improving the reproductive performance of the herd. Today, the most accurate method for making an early diagnosis of gestation from 28 to 30 days post-insemination is B-mode ultrasound. In recent years, indirect methods have included techniques that allow non-pregnant cows to be identified with a minimum margin of error, the most widely utilized of which is the colour Doppler. This technique is rapidly becoming established for the diagnosis of non-pregnancy, which allows for the identification of non-pregnant animals earlier compared with the pregnancy diagnosis. Some limitations of this technique in dairy cow have been presented.

Do acne treatments affect insulin-like growth factor-1 serum levels? A clinical and laboratory study on patients with acne vulgaris.

Acne is a chronic inflammatory disease affecting sebaceous gland follicles. Lately, acne has considered an insulin-like growth factor-1 (IGF-1) mediated disease. Recent research demonstrated that IGF-1 levels decrease after 3 months of isotretinoin. The purpose of our study is evaluating the influence of acne treatments on IGF-1 serum levels. Forty-six subjects with acne vulgaris aged 14 to 30 years were subdivided into three groups according to their severity of acne and treated following the European Dermatology Forum guidelines. IGF-1 was measured in patients before and after the treatment and then compared to the IGF-1 of a healthy population of the same age. IGF-1 resulted higher in patients than in controls but there was not a statistically significant variation after treatment. To the best of our knowledge, this is the first study evaluating the influence of topical and systemic acne treatment on IGF-1 serum levels. In contrast with the literature, our results suggest that common therapies for acne are not able to significantly modify IGF-1 serum levels.

Impact of maternally derived immunity on piglets' immune response and protection against porcine circovirus type 2 (PCV2) after vaccination against PCV2 at different age.

BACKGROUND: This study was aimed at evaluating the clinical protection, the level of Porcine circovirus type 2 (PCV2) viremia and the immune response (antibodies and IFN-γ secreting cells (SC)) in piglets derived from PCV2 vaccinated sows and themselves vaccinated against PCV2 at different age, namely at 4, 6 and 8 weeks. The cohort study has been carried out over three subsequent production cycles (replicates). At the start/enrolment, 46 gilts were considered at first mating, bled and vaccinated. At the first, second and third farrowing, dams were bled and re-vaccinated at the subsequent mating after weaning piglets. Overall 400 piglets at each farrowing (first, second and third) were randomly allocated in three different groups (100 piglets/group) based on the timing of vaccination (4, 6 or 8 weeks of age). A fourth group was kept non-vaccinated (controls). Piglets were vaccinated intramuscularly with one dose (2 mL) of a commercial PCV2a-based subunit vaccine (Porcilis® PCV). Twenty animals per group were bled at weaning and from vaccination to slaughter every 4 weeks for the detection of PCV2 viremia, humoral and cell-mediated immune responses. Clinical signs and individual treatments (morbidity), mortality, and body weight of all piglets were recorded. RESULTS: All vaccination schemes (4, 6 and 8 weeks of age) were able to induce an antibody response and IFN-γ SC. The highest clinical and virological protection sustained by immune reactivity was observed in pigs vaccinated at 6 weeks of age. Overall, repeated PCV2 vaccination in sows at mating and the subsequent higher levels of maternally derived antibodies did not significantly interfere with the induction of both humoral and cell-mediated immunity in their piglets after vaccination. CONCLUSIONS: The combination of vaccination in sows at mating and in piglets at 6 weeks of age was more effective for controlling PCV2 natural infection, than other vaccination schemas, thus sustaining that some interference of MDA with the induction of an efficient immune response could be considered. In conclusion, optimal vaccination strategy needs to balance the levels of passive immunity, the management practices and timing of infection.

Tactile stimulation of the perigenital region during manual bladder expression improved the urine stream in cats affected by upper motor neuron injury.

OBJECTIVE: The aim of the study was to evaluate whether the tactile stimulation of the perigenital region together with manual bladder expression (MBE) facilitated the urine stream in cats with acute or chronic upper motor neuron injury (UMNI). ANIMALS: 34 cats with UMNI having urinary retention. METHODS: All the cats had a complete neurologic examination, which determined the localization of the UMNI between T3 and L3. They were classified as chronic UMNI if the injury had occurred more than 3 days previously. The cats were divided equally into 2 groups: the M group (n = 17) managed with only MBE, and the MT group (17) managed with MBE and tactile stimulation. RESULTS: In both groups, all the cats affected by chronic UMNI resumed urination. The time required to obtain a urine stream in the chronic UMNI was 9.3 seconds in the M group and 3.1 seconds in the MT group (P < .05). In the cats affected by acute UMNI, a urine stream was achieved in 54% of the M group and 100% of the MT group (P < .05). The time to obtain a urine stream in the acute UMNI cats was 7.8 seconds in the M group and 3.75 seconds in the MT group (P < .05). CLINICAL RELEVANCE: Adding tactile stimulation of the perigenital region to the MBE improved the urine stream in cats affected by UMNI.

Bioactive Peptides in Dairy Milk: Highlighting the Role of Melatonin.

Melatonin, an endogenous indolamine derived from tryptophan, is primarily synthesized by the pineal gland in mammals and regulated by a complex neural system. Its release follows a circadian rhythm, which is crucial for regulating physiological processes in response to light-dark cycles in both humans and animals. In this review, we report that the presence of this hormone in bovine milk, with significant differences in concentration between daytime and nighttime milking, has increased interest in milk as a natural source of bioactive molecules. Melatonin lowers cortisol levels at night, reduces body temperature and blood pressure, coinciding with decreased alertness and performance, acts as an antioxidant and anti-inflammatory agent, modulates the immune system, offers neuroprotective benefits, and supports gastrointestinal health by scavenging free radicals and reducing oxidative stress in dairy cows. Many factors influence the release of melatonin, such as the intensity of artificial lighting during nighttime milking, the frequency of milkings, milk yield, and genetic differences between animals. Nocturnal milking under low-intensity light boosts melatonin, potentially reducing oxidative damage and mastitis risk. Additionally, ultra-high temperature (UHT) treatment does not significantly affect the melatonin content in milk. However, further research on its stability during milk processing and storage is crucial for ensuring product efficacy. In some countries, nighttime milk with naturally elevated melatonin content is already commercialized as a natural aid for sleep. Thus, naturally melatonin-rich milk may be a promising alternative to synthetic supplements for promoting better sleep and overall well-being.

Hyperosmotic Stress Induces the Expression of Organic Osmolyte Transporters in Porcine Intestinal Cells and Betaine Exerts a Protective Effect on the Barrier Function.

Background/objectives: The porcine intestinal epithelium plays a fundamental role as a defence interface against pathogens. Its alteration can cause severe inflammatory conditions and diseases. Hyperosmotic stress under physiological conditions and upon pathogen challenge can cause malabsorption. Different cell types counteract the osmolarity increase by accumulating organic osmolytes such as betaine, taurine, and myo-inositol through specific transporters. Betaine is known for protecting cells from hyperosmotic stress and has positive effects when fed to pigs. The aim of this study is to demonstrate the modulation of osmolyte transporters gene expression in IPEC-J2 during osmolarity changes and assess the effects of betaine. Methods: IPEC-J2 were seeded in transwells, where differentiate as a polarized monolayer. Epithelial cell integrity (TEER), oxidative stress (NO) and gene expression of osmolyte transporters, tight junction proteins (TJp) and pro-inflammatory cytokines were evaluated. Results: Cells treated with NaCl hyperosmolar medium (500 mOsm/L) showed a TEER decrease at 3 h and detachment within 24 h, associated with an osmolyte transporters reduction. IPEC-J2 treated with mannitol hyperosmolar medium (500 mOsm/L) upregulated taurine (TauT), myo-inositol (SMIT) and betaine (BGT1) transporters expression. A decrease in TJp expression was associated with a TEER decrease and an increase in TNFα, IL6, and IL8. Betaine could attenuate the hyperosmolarity-induced reduction in TEER and TJp expression, the NO increase and cytokines upregulation. Conclusions: This study demonstrates the expression of osmolyte transporters in IPEC-J2, which was upregulated upon hyperosmotic treatment. Betaine counteracts changes in intracellular osmolarity by contributing to maintaining the epithelial barrier function and reducing the inflammatory condition. Compatible osmolytes may provide beneficial effects in therapies for diseases characterized by inflammation and TJp-related dysfunctions.

Interval from Oestrus to Ovulation in Dairy Cows-A Key Factor for Insemination Time: A Review.

This review describes the oestrus-to-ovulation interval, the possibility of predicting the time of ovulation, and the optimum time for insemination relative to oestrus in dairy cows. The duration of oestrus in dairy cows is approximately 8-20 h, with differences possibly related to the methods of oestrus detection and the frequency of observations. Most cows ovulate approximately 24-33 h after the onset of oestrus and 15-22 h after the end of oestrus. The interval from the preovulatory luteinising hormone (LH) surge to ovulation is approximately 4-30 h. Ovulation occurs when follicle diameter averages 18-20 mm. When it is possible to correctly determine the beginning of oestrus, artificial insemination can be performed utilizing the "a.m.-p.m. rule", and only one insemination may be applied. In cows with too long or too short oestrus-to-ovulation intervals, fertility can be compromised. One important factor that can alter the oestrus-to-ovulation interval is acute or chronic heat stress during the warm season. When there is a risk that insemination may occur too early or too late with respect to the time of ovulation, GnRH administration can be considered.

Short-chain fatty acids modulate the IPEC-J2 cell response to pathogenic E. coli LPS-activated PBMC.

Intestinal disorders can affect pigs of any age, especially when animals are young and more susceptible to infections and environmental stressors. For instance, pathogenic E. coli can alter intestinal functions, thus leading to altered nutrient adsorption by interacting with local cells through lipopolysaccharide (LPS). Among several compounds studied to counteract the negative effects on the intestine, short-chain fatty acids (SCFA) were demonstrated to exert beneficial effects on gut epithelial cells and resident immune cells. In this study, acetate and propionate were tested for their beneficial effects in a co-culture model of IPEC-J2 and porcine PBMC pre-stimulated with LPS from E. coli 0111:B4 aimed at mimicking the interaction between intestinal cells and immune cells in an inflammatory/activated status. IPEC-J2 viability was partially reduced when co-cultured with activated PBMC and nitric oxide concentration increased. IPEC-J2 up-regulated innate and inflammatory markers, namely BD-1, TLR-4, IL-8, TNF-α, NF-κB, and TGF-ß. Acetate and propionate positively modulated the inflammatory condition by sustaining cell viability, reducing the oxidative stress, and down-regulating the expression of inflammatory mediators. TNF-α expression and secretion showed an opposite effect in IPEC-J2 depending on the extent of LPS stimulation of PBMC and TGF-ß modulation. Therefore, SCFA proved to mediate a differential effect depending on the degree and duration of inflammation. The expression of the tight junction proteins (TJp) claudin-4 and zonula occludens-1 was up-regulated by LPS while SCFA influenced TJp with a different kinetics depending on PBMC stimulation. The co-culture model of IPEC-J2 and LPS-activated PBMC proved to be feasible to address the modulation of markers related to anti-bacterial immunity and inflammation, and intestinal epithelial barrier integrity, which are involved in the in vivo responsiveness and plasticity to infections.

Acetate and propionate effects in response to LPS in a porcine intestinal co-culture model.

BACKGROUND: The interest in acetate and propionate as short chain fatty acids (SCFA) derives from research on alternative strategies to the utilization of antibiotics in pig farms. SCFA have a protective role on the intestinal epithelial barrier and improve intestinal immunity by regulating the inflammatory and immune response. This regulation is associated with an increase in intestinal barrier integrity, mediated by the enhancement of tight junction protein (TJp) functions, which prevent the passage of pathogens through the paracellular space. The purpose of this study was to evaluate the effect of in vitro supplementation with SCFA (5 mM acetate and 1 mM propionate) on viability, nitric oxide (NO) release (oxidative stress), NF-κB gene expression, and gene and protein expression of major TJp (occludin [OCLN], zonula occludens-1 [ZO-1], and claudin-4 [CLDN4]) in a porcine intestinal epithelial cell (IPEC-J2) and peripheral blood mononuclear cell (PBMC) co-culture model upon LPS stimulation, through which an acute inflammatory state was simulated. RESULTS: Firstly, the inflammatory stimulus induced by LPS evaluated in the IPEC-J2 monoculture was characterized by a reduction of viability, gene expression of TJp and OCLN protein synthesis, and an increase of NO release. The response evaluated in the co-culture showed that acetate positively stimulated the viability of both untreated and LPS-stimulated IPEC-J2 and reduced the release of NO in LPS-stimulated cells. Acetate also promoted an increase of gene expression of CLDN4, ZO-1, and OCLN, and protein synthesis of CLDN4, OCLN and ZO-1 in untreated and LPS-stimulated cells. Propionate induced a reduction of NO release in both untreated and LPS-stimulated IPEC-J2. In untreated cells, propionate induced an increase of TJp gene expression and of CLDN4 and OCLN protein synthesis. Contrarily, propionate in LPS-stimulated cells induced an increase of CLDN4 and OCLN gene expression and protein synthesis. PBMC were influenced by acetate and propionate supplementation, in that NF-κB expression was strongly downregulated in LPS-stimulated cells. CONCLUSIONS: The present study demonstrates the protective effect of acetate and propionate upon acute inflammation by regulating epithelial tight junction expression and protein synthesis in a co-culture model, which simulates the in vivo interaction between epithelial intestinal cells and local immune cells.

Osmolarity modulates the de-differentiation of horse articular chondrocytes during cell expansion in vitro: implications for tissue engineering in cartilage repair.

Due to the importance of joint disease and ostearthritis (OA) in equine athletes, new regenerative treatments to improve articular cartilage repair after damage are gaining relevance. Chondrocyte de-differentiation, an important pathogenetic mechanism in OA, is a limiting factor when differentiated articular chondrocytes are used for cell-based therapies. Current research focuses on the prevention of this de-differentiation and/or on the re-differentiation of chondrocytes by employing different strategies in vitro and in vivo. Articular chondrocytes normally live in a condition of higher osmolarity (350-450 mOsm/L) compared to normal physiological fluids (~ 300 mOsm/L) and some studies have demonstrated that osmolarity has a chondroprotective effect in vitro and in vivo. Therefore, the response of horse articular chondrocytes to osmolarity changes (280, 380, and 480 mOsm/L) was studied both in proliferating, de-differentiated chondrocytes grown in adhesion, and in differentiated chondrocytes grown in a 3D culture system. To this aim, cell proliferation (cell counting), morphology (optical microscopy), and differentiation (gene expression of specific markers) were monitored along with the expression of osmolyte transporters involved in volume regulation [betaine-GABA transporter (BGT-1), taurine transporter (SLC6A6), and neutral amino acid transporter (SNAT)] real-time qPCR. Proliferating chondrocytes cultured under hyperosmolar conditions showed low proliferation, spheroidal morphology, a significant reduction of de-differentiation markers [collagen type I (Col1) and RUNX2] and an increase of differentiation markers [collagen type II (Col2) and aggrecan]. Notably, a persistently high level of BGT-1 gene expression was maintained in chondrocyte cultures at 380 mOsm/L, and particularly at 480 mOsm/L both in proliferating and differentiated chondrocytes. These preliminary data encourage the study of osmolarity as a microenvironmental co-factor to promote/maintain chondrocyte differentiation in both 2D and 3D in vitro culture systems.

High-Resolution Ultrasonographic Anatomy of the Carpal Tendons of Sporting Border Collies.

Recent literature has demonstrated that high-resolution ultrasonographic anatomy of the canine carpus is possible; however, only the structures of the dorsal face were described. The aims of this prospective study were: (1) to describe the normal ultrasonographic appearance of the carpal tendons in sporting Border Collies; (2) to measure the height, length, and thickness of the tendon at the radial ulnar notch level in order to create a baseline reference for the breed, and (3) to describe a standardised protocol to ultrasonographically evaluate the carpal faces and visible tendinous structures. A pilot study based on ten cadaveric front limbs was used to identify the structures. A subsequent clinical phase of the study using twenty-six Border Collies was recorded. The tendons of the Extensor Carpi Radialis, Extensor Digitorum Communis, and Extensor Digitorum Lateralis were identified and followed from the tenomuscular junction to the distal insertion on the dorsal face of the digits. On the lateral face, the tendon of the Extensor Carpi Ulnaris was recognised and followed. On the palmar face, the two heads of the Flexor Carpi Ulnaris tendon ending on the accessory carpal bone, the adjacent Flexor Digitorum Superficialis tendon, and the deep and medially located Flexor Digitorum Profundus tendon were seen and followed. The Flexor Carpi Radialis and the Abductor Pollicis Longus tendons were seen in the medial carpal face. The ulnar notch of the radius was used as the measurement and starting point of the ultrasonography. These data could be used as a standard reference in the case of chronic overuse and trauma-induced changes in the canine carpus.

Development and single laboratory validation of a targeted liquid chromatography-triple quadrupole mass spectrometry-based method for the determination of insulin like growth factor-1 in different types of milk samples.

A simple and reliable targeted liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS) method was developed and validated through the selection of two biomarker peptides for the identification and determination of bovine insulin like growth factor-1 (IGF-1) in milk samples. Two urea-based sample extraction procedures were tested. The validation results provided detection limits at the 1-5 ng IGF-1/mL level as a function of the milk matrix, precision ranged from 3 to 8% and the method accuracy in the different milk matrices was assured. Finally, IGF-1 was measured in milk samples obtained by treatment with eleven different technological processes: IGF-1 concentrations were spread over a wide range from 11.2 ± 0.3 ng/mL to 346 ± 8 ng/mL with a median of 57.0 ± 0.2 ng/mL. The highest amount of IGF-1 was found in fresh whole milk samples and no significant correlation was found between the total milk protein content and the IGF-1 concentration level.

Effects of different short-chain fatty acids (SCFA) on gene expression of proteins involved in barrier function in IPEC-J2.

BACKGROUND: Gut microbial anaerobic fermentation produces short-chain fatty acids (SCFA), which are important substrates for energy metabolism and anabolic processes in mammals. SCFA can regulate the inflammatory response and increase the intestinal barrier integrity by enhancing the tight junction protein (TJp) functions, which prevent the passage of antigens through the paracellular space. The aim of this study was to evaluate the effect of in vitro supplementation with SCFA (acetate, propionate, butyrate, and lactate) at different concentrations on viability, nitric oxide (NO) release (oxidative stress parameter) in cell culture supernatants, and gene expression of TJp (occludin, zonula occludens-1, and claudin-4) and pro-inflammatory pathway-related mediators (ß-defensin 1, TNF-α, and NF-κB) in intestinal porcine epithelial cell line J2 (IPEC-J2). RESULTS: The SCFA tested showed significant effects on IPEC-J2, which proved to be dependent on the type and specific concentration of the fatty acid. Acetate stimulated cell viability and NO production in a dose-dependent manner (P < 0.05), and specifically, 5 mM acetate activated the barrier response through claudin-4, and immunity through ß-defensin 1 (P < 0.05). The same effect on these parameters was shown by propionate supplementation, especially at 1 mM (P < 0.05). Contrarily, lactate and butyrate showed different effects compared to acetate and propionate, as they did not stimulate an increase of cell viability and regulated barrier integrity through zonula occludens-1 and occludin, especially at 30 mM and 0.5 mM, respectively (P < 0.05). Upon supplementation with SCFA, the increase of NO release at low levels proved not to have detrimental effects on IPEC-J2 proliferation/survival, and in the case of acetate and propionate, such levels were associated with beneficial effects. Furthermore, the results showed that SCFA supplementation induced ß-defensin 1 (P < 0.05) that, in turn, may have been involved in the inhibition of TNF-α and NF-κB gene expression (P < 0.05). CONCLUSIONS: The present study demonstrates that the supplementation with specific SCFA in IPEC-J2 can significantly modulate the process of barrier protection, and that particularly acetate and propionate sustain cell viability, low oxidative stress activity and intestinal barrier function.

Hypoxia and platelet lysate sustain differentiation of primary horse articular chondrocytes in xeno-free supplementation culture.

Currently, the main limitation for the use of adult differentiated chondrocytes in cell-based therapy and tissue engineering for the repair of articular cartilage is the difficulty of maintaining their state of differentiation during cell expansion. The adult articular cartilage has no direct blood supply, and local oxygen concentrations range from 5%-10% at the surface near the synovial fluid to less than 1% in the deep layer. Low oxygen tension is currently considered an important environmental condition for chondrocytes, and hypoxia has been explored as a signal potentially promoting differentiation and matrix deposition. In the present study, hypoxia and PL supplementation were studied to maintain differentiation in adult articular chondrocytes. Freshly isolated equine articular chondrocytes were grown in monolayer culture at a low seeding density (condition favoring proliferation and dedifferentiation) and in alginate beads (3D culture condition maintaining chondrocyte differentiation) both in normoxic and hypoxic conditions and in various conditions of supplementation or deprivation (fetal bovine serum [FBS]- and PL-free; 10% FBS; 5% PL; 10% PL). Results demonstrated that hypoxia is a micro-environmental condition that reduces chondrocyte dedifferentiation or maintains differentiation during in vitro expansion, as shown by the sustained expression of differentiation markers (COL2, ACAN, SOX9, HIF1a) and the reduction of dedifferentiation marker expression (COL1, RUNX2). In association with hypoxia, PL supplementation demonstrated a positive effect on chondrocyte differentiation in association with hypoxia. This promising result should be confirmed in other conditions of chondrocyte differentiation before proposing PL as a complete alternative to xenogenic serum for the expansion of articular chondrocytes.

The influence of Mediterranean diet in acne pathogenesis and the correlation with insulin-like growth factor-1 serum levels: Implications and results.

Acne is a chronic inflammatory disease of the pilosebaceous unit, and its etiology is complex and multifactorial. The role of the diet in its pathogenesis is still debated. The purpose of this study was to assess the association between MD and IGF-1 in acne patients and, as secondary objective, the role of systemic treatment on IGF-1 serum levels, in accordance with the patients' diet. This study included 35 patients aged 14-30 years affected by acne and treated in line with the EDF guidelines. Patients were divided into 2 groups based on a questionnaire score assessing the adherence to the Mediterranean diet: the Mediterranean Group (score ≥6) and the Western Group (score< 5). IGF-1 serum levels were measured in all patients before and after treatment and then compared to healthy population. IGF-1 levels were higher in patients than in controls and in the Western group than in the Mediterranean group. We speculate that the Mediterranean diet can have a protective role in the pathogenesis of acne by acting on the systemic route of IGF-1.

Effects of Heat Stress on Follicular Physiology in Dairy Cows.

Follicular organization starts during mid-to-late fetal life with the formation of primordial follicles. The bilateral interplay between the oocyte and adjoining somatic cells during follicular growth and ovulation may be sensitive to heat stress (HS). Mechanisms giving rise to pre-ovulatory temperature gradients across reproductive tissues are mostly regulated by the pre-ovulatory follicle, and because the cooling of the gonads and genital tract depends on a counter-current transfer system of heat, HS may be considered a major factor impairing ovulation, fertilization and early embryo development. There is evidence of a long-lasting influence of HS on oogenesis and final follicular maturation. Follicular stages that are susceptible to HS have not been precisely determined. Therefore, the aim of this review was to describe the influence of HS during the staged follicular development in dairy cattle, from the activation of primordial follicles to ovulation. Some clinical prospects are also considered.

Immune B cell responsiveness to single-dose intradermal vaccination against Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a major pathogen affecting pig herds and vaccination is the most utilized approach, despite providing partial protection. Age at vaccination, the delivery route, and vaccination protocol can influence vaccine efficacy. The influence of age and the presence of maternally-derived antibodies at vaccination on single-dose needle-less intradermal (ID) administration of an inactivated bacterin-based vaccine (Porcilis® M Hyo ID Once) were assessed in conventional pigs under field conditions. The induction of IgA+ and IgG+ B cell responses and the expression of the activation markers TLR2, TLR7, CCR9, and CCR10 were determined in PBMC. Vaccination at 4 weeks efficiently elicited an anamnestic antibody response associated with TLR2 and TLR7 upregulation. Although animals vaccinated at 1 week did not show seroconversion and a recall response upon infection, the responsiveness of Mycoplasma-recalled IgA+ B cells suggests the activation of mucosal immune cells after vaccination and infection. Vaccination at 1 week induced TLR2, TLR7, and CCR9 upregulation, suggesting the potential for systemic and local activation of immune cell trafficking between blood and target tissues. Vaccination at 4 weeks induced a CCR10 increase, suggesting that recalled IgA+ and IgG+ B cells can display an activated status upon infection. The antibody response after Mycoplasma infection in 4-week-old ID-vaccinated pigs was associated with TLR2 and CCR10 increases, confirming the potential use of this vaccination schedule for the safe and efficient delivery of single-dose M. hyopneumoniae vaccines. ID vaccination, especially at 4 weeks, was associated with a great degree of protection against enzootic pneumonia (EP)-like lung lesions.

A Co-Culture Model of IPEC-J2 and Swine PBMC to Study the Responsiveness of Intestinal Epithelial Cells: The Regulatory Effect of Arginine Deprivation.

Arginine is a semi-essential amino acid, supplementation with which induces a reduction of intestinal damage and an improvement of intestinal immunity in weaned piglets, but the mechanism is not yet entirely clear. The aim of this study was to characterise a co-culture model by measuring changes in gene expression over time (24 and 48 h) in intestinal IPEC-J2 cells in the presence of immune cells activated with phytohemagglutinin and, consequently, to assess the effectiveness of arginine deprivation or supplementation in modulating the expression of certain cytokines related to the regulation of intestinal cells' function. The main results show the crucial role of arginine in the viability/proliferation of intestinal cells evaluated by an MTT assay, and in the positive regulation of the expression of pro-inflammatory (TNF-α, IL-1α, IL-6, IL-8) and anti-inflammatory (TGF-ß) cytokines. This experimental model could be important for analysing and clarifying the role of nutritional conditions in intestinal immune cells' functionality and reactivity in pigs as well as the mechanisms of the intestinal defence system. Among the potential applications of our in vitro model of interaction between IEC and the immune system there is the possibility of studying the effect of feed additives to improve animal health and production.

Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate.

Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.