Relationship between residual feed intake and digestive traits of fattening bulls fed grass silage- or maize silage-based diets.

Several studies tried to identify digestive determinants of individual variation in feed efficiency between fattening bulls, because of their importance for breeding and management strategies. Most studies focused on single traits or single diet. Little is known about diet-dependent differences in digestive determinants and on their relative importance in distinguishing divergent residual feed intake (RFI) bulls. This research aimed (i) to identify digestive traits that differed between bulls diverging in RFI and fed a maize silage- or a grass silage-based diets; (ii) to highlight the relationships between RFI and digestive traits, and (iii) to explore the hierarchy among digestive traits in discriminating RFI divergent bulls. After an initial RFI test of 84 days on 100 Charolais growing bulls fed two different diets based on grass silage (GS), or maize silage (MS), the 32 most RFI divergent bulls were selected (eight efficient RFI- and eight inefficient RFI+ bulls per diet) and measured thereafter for total tract apparent digestibility and transit rate, enteric gas emissions (CH4 and H2), rumen pH, and feeding behaviour. Rumen particle size and visceral organ and reticulo-omasal orifice (ROO) sizes and rumen and ileum histology were measured at slaughter on the 32 selected extreme RFI bulls. Irrespective of the diet, efficient bulls (RFI-) had lower rumen size, CH4 yield (g/kg DM intake; tendency), lower number of cells in the ileal crypts, tended to have longer time of rumen pH below 5.8 and lower proportion of small size particles in rumen content than non-efficient bulls (RFI+). A long-term test for feed efficiency (197 d on average) was performed on the whole experimental period until slaughter for the 100 animals. The long-term RFI value was negatively related to time spent in activity other than ingestion, rumination, and resting, and positively related (tendency) to the duration of ingestion events, to rumen and abomasum size, irrespective of the diet. Diet-dependent effects were noted: with GS, efficient (RFI-) bulls showed a slower transit rate, whereas with MS, efficient (RFI-) bulls tended to have shorter resting events and a smaller ROO than inefficient bulls (RFI+). The transit rate and the ROO size tended to be positively related, while total tract apparent digestibility of nitrogen was negatively related to long-term RFI value, but only in GS. Rumen size appeared as the most discriminating digestive variable between RFI divergent bulls, but this result should be validated on a larger number of animals and diets.

Biomimetic approach for an articular cartilage patch: Combination of decellularized cartilage matrix and silk-elastin-like-protein (SELP) hydrogel.

Articular cartilage degradation due to injury, disease and aging is a common clinical issue as current regenerative therapies are unable to fully replicate the complex microenvironment of the native tissue which, being avascular, is featured by very low ability to self-regenerate. The extracellular matrix (ECM), constituting almost 90% of the entire tissue, plays a critical role in its function and resistance to compressive forces. In this context, the current tissue engineering strategies are only partially effective in restoring the biology and function of the native tissue. A main issue in tissue regeneration is treatment failure due to scarce integration of the engineered construct, often following a gradual detachment of the graft. In this scenario, we aimed to create an adhesive patch able to adequately support cartilage regeneration as a promising tool for the treatment of cartilage injuries and diseases. For this, we produced an engineered construct composed of decellularized ECM (dECM) obtained from horse joint cartilage, to support tissue regeneration, coupled with a Silk-Elastin-Like Proteins (SELP) hydrogel, which acts as a biological glue, to guarantee an adequate adherence to the host tissue. Following the production of the two biomaterials we characterized them by assessing: 1) dECM morphological, chemical, and ultrastructural features along with its capability to support chondrocyte proliferation, specific marker expression and ECM synthesis; 2) SELP microarchitecture, cytocompatibility and mechanical properties. Our results demonstrated that both materials hold unique properties suitable to be exploited to produce a tailored microenvironment to support cell growth and differentiation providing a proof of concept concerning the in vitro biological and mechanical efficacy of the construct. The SELP hydrogel displayed a very interesting physical behavior due to its high degree of resistance to mechanical stress, which is generally associated with physiological mechanical load during locomotion. Intriguingly, the shear-thinning behavior of the hydrogel may also make it suitable to be applied and spread over non-homogeneous surfaces, therefore, we hypothesize that the hybrid biomaterial proposed may be a real asset in the treatment of cartilage defects and injuries.

Orexin B inhibits viability and differentiation of stromal cells from swine adipose tissue.

Adipose tissue is recognized as a fundamental endocrine organ. Nowadays, we are also aware that it contains the highest number of stromal cells (ASCs) per unit of volume. These cells can differentiate between different phenotypes among which the adipocytes. The aim of this work was to verify whether orexin B, crucial mediator of the energy balance, modifies the differentiation of cultured ASCs. We used the pig as a model. Our data demonstrate that swine ASCs express prepro-orexin. Orexin B treatment inhibits ASCs proliferation (P < 0.05) and adipogenic differentiation (P < 0.05). Data collected could be interesting both in animal production field because consumers require lean meat, and in human medicine study about obesity because pig can be considered a valuable animal model for translational studies.

The myokine irisin: localization and effects in swine late medium and large antral ovarian follicle.

Irisin is mainly synthesized by skeletal muscle tissue, where it is believed to be responsible for the benefits of exercise on metabolism and cardiovascular system. In adipose tissue, its best-known effect is the browning of white adipocytes, resulting in the increase of thermogenesis and energy expenditure. As it has been largely documented that metabolic dysfunctions can frequently be associated with reductions in fertility, the possible involvement of this molecule in the regulation of reproductive processes represents an issue to be addressed. On this basis, the first aim of this work was the evaluation of the presence of irisin in the swine ovary; then, we investigated the expression of the associated molecules FNDC5, PGC-1α, and PPAR-γ. To verify a potential modulatory role both on ovarian function and on redox status, cell growth, steroidogenesis, production of superoxide anion and nitric oxide, the nonenzymatic antioxidant scavengers, were assessed in vitro on granulosa cells treated with increasing concentrations of irisin (50, 100, and 150 ng/mL). The data collected demonstrate the presence of irisin in swine ovarian follicle. Moreover, the highest concentrations tested stimulated metabolic activity and inhibited cell proliferation (P < 0.05); the peptide exerted a biphasic effect on progesterone (P < 0.01) production and, at the highest concentrations, inhibited nitric oxide while stimulated the nonenzymatic antioxidant power (P < 0.05). Superoxide anion and estradiol 17ß were unaffected. The demonstration of the local presence of irisin at the ovarian level and the highlighted effects allow us to qualify this molecule as a potential physiological regulator of follicular function.

Expression and function of the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in the swine ovarian follicle.

The most characterized stromal cell-derived factor-1 (SDF-1) variants are the isoform α, which is the predominant one but undergoes rapid proteolysis, and the ß isoform, which is more resistant. Through the interaction with a specific chemokine receptor called CXCR4, SDF-1 is able to regulate different physiological processes. The aim of this study was to verify the expression and potential functional role of SDF-1 and CXCR4 in the porcine ovary. Firstly, the expression of SDF-1 and its receptor in different ovarian districts was verified for the first time. Thereafter, the effect of SDF-1 ß isoform (51-72) fragment on functional parameters, such as proliferation, metabolic activity, redox status, nitric oxide production, and steroidogenic activity, was assessed on granulosa cells collected from follicles. In addition, the potential effect of this protein in vascular events was verified through investigations on porcine aortic (AOC) endothelial cells, such as the production of nitric oxide and viability tests. The proliferation and metabolic activity were not affected by treatment with the cytokine. As regard to steroidogenesis, the peptide stimulated both estrogen (P = 0.049) and progesterone production (P = 0.039). Redox status was affected by the examined substance since superoxide anion was inhibited (P = 0.001), while antioxidant power (P = 0.034), as well as nitric oxide generation, were stimulated (P = 0.034). Tests performed on AOCs showed significant stimulation of nitric oxide production (P = 0.004) by the examined peptide, while cell viability was unaffected. Therefore, the potential role of cytokine in the mechanisms involved in the regulation of follicular function can be hypothesized.

Orexin A in swine corpus luteum.

Orexin A (OXA) is a hypothalamic neuropeptide which acts on 2 known G-protein-coupled receptors. It has been demonstrated that OXA is a central molecular link between food intake and reproduction. More recently, its peripheral role has been investigated, and we demonstrated its involvement in regulating ovarian follicle function. The present study was undertaken to explore a potential physiological role of orexin system in swine corpus luteum, a transient ovarian endocrine organ. Our aim was, first, to analyze the localization and eventual colocalization of OXA and its 2 receptors within the different cell types composing the corpus luteum structure. Second, we wanted to explore the effects of OXA on isolated luteal cells, and finally to verify a potential involvement of OXA in angiogenesis, a crucial event in corpus luteum development. Our data demonstrate the local expression of OXA and its receptors in swine corpus luteum. Luteal cell functions were affected by treatment with OXA. In particular, progesterone production was inhibited (P < 0.05) and nonenzymatic scavenging activity was increased (P < 0.05). Moreover, OXA inhibited (P < 0.05) new vessel growth. Our results suggest that OXA could act locally to play a role in corpus luteum demise.

Orexin system in swine ovarian follicles.

Successful reproduction is strictly linked to metabolic cues. The orexins are a family of hypothalamic neurohormones, well known for their key role in the control of food intake and the involvement in several aspects of the reproductive process. The biological actions of both orexins are carried out through binding to the related Orexin 1 (OX1R) and Orexin 2 (OX2R) G-protein-coupled receptors. The purpose of this study was to investigate the presence of orexin system components in the porcine ovaries, to contribute to expand the knowledge about their pleiotropic role. First, we investigated the localization of orexin A (OXA) and its receptors by immunochemistry in different ovarian districts. Thereafter, we evaluated the expression of the prepro-orexin (PPO) gene and OXA effects on granulosa cell functions. Immunohistochemical study revealed the presence of orexinergic system components in porcine ovarian follicles. Moreover, our data show the expression of PPO messenger RNA in swine ovarian follicles >5 mm. In addition, OXA influences proliferation (P < 0.05), steroidogenic activity (P < 0.05), and redox status of granulosa cells (P < 0.05). Therefore, we hypothesize that OXA could exert a local physiological role in swine ovarian follicles even if further studies are required to deeply define the function of this pleiotropic system.

Effect of the supplementation with a blend containing short and medium chain fatty acid monoglycerides in milk replacer on rumen papillae development in weaning calves.

Feeding of neonates with artificial milk formulas is a popular trend toward early weaning of newborn dairy calves. These milk replacers (MR) should accelerate the rumen development, determining early solid feed intake and leading to better performances in cattle. Previous research demonstrated that sodium butyrate supplementation in MR can affect both small intestine and rumen development. Also acetate and propionate showed similar properties, while only a few studies indicate some potential benefit of monoglycerides on gut functions. The present study is aimed to determine the effect of the supplementation of a blend containing short and medium chain fatty acids monoglycerides (SMCFA) in milk replacer on rumen papillae development and growth performances in weaning calves. Twenty bull calves (about 2 weeks old, weighing around 43kg) were randomly allocated into two groups: control (C) and treated (T). Besides MR and starter diet, the latter offered at libitum, T calves received 0.2% SMCFA in MR. Animals were slaughtered after 56 days from the beginning of the trial. No difference was found between groups either in growth performances or in mean number of papillae/cm(2) of mucosa, total surface of papillae (mm(2))/cm(2) of mucosa or papillary size. In both groups, the morphology of the rumen epithelium was typical of parakeratosis. The cells of the stratum spinosum were directly transformed into swollen, ovoid, still nucleated keratinocytes, particularly at the papillary tip, probably as a result of unphysiological osmolarities caused by high concentrate intake. Degenerated squamous horn cells covered the "balloon like" cells forming several layers, particularly in the places of the rumen mucosa more protected from an abrasive action of solid feed. This was more evident in C animals. The squamous cells covering the papillary tip showed cytoplasmic protrusion, representing remains of the attachment sites of desmosomes, which increased the total absorptive surface and were more numerous and higher in T compared to C animals. It might be hypothesized that SMCFA supplementation in MR could better regulate epithelial cell proliferation and probably have an "emollient effect" leading to an easier "peeling" that might increase efficiency for nutrient transport across the epithelium.

Localization of peripheral autonomic neurons innervating the boar urinary bladder trigone and neurochemical features of the sympathetic component.

The urinary bladder trigone (UBT) is a limited area through which the majority of vessels and nerve fibers penetrate into the urinary bladder and where nerve fibers and intramural neurons are more concentrated. We localized the extramural post-ganglionic autonomic neurons supplying the porcine UBT by means of retrograde tracing (Fast Blue, FB). Moreover, we investigated the phenotype of sympathetic trunk ganglion (STG) and caudal mesenteric ganglion (CMG) neurons positive to FB (FB+) by coupling retrograde tracing and double-labeling immunofluorescence methods. A mean number of 1845.1±259.3 FB+ neurons were localized bilaterally in the L1-S3 STG, which appeared as small pericarya (465.6±82.7 µm2) mainly localized along an edge of the ganglion. A large number (4287.5±1450.6) of small (476.1±103.9 µm2) FB+ neurons were localized mainly along a border of both CMG. The largest number (4793.3±1990.8) of FB+ neurons was observed in the pelvic plexus (PP), where labeled neurons were often clustered within different microganglia and had smaller soma cross-sectional area (374.9±85.4 µm2). STG and CMG FB+ neurons were immunoreactive (IR) for tyrosine hydroxylase (TH) (66±10.1% and 52.7±8.2%, respectively), dopamine beta-hydroxylase (DßH) (62±6.2% and 52±6.2%, respectively), neuropeptide Y (NPY) (59±8.2% and 65.8±7.3%, respectively), calcitonin-gene-related peptide (CGRP) (24.1±3.3% and 22.1±3.3%, respectively), substance P (SP) (21.6±2.4% and 37.7±7.5%, respectively), vasoactive intestinal polypeptide (VIP) (18.9±2.3% and 35.4±4.4%, respectively), neuronal nitric oxide synthase (nNOS) (15.3±2% and 32.9±7.7%, respectively), vesicular acetylcholine transporter (VAChT) (15±2% and 34.7±4.5%, respectively), leu-enkephalin (LENK) (14.3±7.1% and 25.9±8.9%, respectively), and somatostatin (SOM) (12.4±3% and 31.8±7.3%, respectively). UBT-projecting neurons were also surrounded by VAChT-, CGRP-, LENK-, and nNOS-IR fibers. The possible role of these neurons and fibers in the neural pathways of the UBT is discussed.

Neurochemical features of boar lumbosacral dorsal root ganglion neurons and characterization of sensory neurons innervating the urinary bladder trigone.

Porcine lumbosacral dorsal root ganglion (DRG) neurons were neurochemically characterized by using six neuronal markers: calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS), neurofilament 200kDa (NF200), transient receptor potential vanilloid 1 (TRPV1), and isolectin B4 (IB4) from Griffonia simplicifolia. In addition, the phenotype and cross-sectional area of DRG neurons innervating the urinary bladder trigone (UBT) were evaluated by coupling retrograde tracer technique and immunohistochemistry. Lumbar and sacral DRG neuronal subpopulations were immunoreactive (IR) for CGRP (30 ± 3% and 29 ± 3%, respectively), SP (26 ± 8% and 27 ± 12%, respectively), nNOS (21 ± 4% and 26 ± 7%, respectively), NF200 (75 ± 14% and 81 ± 7%, respectively), and TRPV1 (48 ± 13% and 43 ± 6%, respectively), and labeled for IB4 (56 ± 6% and 43 ± 10%, respectively). UBT sensory neurons, which were distributed from L2 to Ca1 DRG, had a segmental localization, showing their highest density in L4-L5 and S2-S4 DRG. Lumbar and sacral UBT sensory neurons expressed similar percentages of NF200 immunoreactivity (64 ± 33% and 58 ± 12%, respectively) but showed a significantly different immunoreactivity for CGRP, SP, nNOS, and TRPV1 (56 ± 9%, 39 ± 15%, 17 ± 13%, 62 ± 10% vs. 16 ± 6%, 16 ± 11%, 6 ± 1%, 45 ± 24%, respectively). Lumbar and sacral UBT sensory neurons also showed different IB4 labeling (67 ± 19% and 48 ± 16, respectively). Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the UBT. The findings related to cell size also reinforced this hypothesis, because lumbar UBT sensory neurons were significantly larger than sacral ones (1,112 ± 624 µm(2) vs. 716 ± 421 µm(2) ).

MHC genotype predicts mate choice in the ring-necked pheasant Phasianus colchicus.

Females of several vertebrate species selectively mate with males on the basis of the major histocompatibility complex (MHC) genes. As androgen-mediated maternal effects have long-lasting consequences for the adult phenotype, both mating and reproductive success may depend on the combined effect of MHC genotype and exposure to androgens during early ontogeny. We studied how MHC-based mate choice in ring-necked pheasants (Phasianus colchicus) was influenced by an experimental in ovo testosterone (T) increase. There was no conclusive evidence of in ovo T treatment differentially affecting mate choice in relation to MHC genotype. However, females avoided mating with males with a wholly different MHC genotype compared with males sharing at least one MHC allele. Females also tended to avoid mating with MHC-identical males, though not significantly so. These findings suggest that female pheasants preferred males with intermediate MHC dissimilarity. Male MHC heterozygosity or diversity did not predict the expression of ornaments or male dominance rank. Thus, MHC-based mating preferences in the ring-necked pheasant do not seem to be mediated by ornaments' expression and may have evolved mainly to reduce the costs of high heterozygosity at MHC loci for the progeny, such as increased risk of autoimmune diseases or disruption of coadapted gene pools.

Experimental study on the location of neurons associated with the first sacral sympathetic trunk ganglion of the pig.

The neurons associated with the left first sacral sympathetic trunk ganglion (STG S1), an autonomic ganglion particularly concerned in the innervation of the smooth and striated musculature associated with pelvic organs, were identified in the pig, using the non-trans-synaptic fluorescent retrograde neuronal tracer Fast Blue. The labelled neurons were located mostly ipsilaterally, in the intermediolateral nucleus of the spinal cord segments T10-L5, in the sympathetic trunk ganglia L3-Co1, in the caudal mesenteric ganglia, in the pelvic ganglia, and in the spinal ganglia T13-S4. Our results could indicate the existence of visceral neuronal circuits concerning the ganglia of the sympathetic trunk and the caudal mesenteric, pelvic and spinal ganglia with or without the intervention of the central nervous system, whose identification and preservation during surgical treatments could be helpful in reducing the risk of subsequent urinary and sexual disfunctions.

Double labelling immunohistochemical characterization of autonomic sympathetic neurons innervating the sow retractor clitoridis muscle.

Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM). Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leu-enkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances. Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.

Bone response to polymers based on poly-lactic acid and having different degradation times.

Authors studied two degradable and resorbable polymers derived from lactic acid: poly-L-Lactic acid (PLLA), with a relatively long time of degradation (longer than 6 months, PL10 Purac NL); poly-DL-Lactic acid (PDLLA), with a relatively short time of degradation (shorter than 6 months, PDL Purac NL). The animal species was the young adult New Zealand White rabbit. The in-vivo study was performed by implantation of small cylinders of 10 x 3 mm in size (length x diameter) in the distal metaepiphysis of the femur; 34 cylinders have been implanted. Retrievals of PLLA specimens took place at 3, 6, 9, 12 and 24 months; for PDLLA specimens at 1, 2, 4 months. Polarized light microscopy of undecalcified tissue sections was performed. The analysis for PLLA and PDLLA has shown a favorable response of bone tissue: alterations in the bone repair, growth and remodeling have not been observed. PLLA is persistent at the times studied; there is never a tight apposition between bone and PLLA implant and an intervening fibrous layer has often been observed. PDLLA is not persistent at the times studied and it degrades quite fast; bone repair of the empty implantation's hole occurs by bony growth from the endosteal trabeculae. The newly formed bone covers the hole's walls with an elongation parallel to them. For both polymers, whether the degradation is fast or slow, the material's substitution by newly formed bone never starts from the walls of the implantation hole. Only after the complete disappearance of the polymeric material newly formed bone begins to fill the hole.

X-ray diffraction and polarizing optical microscopy investigation of the structural organization of rabbit tibia.

X-ray diffraction and polarized optical microscopy investigations were carried out on thin sections of rabbit tibia in order to study the morphological organization of the structural components of this tissue, which often is utilized to test bone response to implants. In the optical microscope, the lateral face as well as the lateral portion of the caudal face exhibit a lamellar structure with an alternation of dark and bright lamellae running parallel to the long axis of the tibia. In contrast, both in the medial face and in the medial portion of the caudal face there are numerous osteonic structures. In spite of the complexity of this morphological organization, the results of small- and high-angle X-ray diffraction analyses indicate that the structural relationship between collagen fibrils and inorganic crystals is quite similar to that observed in single osteons and allows evaluation of the orientation of the two main structural components. Both collagen fibrils and apatitic crystallites are preferentially oriented parallel to the long axis of the tibia. The degree of orientation is greater in the thickness than in the plane of the lamellae, suggesting that collagen fibrils and inorganic crystallites lie preferentially in the plane of the lamellae, where they follow an oblique course. The degree of orientation of the apatitic crystallites is higher in the lateral face than in the medial and caudal faces, in agreement with the optical microscopic images. The results provide information that must be taken into account when evaluating the structural modifications of bone due to the insertion of a prosthetic device.