Three-dimensional microvascolature remodeling study of pig periovulatory follicles using vascular corrosion casts

Veterinary Research Communications -     

Intrinsic innervation of the ileocaecal junction in the horse: Preliminary study

Reason for performing study: In horses, morpho‐functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. Objectives: To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). Methods: Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene‐related peptide (CGRP) and neurofilament NF200kDa (NF). Results: Ileal FB‐labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 ± 11% and 68 ± 4%, respectively), cholinergic (60 ± 19% and 82 ± 11%, respectively), NF‐IR (54 ± 9% and 78 ± 21%, respectively), and SP‐IR (about 20% in both the plexuses). CGRP‐IR was expressed only by SMP descending neurons (45 ± 21%). In both the plexuses descending neurons coexpressing nNOS‐and ChAT‐IR were also observed (25 ± 11% and 61 ± 27%, respectively). Conclusions: The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. Potential relevance: The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.     

Identification and association analysis of several hundred single nucleotide polymorphisms within candidate genes for back fat thickness in Italian Large White pigs using a selective genotyping approach

Combining different approaches (resequencing of portions of 54 obesity candidate genes, literature mining for pig markers associated with fat deposition or related traits in 77 genes, and in silico mining of porcine expressed sequence tags and other sequences available in databases), we identified and analyzed 736 SNP within candidate genes to identify markers associated with back fat thickness (BFT) in Italian Large White sows. Animals were chosen using a selective genotyping approach according to their EBV for BFT (276 with most negative and 279 with most positive EBV) within a population of ≈ 12,000 pigs. Association analysis between the SNP and BFT has been carried out using the MAX test proposed for case-control studies. The designed assays were successful for 656 SNP: 370 were excluded (low call rate or minor allele frequency <5%), whereas the remaining 286 in 212 genes were taken for subsequent analyses, among which 64 showed a P(nominal) value <0.1. To deal with the multiple testing problem in a candidate gene approach, we applied the proportion of false positives (PFP) method. Thirty-eight SNP were significant (P(PFP) < 0.20). The most significant SNP was the IGF2 intron3-g.3072G>A polymorphism (P(nominal) < 1.0E-50). The second most significant SNP was the MC4R c.1426A>G polymorphism (P(nominal) = 8.0E-05). The third top SNP (P(nominal) = 6.2E-04) was the intronic TBC1D1 g.219G>A polymorphic site, in agreement with our previous results obtained in an independent study. The list of significant markers also included SNP in additional genes (ABHD16A, ABHD5, ACP2, ALMS1, APOA2, ATP1A2, CALR, COL14A1, CTSF, DARS, DECR1, ENPP1, ESR1, GH1, GHRL, GNMT, IKBKB, JAK3, MTTP, NFKBIA, NT5E, PLAT, PPARG, PPP2R5D, PRLR, RRAGD, RFC2, SDHD, SERPINF1, UBE2H, VCAM1, and WAT). Functional relationships between genes were obtained using the Ingenuity Pathway Analysis (IPA) Knowledge Base. The top scoring pathway included 19 genes with a P(nominal) < 0.1, 2 of which (IKBKB and NFKBIA) are involved in the hypothalamic IKKβ/NFκB program that could represent a key axis to affect fat deposition traits in pigs. These results represent a starting point to plan marker-assisted selection in Italian Large White nuclei for BFT. Because of similarities between humans and pigs, this study might also provide useful clues to investigate genetic factors affecting human obesity.     

Purification of secretory immunoglobulin A from milk of the brushtail possum (Trichosurus vulpecula)

AIM: To identify and purify secretory immunoglobulin A (sIgA), a key effecter molecule in mucosal immune responses, from milk of the brushtail possum (Trichosurus vulpecula).

METHODS: Milk samples were collected from female possums with pouch young, and clarified by centrifugation and precipitation methods. The clarified fraction was purified by gel filtration and affinity chromatography to yield sIgA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques were used to assess the purity of the final product, and to identify the heavy (H) chain, light (L) chain and secretory component (SC) of possum sIgA.

RESULTS: Immunoblotting, using antibodies raised against cloned possum sIgA SC and H-chain, and a synthetic peptide fragment of the H-chain, confirmed the identity of the purified protein. The N-terminal amino acid sequence of purified possum sIgA showed strong homology to reported sequences of H-chain variable regions of marsupial immunoglobulins.

CONCLUSIONS: Milk was shown to be a convenient source of mucosal secretion containing sIgA, and a process involving 2 precipitation and 2 chromatography steps produced purified sIgA. This IgA preparation will prove useful for the generation of sIgA-specific immunological reagents for measurement of immune responses in the development of mucosal-based vaccines for biological control of possums.     

Experimental study on allografts of amniotic epithelial cells in calcaneal tendon lesions of sheep

An experimental protocol was designed to study the survival and behaviour of an allograft of amniotic epithelial cells (AECs) in an ovine model. The study was conducted on three healthy adult sheep. A core lesion was created in both calcaneal tendons under ultrasound (US) guidance by injecting 400 UI of Type 1A collagenase diluted in 0.6 ml saline. The AECs were obtained from a 60–80-day-old fetus and cultured under standard conditions. After 15 days of collagenase treatment, 2 × 10⁶ AECs stained with a vital membrane fluorescent probe (PHK26) were injected under US guidance in 500 μl saline solution into the lesion of one limb. The contralateral untreated limb was used as a control. Animals were euthanatized 7 (1) and 30 (2) days later. Histological analyses performed on explanted tendons clearly demonstrate that AECs survived for at least 1 month inside the lesion without any adverse reactions. The damaged tissue of the treated tendons showed a high number of reparative cells in active proliferation that were accumulating collagen within the extracellular matrix. In addition, after 1 month, the neo-collagen began to be organized into parallel arrays of fibers oriented along the longitudinal axis of the tendon.

     

Bovine papillomavirus type-2 (BPV-2) infection and expression of uroplakin IIIb, a novel urothelial cell marker, in urinary bladder tumors of cows

Several immunohistochemical markers have been used to define the differentiation pattern of urothelial cell tumors of the urinary bladder. We investigated the expression of the recently characterized uroplakin (UP) IIIb, an urothelium-specific and differentiation-dependent protein, in 39 urothelial tumors of the urinary bladder in cows that had suffered from chronic enzootic hematuria for several years. Bovine papillomavirus type 2 DNA was amplified and UP IIIb protein was detected in all these tumors. In papillomas and papillary carcinomas, UP IIIb expression was mostly seen as superficial staining; luminal and peripheral patterns were also observed. In nonpapillary carcinomas, UP IIIb appeared to define clearly the cell membrane lining intercellular and intracellular lumina as well as the cell borders in deeper cell layers. In benign and malignant lesions, an intracytoplasmic immunoreactivity was also detected. Coarse intracytoplasmic UP IIIb-positive material close to the nucleus occurred in some malignant cells. Focally strong membraneous immunostaining that marked single cells with complete ringlike peripheral pattern was seen. Although UP IIIb expression does not seem to correlate with the biological behavior of urothelial tumors, it appears to be a highly sensitive marker for bovine urothelial tumors.     

Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, several porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cystatin B) were investigated. Single nucleotide polymorphisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand conformation polymorphism analysis. Sequencing and PCR-RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Piétrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously reported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested population (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glycogen, lactate, and glycolytic potential of semimembranosus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ratio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lysosomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs.