Transforming growth factor type beta (TGF-beta) and adipogenesis in pigs

The present study was performed on s.c. adipose tissue of fetal pigs at 35 to 110 d of gestation to examine the distribution of TGF-beta-positive cells, to localize TGF-beta immunoreactivity at the cellular level using electron microscopy (EM), and to determine the effect of TGF-beta on primary cultures of pig adipose tissue cells. Tissues for EM were fixed and embedded in LR white resin. Sections then were incubated with a polyclonal antibody specific for TGF-beta and TGF-beta was located using 20 nm colloidal gold conjugated second antibody. Tissues were fixed and embedded in paraffin for localization of TGF-beta at the light microscope (LM) level. Tissues were incubated with anti-TGF-beta followed by localization using biotinylated second antibody. Using LM, only a few cells stained positively for TGF-beta within developing blood vessels at 35 d. By 50 d, more TGF-beta-positive cells were associated with forming capillary networks. Between 70 d and 110 d, positively stained adipocytes usually were clustered around blood vessels. Cells surrounding hair follicles stained positive for TGF-beta between 90 to 110 d. Electron microscopy revealed TGF-beta labeling within fat cells. Fibroblasts and endothelial cells did not exhibit TGF-beta immunoreactivity. The addition of TGF-beta to primary cultures of s.c. adipose tissue cells from newborn pigs prevented lipid filling in fat cells. This effect was dose-dependent, with half-maximal inhibition occurring at 3 pM maximum inhibition occurred at 40 pM. These results indicate that TGF-beta may regulate angiogenic activity and lipid filling in s.c. adipose tissue of fetal pigs. Although TGF-beta was present in adipocytes and in cells associated with developing capillary networks, the physiological role of TGF-beta during early adipose tissue development is not known.     

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Foetal Fluids

Plasma concentrations of PAG‐1 are used for pregnancy diagnosis and as a marker of placental/foetal well‐being, while those of PAG‐2 may be an indicator of abortion risk in Neospora caninum‐infected cows. Studies have shown that N. caninum infection modifies PAG‐1 and PAG‐2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG‐1, PAG‐2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG‐2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non‐infected foetuses. In infected foetuses, significantly higher concentrations of PAG‐1 (p < 0.001) and PAG‐2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non‐infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG‐1 and PAG‐2 levels and pH values than fluids from non‐infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.     

Reproductive biology of pond reared Nile tilapia, Oreochromis niloticus L.

The reproductive performance of female Nile tilapia, Oreochromis niloticus L., reared in ponds under tropical conditions was studied. The experiments used stock matured to the age of 2–4 months and a size of 30–50 g. Oocyte development proved a better indicator for the determination of the size at first maturation than the development of the gonado-somatic index. The Nile tilapia became ‘stunted' in the sense that early breeding took place, but the somatic growth of the stock still continued. This result indicates that the earlier breeding of Nile tilapia is not a real bottleneck for the expansion of this production sector. No relationship was found between the condition factor and the size at first maturation or the relative fecundity. This led to the conclusion that aggravation of the living conditions is not a major cause of earlier breeding of pond-reared O. niloticus.     

Biology of leptin in the pig

The recently discovered protein, leptin, which is secreted by fat cells in response to changes in body weight or energy, has been implicated in regulation of feed intake, energy expenditure and the neuroendocrine axis in rodents and humans. Leptin was first identified as the gene product found deficient in the obese ob/ob mouse. Administration of leptin to ob/ob mice led to improved reproduction as well as reduced feed intake and weight loss. The porcine leptin receptor has been cloned and is a member of the class 1 cytokine family of receptors. Leptin has been implicated in the regulation of immune function and the anorexia associated with disease. The leptin receptor is localized in the brain and pituitary of the pig. The leptin response to acute inflammation is uncoupled from anorexia and is differentially regulated among swine genotypes. In vitro studies demonstrated that the leptin gene is expressed by porcine preadipocytes and leptin gene expression is highly dependent on dexamethasone induced preadipocyte differentiation. Hormonally driven preadipocyte recruitment and subsequent fat cell size may regulate leptin gene expression in the pig. Expression of CCAAT-enhancer binding protein (C/EBP) mediates insulin dependent preadipocyte leptin gene expression during lipid accretion. In contrast, insulin independent leptin gene expression may be maintained by C/EBP auto-activation and phosphorylation/dephosphorylation. Adipogenic hormones may increase adipose tissue leptin gene expression in the fetus indirectly by inducing preadipocyte recruitment and subsequent differentiation. Central administration of leptin to pigs suppressed feed intake and stimulated growth hormone (GH) secretion. Serum leptin concentrations increased with age and estradiol-induced leptin mRNA expression in fat was age and weight dependent in prepuberal gilts. This occurred at the time of expected puberty in intact contemporaries and was associated with greater LH secretion. Further work demonstrated that leptin acts directly on pituitary cells to enhance LH and GH secretion, and brain tissue to stimulate gonadotropin releasing hormone secretion. Thus, development of nutritional schemes and (or) gene therapy to manipulate leptin secretion will lead to practical methods of controlling appetite, growth and reproduction in farm animals, thereby increasing efficiency of lean meat production.     

Effect of the Type and Number of Prostaglandin Treatments on Corpus Luteum, the Largest Follicle and Progesterone Concentration in Dairy Cows

Lactating dairy cows (n = 72) with a mature corpus luteum (CL) (diameter of > or = 17 mm) determined by ultrasonography and having a follicle with a diameter of > or = 10 mm were randomly assigned to four groups. Cows were treated with cloprostenol i.m. once or twice, or with dinoprost i.m. once or twice with an 8-h interval. The ovaries of each cow were scanned daily by transrectal ultrasonography to measure the changes in the areas of CL and the largest follicle and to determine the occurrence of ovulation. Oestrus was verified twice daily. In addition, blood sample was withdrawn from each cow daily for measuring progesterone (P4) concentrations. Significant decreases in the percentage changes relative to areas of CL and P4 concentrations or increases in the percentage changes in the area of the largest follicle on day 0 were detected in each group during the experiment. However, the type of the drug and the number of the treatments had no significant effect on those parameters. Cows ovulated with or without showing oestrus (group A) and cows exhibiting no oestrus and ovulation (group B) were also evaluated. In contrast to the mean area of the CL and the mean concentration of P4 on day 0, the mean area of the largest follicles between the two groups on day 0 differed significantly. Significant decreases in the percentage changes relative to the area of the CL and P4 concentration or increases in the percentage changes relative to the area of the largest follicle during the experiment were detected in both groups; however, there were no group differences. Treatment of dairy cows with two injections of prostaglandins (cloprostenol or dinoprost) at an 8-h interval resulted in more cows being observed in oestrus within 5 days after treatment and having significantly higher pregnancy rate than those treated with a single prostaglandin injection.     

In Vitro Fertilization and Development of OPU Derived Goat and Sheep Oocytes

The recent upgrade in IVP technology seen in cattle can be adapted to embryo production in small ruminants to overcome limitations exhibited by surgical procedures on preserving the reproductive potential of donors and the efficiency of embryo production. The aim of the present study was to assess the current procedures used in cattle for the production of IVP embryos in goats and sheep based on laparoscopic-aided ovum pick-up (LOPU) supplied oocytes. Sexually matured goat and sheep donors were treated during the breeding season with FSH and subjected to laparoscopic-guided follicular puncture under general anaesthesia. The collected cumulus-oocyte complexes were matured in medium 199 and fertilized by frozen-thawed spermatozoa using Talp medium supplemented with heparin and oestrus-sheep serum. Cleaved ova were either cultured in sheep in vitro fertilization medium plus amino acids or transferred to sheep oviducts. Blastocyst rate, hatching rate and development rate up to term were used as markers of embryo function. The results obtained for goat and sheep involving 30 and 35 donors respectively (10 and 9 LOPU sessions) were 81.2% and 85.2% of oocyte collection rate; 88.3% and 98.6% oocyte incubation rate; 85.6% and 76.0% fertilization rate; 82.4% and 93.4% of cleavage rate; 50.0% and 61.5% IVP blastocyst rate; 42.1% and 45.5% blastocyst rate in oviducts; 73.0% and 66.7% embryo survival up to term, respectively. The results are comparable to those obtained in small ruminants and in bovines suggesting that requirements for embryo production and development are similar.     

Aspartic Proteinase Members Secreted by the Ruminant Placenta: Specificity of Three Radioimmunoassay Systems for the Measurement of Pregnancy-associated Glycoproteins

Pregnancy‐associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml – 1 mg/ml). Pepsinogen cross‐reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 μg/ml and 500 μg/ml concentrations, respectively. In the presence of pepsin, cross‐reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 μg/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross‐react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross‐reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids.     

Vacuum Squeezing of Solids: Macroscopic Quantum States Driven by Light Pulses

Femtosecond laser pulses and coherent two-phonon Raman scattering were used to excite KTaO3 into a squeezed state, nearly periodic in time, in which the variance of the atomic displacements dips below the standard quantum limit for half of a cycle. This nonclassical state involves a continuum of transverse acoustic modes that leads to oscillations in the refractive index associated with the frequency of a van Hove singularity in the phonon density of states.     

Histochemical studies of muscle development in decapitated and hypophysectomized pig fetuses: blood vessel development

In Exp. 1, fetuses were decapitated in utero on d 45 of gestation and examined at 110 d of gestation; in Exp. 2, fetuses were hypophysectomized (hypox) in utero with a cauterizing needle at d 70 of gestation and examined at 110 d of gestation. Semitendinosus muscles were sampled and transverse cryostat sections were cut from the medial portion of the muscle. Quantitative analysis of lectin-stained sections showed that capillary:fiber ratios (C/F) were lower (P less than .01) for sections from decapitated and hypox fetuses than for sections from control fetuses. The superficial and deep aspects of muscle sections from control fetuses had markedly different C/F ratios (deep region, 24.1 +/- 2; superficial region, 11.2 +/- .5). Fetal decapitation and hypophysectomy abolished differences in C/F ratios between deep and superficial regions of muscle sections (hypox fetuses; deep region, 6.9 +/- 1; superficial region, 6.4 +/- .6). Capillaries in sections from control fetuses were reactive for several enzymes, whereas capillaries in sections from experimental fetuses (decapitated and hypox) were not reactive for these enzymes. The number of small arterioles was reduced in sections from experimental fetuses (decapitated and hypox) compared with sections from control fetuses. These studies demonstrate that lowering the levels of pituitary hormones in the fetus retards the development of blood vessels in muscle tissue both quantitatively and qualitatively.