The morphology and location of atrial specific granules and the demonstration of atrial natriuretic factor in porcine,lapine and bovine heart by immunoelectronmicroscopy

The atrial specific granules (ASGs) were studied in samples collected from the right and left auricles of conventionally slaughtered cows (10), pigs (16) and rabbits (8). In addition, the presence of atrial natriuretic factor (ANF) was detected by immunocytochemistry. Mature ASGs, characterized by the presence of highly osmiophilic and electron-dense material surrounded by a membrane, were present in all atrial myoendocrine cells and their diameters ranged from 100 to 470 nm in pigs, from 100 to 235 nm in cattle, and from 125 to 275 nm in rabbits. Immunoelectronmicroscopical studies revealed the presence of ANF in the ASGs of pigs and cattle, whereas anti-ANF polyclonal serum failed to detect any significative reaction in lapine ASGs. The ultrastructural features of the ASGs of pigs, cattle and rabbits described may be useful in comparing the morphological picture of several cardiac endocrine pathological conditions.     

Evaluation of beta3-adrenoceptor-mediated relaxation in intact and endotoxin-treated equine digital veins

OBJECTIVE: To investigate the functional expression of beta3-adrenoceptors (beta3-ARs) in equine digital veins (EDVs) and to examine whether beta3-AR relaxation was altered in EDVs incubated with endotoxin. SAMPLE POPULATION: Forelimbs obtained from 30 horses. PROCEDURE: Forelimbs were obtained from horses in an abattoir. Equine digital veins were carefully removed from distal portions of the forelimbs. Rings of dissected EDVs were mounted in 5-mL organ baths to record isometric tension in the presence of various beta3-AR agonists (SR 58611A, ZD 2079, and ZM 215001). RESULTS: In intact EDVs, isoprenaline, SR 58611A, ZD 2079, and ZM 215001 induced concentration-dependent relaxation. Isoprenaline and SR 58611A-induced relaxations were reduced or unaffected by nadolol, respectively. In intact EDVs, SR 58611A-induced relaxation was significantly reduced in the presence of 2 microM ZM 215001 (used as a beta3-AR antagonist). In endothelium-denuded EDVs or intact EDVs in the presence of a nitric oxide synthase inhibitor, isoprenaline and SR 58611A-induced relaxations were significantly decreased. The endothelium-independent relaxation to SR 58611A was significantly inhibited in the presence of ZM 215001. In endotoxin-treated EDV, isoprenaline- and SR 58611A-induced relaxations were significantly reduced. In these conditions, cycloheximide (a protein synthesis inhibitor) and ibuprofen (a cyclooxygenase inhibitor) restored the relaxant response to SR 58611A. CONCLUSIONS AND CLINICAL RELEVANCE: Beta3-adrenoceptors are functionally expressed in EDVs. Incubation in the presence of endotoxin, used as an in vitro model of laminitis, induced an alteration of beta-AR-mediated relaxations in EDVs, which could be the consequence of cyclooxygenase induction and subsequent prostanoid production.     

Angiostatin and integrin alphavbeta3 in the feline, bovine, canine, equine, porcine and murine retina and cornea

PURPOSE: Angiogenesis is tightly controlled in the ocular tissues of domestic animals but its mechanisms are not fully understood. This is largely because of insufficient data on the expression of molecules that impact angiogenesis. Because angiostatin and one of its receptors integrin alphavbeta3 inhibit and promote angiogenesis, respectively, we hypothesized that the normal retina and cornea of domestic animals would express angiostatin but not integrin alphavbeta3. PROCEDURE: Normal eyes of the cat, cow, dog, horse, pig and rat were evaluated for angiostatin and integrin alphavbeta3 by light and electron immunocytochemistry and estern blots. RESULTS: Angiostatin was detected in the corneal epithelium of the cat, dog, horse, pig and rat, but was not found in cow corneal epithelium. Angiostatin was localized in the nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and the photoreceptor layer of the cat, cow, dog and rat. Horse and pig retinas showed additional staining in the matrix of the inner nuclear layer. Immunogold electron microscopy further confirmed angiostatin in cat retina. Western blots showed angiostatin in corneal and retinal homogenates. Integrin alphavbeta3 was absent in cornea and retina of all the species studied. CONCLUSION: These data show that angiostatin, an inhibitor of angiogenesis, is present while integrin alphavbeta3, which promotes angiogenesis, is absent in normal cornea and retina of the domestic animals in this study with the exception being angiostatin absence in cow corneal epithelium. Therefore, angiostatin may contribute to the anti-angiogenic environment in the normal domestic animal eye while its absence in the cow may contribute to greater propensity for corneal vascularization. Because integrin alphavbeta3 is one of the receptors for angiostatin, its absence may prevent angiostatin from killing normal retinal and corneal cells.     

Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid

The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro‐produced zygotes were stored at 25°C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA‐containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 hr was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non‐stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non‐stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.     

2D versus 3D in the kinematic analysis of the horse at the trot

The handled trot of three Lusitano Purebred stallions was analyzed by using 2D and 3D kinematical analysis methods. Using the same capture and analysis system, 2D and 3D data of some linear (stride length, maximal height of the hoof trajectories) and angular (angular range of motion, inclination of bone segments) variables were obtained. A paired Student T-test was performed in order to detect statistically significant differences between data resulting from the two methodologies With respect to the angular variables, there were significant differences in scapula inclination, shoulder angle, cannon inclination and protraction-retraction angle in the forelimb variables, but none of them were statistically different in the hind limb. Differences between the two methods were found in most of the linear variables analyzed.     

Body growth,intestinal morphology and microflora of quail on diets supplemented with micronised wheat fibre

1. Particle size reductions of fibre-rich materials alter structure, functional and digestive properties. To determine the effects of using fibre as an additive in Japanese quail rations on performance and gut physiology, a trial using micronised wheat fibre (MWF) at levels of 0.0, 5, 10 and 15 g/kg in feed was conducted.

2. Growth rate and feed efficiency were significantly improved when diets contained MWF while feed intake was not affected by levels of the fibre. As MWF content increased, the relative weight of gizzard and gastrointestinal tract (GIT) significantly increased whereas liver relative weight significantly decreased.

3. MWF inclusion significantly increased relative length of gut segments, villi height, villus thickness, the villi height to crypt depth proportion in jejunum and ileum and the number of goblet cells in different parts of intestine.

4. Tibia weight, length and ash content were increased linearly with rising MWF inclusion. Litter moisture was affected by MWF inclusions in a quadratic manner. The colony forming unit (CFU/g) of Streptococci spp. in ileal digesta was decreased with increasing MWF inclusion levels in the diet.

5. In conclusion, MWF can be used as a feed additive in quail diets and its inclusion in feed resulted in better performance, beneficial changes in intestinal microbial counts and improvements in small intestine morphology.     

Supplementing media with NAD+ precursors enhances the in vitro maturation of porcine oocytes

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD⁺-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD⁺ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.     

3D reconstruction of the rat adrenal medulla

We performed 3D reconstruction of the microscopic structure of the adrenal medulla of the adult rats using serial histological sections with histochemical differentiation of adrenaline-storing (A) and noradrenaline-storing (NA) cells. Medulla volume is 1.18 ± 0.17 mm³. Chromaffin tissue consists of 82.9 ± 2.6% of A and 17.1 ± 2.6% of NA cells. Cords of the chromaffinocytes run along the nerves in the adrenal cortex and form cones when merging with medulla bulk. There is no unambiguously greater prevalence of A cells over NA in the areas of the medulla bordering on the cortex as compared to deep layers of medulla. NA cells form a network of beams. Their concentration increases with distance from the entry site of the nerves and is maximal on the opposite side. This testifies to the fallacy of the point of view about the disordered distribution of NA cells in the medulla. Based on the polar asymmetric arrangement of the adrenal chromaffin tissue, if it is necessary to completely remove the medulla with the keeping or reimplantation of the cortex, the subcapsular cortex zone located on the pole opposite to the entrance of nerves should be chosen. In addition, comparable results in the stereological examination of the medulla can be obtained only if taking its areas similar in location. The pronounced relationship in the arrangement of A and NA cells with nerves clearly indicates that in vivo nerve factors play a key role in differentiation and stabilization of the A and NA cells phenotypes.     

Endothelin-1, endothelin converting enzyme-1 and endothelin receptors in the porcine corpus luteum

Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2α (PGF-2α) (ie, luteolytic sensitivity [LS]) until about day 12-13 of the estrous cycle. Although little is known of the control of LS in any species, endothelin-1 (EDN1) is believed to play a role in LS control in ruminants. Therefore, we measured mRNA and protein expression and examined the cellular localization of EDN1 precursor (pre-pro EDN1, or ppEDN1), EDN-converting enzyme-1 (ECE1), and EDN receptors (A, EDNRA and B, EDNRB) in porcine CLs collected on days 4, 7, 10, 13, and 15 of the estrous cycle to look for differences between CLs displaying (days 13-15) versus those lacking (days 4-10) LS. Abundance of ppEDN1 mRNA was greatest (and significant vs all other days) on day 7 of the cycle, whereas EDN1 protein expression did not vary during the cycle and was localized exclusively to endothelial cells (EC). Abundance of ECE1 mRNA was also greatest on day 7 (vs all other days), but ECE1 protein was significantly elevated on day 10 (vs day 4) and was immunolocalized to ECs and large luteal cells (LLC). Abundance of EDNRA mRNA was also maximal on day 7 (vs all other days) of the cycle, whereas EDNRA protein expression was not significantly changed during the cycle and was observed in LLCs, ECs, and small luteal cells (SLC). On day 13, EDNRB mRNA was significantly decreased (versus day 7). Expression of EDNRB protein was decreased on day 10 (versus all other days), and on days 13-15 (vs day 4), and was primarily localized to ECs. In conclusion, the observed elevation in ECE1 protein concentrations on day 10 and the presence of EDNRA on LLC suggests a possible role for EDN1 (resulting from the actions of ECE1) acting via EDNRA in the control of LS in the pig.     

Undetectable vitamin D3 in equine skin irradiated with ultraviolet light

Vitamin D requirements for most animals are expected to be fulfilled through daily exposure of the skin to solar ultraviolet B radiation. The synthesis of vitamin D₃ in skin depends on different factors including melanin pigmentation, the amount of UVB radiation reaching the skin, type of clothing/hair coat, latitude and altitude, season, and time of day. Alternatively vitamin D₂ may be obtained from UVB irradiated pasture species. Recent studies have shown that in unsupplemented grazing horses 25-hydroxyvitamin D₂ is the predominant form of vitamin D in plasma, and that 25OHD₃ is undetectable suggesting horses may rely on diet to obtain vitamin D. In order to mimic the natural environment of skin to sunlight exposure, five equine and two ovine devitalized skin samples were irradiated with 5 J/cm² of UVB light followed by measurement of 7-dehydrocholesterol (7-DHC) and vitamin D₃ concentrations using reverse-phase high pressure liquid chromatography (HPLC). HPLC revealed the presence of 7-DHC in the skin of both horses and sheep. Vitamin D₃ was undetectable in both ovine and equine skin prior to irradiation, but after irradiation with UVB light, ovine skin showed an increase in vitamin D₃ concentration (mean 0.16 ± 0.07 µg/g), whereas vitamin D₃ was undetectable in equine skin. These results provide additional evidence that horses make negligible quantities of vitamin D₃ in their skin after exposure to UVB light and may therefore rely on their diet as a primary source of vitamin D.     

Cross-reactivity of a monoclonal antibody with bovine, equine, ovine, and porcine peripheral blood B lymphocytes

Ficoll-thrombin purified suspensions of bovine, equine, ovine, and porcine peripheral blood lymphocytes were fractionated on nylon-wool columns. The percentages of surface immunoglobulin (SIg+)-bearing lymphocytes in the adherent (B-cell enriched) and nonadherent (T-cell enriched) fractions were determined for individual animals using fluorescein isothiocyanate conjugated species-specific anti-Ig sera. Subsequently, the human leukocyte antigen DR-specific monoclonal antibody, H4, was tested for its ability to recognize a cross-reactive antigen on the fractionated lymphocytes, using the microcytotoxicity technique. The H4 plus complement killed a percentage of lymphocytes equivalent to the percentage of SIg+ lymphocytes in the adherent and nonadherent fractions. In a parallel experiment, a 2 fluorochrome technique was used to visualize bovine lymphocytes that were SIg+ and H4+. Lymphocytes that were SIg+ also stained with ethidium bromide (orange fluorescence) after complement-mediated cytotoxicity. Seemingly, H4 recognizes an evolutionarily conserved major histocompatibility complex encoded class-II-like determinant on the B lymphocytes of cattle, horses, sheep, and swine.