Morphologic evaluation of IgM cells of the canine small intestine by fluorescence microscopy.

Biopsies of small intestine from 7 dogs were examined by fluorescence microscopy to determine the number of IgM-containing cells in the lamina propria. Biopsies were taken from duodenum, jejunum, and ileium. (Cell counts were made by 2 persons to demonstrate reproducibility.) There were 452.24 +/- 60.09 cells per mm2 in the duodenum 572.68 +/- 62.13 cells per mm2 in the jejunum, and 107.47 +/- 59.57 cells per mm2 in the ileum. All sections were cut at 6 micrometer. The ileum had fewer cells than either duodenum or jejunum (P = 0.000038 and 0.00001, respectively), whereas duodenum and jejunum did not differ significantly in numbers of cells (P = 0.17528). Quantifying autofluorescent cells in the same sites showed no significant differences among the 3 tissues (P = 0.24697). The autofluorescent cells differed in intensity and morphology from the IgM cells. These two observations tend to support the contention that the autofluorescent cells did not bias the IgM cell counts at the 3 sites. Total autofluorescence (cells, collagen, and vessels) was higher in the ileum than in either the jejunum or the duodenum (P = 0.04967 and 0.03050, respectively). However, all 3 categories counted (IgM cells, autofluorescent cells, and autofluorescent structures) had significant dog-tissue interactions. This will necessitate determining normals for each age-sex-breed category of dog studied.     

Potential role of G-protein-coupled receptor 30 (GPR30) in estradiol-17beta-stimulated IGF-I mRNA expression in bovine satellite cell cultures

Androgenic and estrogenic steroids enhance muscle growth in animals and humans. Estradiol-17beta (E2) and trenbolone acetate (TBA) (a synthetic testosterone analog) increased IGF-I mRNA expression in bovine muscle satellite cell (BSC) cultures. The goal of this study was to evaluate the mechanisms responsible for this increase by evaluating the effects of ICI 182 780 (an E2 receptor antagonist), flutamide (an androgen receptor inhibitor), G1 (a GPR30 agonist), and BSA-conjugated E2 on E2 and/or TBA-stimulated IGF-I mRNA expression in BSC cultures. Flutamide completely suppressed TBA-stimulated IGF-I mRNA expression in BSC cultures. ICI 182 780 did not suppress E2-stimulated IGF-I mRNA expression and 100nM ICI 182 780 enhanced (93%, p<0.05) IGF-I mRNA levels in BSC cultures. G1 (100nM) stimulated IGF-I mRNA expression (100%, p<0.05) but had no effect on proliferation in BSC cultures. E2-BSA, which cannot cross the cell membrane, stimulated IGF-I mRNA expression (approximately 100%, p<0.05) in BSC but even at extremely high concentrations had no effect on proliferation. In summary, our data indicate the E2-stimulation of proliferation and E2-stimulation of IGF-I mRNA expression in BSC cultures occur via different mechanisms. Our previous results showing that ICI 182 780 inhibited BSC proliferation and results of the current study showing lack of response to E2-BSA or G1 suggest that E2-stimulated proliferation in BSC cultures is mediated through classical estrogen receptors. Stimulation by ICI 182 780, G1 and E2-BSA suggests the E2-stimulated IGF-I mRNA expression in BSC cultures is mediated through the GPR30 receptor.     

Calves Born after Direct Transfer of Vitrified Bovine In Vitro-produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super‐cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non‐vitrified control group, (ii) vitrified in normal (?196°C) liquid nitrogen (LN₂) and (iii) vitrified in super‐cooled LN₂ (≤?200°C). Open‐pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro‐matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN₂ state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super‐cooled LN₂, resulted in viable blastocysts and live calves following in vitro embryo production.     

Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-I, and sera on protein turnover in porcine myotube cultures

Porcine myogenic cells isolated from 50 to 55-d porcine fetuses were frozen and stored in liquid nitrogen until they were needed to establish cultures. Approximately 75.8 +/- .59% of the clonal cultures established from these frozen stocks produced myotubes and 60.8 +/- 2.3% of the nuclei in differentiated mass cultures were in myotubes. Differentiated cultures contained higher levels of creatine phosphokinase activity than undifferentiated cultures. Additionally, differentiated cultures incorporated [35S]methionine into putative myosin heavy chain, alpha-actinin, and actin more rapidly than did undifferentiated cultures. Insulin, insulin-like growth factor I, and sera stimulated total protein synthesis rate and decreased total protein degradation rate in myotube cultures. Based on our initial characterization, we believe that we have developed an effective and practical procedure for isolating and culturing fetal porcine myogenic cells.     

Circulating Metabolic Hormones During the Peripubertal Period and Their Association with Testicular Development in Bulls

The objective of the present study was to characterize changes in serum metabolic hormones concentrations from 20 weeks before to 20 weeks post-puberty in bulls and to investigate the associations of metabolic hormones concentrations with testicular development. Leptin concentrations increased from 16 weeks before puberty to 8 weeks post-puberty and insulin concentrations increased from puberty to 8 weeks post-puberty. Growth hormone concentrations decreased after 4 weeks post-puberty, whereas IGF-I concentrations increased from 8 weeks before puberty to 8 weeks post-puberty. During this period, testicular growth was accelerated and testosterone secretion increased substantially, without any significant changes in gonadotropin secretion. Monthly circulating concentrations of leptin, IGF-I and insulin accounted for 63% of the variation in scrotal circumference and 59% of the variation in paired testes volume. In conclusion, the secretion of metabolic hormones was not associated with changes in gonadotropins concentrations. Furthermore, the associations of leptin, IGF-I and insulin concentrations with testes size indicated that these hormones might be involved in a gonadotropin-independent mechanism regulating the testicular development in peripubertal bulls.