Enhancement of enrofloxacin serum antibacterial activity by calcium primed broilers

The aim of this trial was to assess the effect that calcium gluconate priming of 468 broilers has on the antibacterial activity of a standard dose of enrofloxacin. Hence, a series of oral pharmacokinetic studies were carried out in four groups of broilers medicated individually through an oral cannula as follows: group A, medicated only with enrofloxacin 10mg/kg; group B, receiving immediately one after the other, calcium gluconate (200mg/kg) and enrofloxacin 10mg/kg; group C, dosed first with calcium gluconate (200mg/kg) and 1h later enrofloxacin (10mg/kg); and group D, dosed first with calcium gluconate (200mg/kg) and 2h later enrofloxacin (10mg/kg). Broilers were bled at different times after the dose of enrofloxacin and antibacterial activity, measured as concentration of enrofloxacin, was measured by an agar diffusion assay. Results revealed that group D the greatest values of maximum serum concentration (Cs(max)), area under the concentration vs. time curve (AUC) and area under the moment curve (AUMC). These values were statistically higher than the corresponding ones derived from groups A, B and C (P<0.05). Taking Cs(max) and AUC values of group A as reference baseline, an increase of 24% and 50%, respectively, was obtained in group D. Group B had the lowest Cs(max), AUC, AUMC and elimination half life (T(1/2)beta) and these values were statistically different from groups A, C and D (P<0.05). The T(1/2)beta was statistically longer in groups C and D as compared with A and B, and the former groups were also different between each other (P<0.05). These results show that if calcium gluconate is first dosed to broilers and 2h later enrofloxacin is administered (as in group D), a more pronounced antibacterial activity of enrofloxacin can be obtained. A challenge of this sequential dosing scheme in a field trial may reveal its clinical value.     

Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

Evaluation of Methods to Determine Sperm Density for the European eel,Anguilla anguilla

European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer‐assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G‐forces, were tested and the best G‐force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 ×  g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA‐1 and CASA‐2), CASA‐2 showed a very high accuracy to haemocytometer counts (r  =  0.93), but low precision (CV: CASA‐2 = 28.4%). FCM was tested with and without microfluorospheres (FCM‐1 and FCM‐2), and relationships to haemocytometer counts were highly accurate (FCM‐1: r  =  0.94; FCM‐2: r  =  0.88) and precise (CV: FCM‐1 = 2.5; FCM‐2 = 2.7%). Overall, CASA‐2 and FCM‐1 feature reliable methods for quantification of European eel sperm, but FCM‐1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.     

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.     

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.     

ORIGINAL ARTICLE: Effects of herbage ingestion upon ileal digestibility of amino acids in heavy Iberian pigs fed on an acorn‐based diet

We conducted two experiments with heavy Iberian pigs to determine the ileal digestibility of amino acids (AA) in acorns and freshly cut herbage, and the effects of adding fresh herbage upon the supply of ileal digestible AA when pigs were fed on holm‐oak acorns. In Experiment 1, carried out in cannulated pigs of 107 kg bodyweight (BW), daily intake of acorns reached 44.9 g DM/kg^0.75 BW. Arg, His and Thr showed the lowest apparent ileal digestibility (AID) values, whereas Met, the branched‐chain AA and Phe had the highest coefficients. The AID of total EAA was 0.716 but only 0.222 for NEAA. Most of the digestive and absorptive processes of acorn protein occurred before the hindgut. Acorn provides (per kg DM) 2.27 g apparent ileal digestible Lys and 22.7 g apparent total digestible AA. Standardized ileal digestibility (SID) values for EAA, NEAA and total AA were 0.924 ± 0.020, 0.784 ± 0.041 and 0.860 ± 0.029. In Experiment 2 fresh herbage was given to six cannulated Iberian pigs of 140 kg either as a single feed (13.7 g DM/kg^0.75 BW) or as a supplement to acorns (28.4 g DM/kg^0.75 BW). When only freshly cut forage was offered the AID of the EAA, NEAA and total AA was close to 0.65 and supplied (per kg DM ingested) 5.61 g AID Lys and 91.7 g digestible AA. Standardized ileal values were 0.744 ± 0.023, 0.912 ± 0.038 and 0.831 ± 0.030 respectively. The addition of fresh forage to the acorns led to a significant decrease in AID of AA in acorn due to digesta transfer to the hindgut: His (p < 0.01), Met (p < 0.001), Phe (p = 0.092), Thr (p < 0.05) and Val (p < 0.05), but Arg, Lys and the branched‐chain AA remained unaffected. The main contribution of herbage to AA nutrition of the grazing Iberian pig relies mainly on increasing the supply of digestible AA for pig tissues.     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.     

Supercritical fluid extraction of pesticides from a table-ready food composite of plant origin (gazpacho)

Supercritical fluid carbon dioxide extraction (SFE) has been evaluated for the extraction of 17 organohalogen and organophosphate pesticides in gazpacho (a table-ready food composite containing crude vegetables, white bread, vegetable oil, water, and other minor components) using anhydrous magnesium sulfate as drying agent. The effects of different parameters, such as fat content in gazpacho composites, magnesium sulfate/gazpacho ratio, supercritical fluid volume, pressure, temperature, and static modifier additions, on SFE recoveries from spiked gazpacho samples have been studied. Analyses were performed by gas chromatography (GC) with flame photometric (FPD), electron capture (ECD), and mass spectrometry (MSD) detectors. In most experiments, recoveries obtained for the nonpolar organohalogen pesticides were lower than those obtained for the most polar organophosphate pesticides, but overall pesticide recoveries determined by using the optimal SFE conditions indicate that SFE could be used to determine pesticide residue levels in gazpacho.