Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Preliminary assessment of somatic cell nuclear transfer in the dromedary (Camelus dromedarius)

Somatic cloning may enable the maintenance/expansion of the population of camels with the highest potential for milk production or the best racing performances. However, there have been no reports of embryonic or somatic nuclear transfer in camels. The aim of this study was to produce dromedary embryos by nuclear transfer using in vitro matured oocytes and two somatic cells from two sources (adult fibroblasts or granulosa cells). A total of 58 adult females were superstimulated by a single dose of eCG (3500 IU). Ten days later, their ovaries were collected postmortem. Cumulus–oocytes-complexes (COCs) were aspirated from stimulated follicles and were matured in vitro for 30 h. Fibroblasts (from live adult male) and granulosa cells (from slaughtered adult females) were used as donor karyoplasts and injected into mature enucleated dromedary oocytes.The cleavage rate was significantly higher (P < 0.05) for embryos reconstructed with fibroblasts (59%) versus those with granulosa cells (45%). However, there was no difference between the two groups in the proportion of cloned embryos reaching the blastocyst stage (fibroblasts: 14% vs. granulosa cells: 15%) or those that hatched (fibroblasts: 10% vs. granulosa cells: 12%). The viability of reconstructed dromedary embryos from the two sources of donor cells (fibroblasts; n = 5 vs. granulosa cells; n = 7) was examined by transferring them to synchronized recipients. Two females (fibroblasts: 1/5; 20%, granulosa cells: 1/7; 14%) were confirmed pregnant by ultrasonography at 15 and 25 days following transfer. Later, the pregnancies were followed by pregnancy empirical-symptoms. These two pregnancies were lost between 25 and 60 days following transfer, respectively.In conclusion, the present study shows for the first time that the development of dromedary NT embryos derived from either adult fibroblasts or granulosa cells can occur in vitro and the transfer of these cloned embryos to recipients can result in pregnancies.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Visceral and Subcutaneous Fat Increased after Treatment of Graves Disease

Objective: Patients with Graves disease (GD) tend to gain weight after treatment, but it remains unknown if weight gain is associated with an increase in the visceral and/or subcutaneous fat areas (VFA, SFA).Methods: We enrolled 25 newly diagnosed GD patients (22 females, median age 33.0 years) and studied their clinical parameters, and VFA and SFA measured by a dual bioelectric impedance analysis. We divided them into 2 groups based on the rates of change in the VFA and SFA, and we compared clinical parameters at the baseline between the groups to evaluate factors that influence increases in the VFA and/or SFA with treatment.Results: The patients' body weight (BW), VFA, and SFA were significantly increased after a 6-month treatment (BW: from 54.3 ± 10.3 kg to 58.0 ± 11.2 kg; P<.001; VFA: from 47.1 ± 21.3 cm² to 54.7 ± 23.4 cm²; P = .004; SFA: from 159.8 ± 85.9 cm² to 182.2 ± 82.9 cm²; P = .008). The percent changes of BW correlated with the SFA (ρ = .591, P = .002), but not with the VFA. The patients with larger VFA increases had significantly less VFA at the baseline compared to those with smaller increases, expressed as median and interquartile range (33.9 cm² [22.7 to 47.5 cm²] versus 54.5 cm² [45.2 to 64.0], respectively; P = .011). A larger increase in the SFA was negatively associated with serum alkaline phosphatase. An increase in the SFA was associated with free triiodothyronine (T3) in a multivariate logistic analysis (odds ratio: 0.80 [0.59 to 0.97]; P = .013).Conclusion: The patients' BW, VFA, and SFA were increased after GD treatment. The increase in SFA seemed to contribute to weight gain and was associated with a low baseline level of free T3.Abbreviations: ALP = alkaline phosphatase; BMI = body mass index; BW = body weight; GD = Graves disease; SFA = subcutaneous fat area; T3 = triiodothyronine; T4 = thyroxine; TG = triglycerides; VFA = visceral fat areas     

Effects of PEGylated porcine glucagon-like peptide-2 therapy in weaning piglets challenged with lipopolysaccharide

This study aims to evaluate the therapeutic effect of polyethylene glycosylated porcine glucagon-like peptide-2 (pGLP-2), a long-acting form of pGLP-2, in lipopolysaccharide (LPS)-challenged piglets. Eighteen 21-day-old weaning piglets were randomly assigned into three groups: control (saline solution), LPS (100 μg/kg LPS), and PEG–pGLP-2 (10 nmol/kg PEG–pGLP-2 + 100 μg/kg LPS). All treatments were administered intraperitoneally. Compared with the control treatment, LPS treatment significantly decreased (P < 0.05) the villus heights of the duodenum and jejunum, as well as the villus height/crypt depth ratio of the jejunum. However, PEG–pGLP-2 therapy reduced these effects (P > 0.05). Specifically, PEG–pGLP-2 infusion significantly increased the villus height/crypt depth ratio of the duodenum (P < 0.05) compared with LPS treatment. Compared with the control treatment, LPS treatment significantly increased (P < 0.05) the mRNA expression levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the jejunum. However, PEG–pGLP-2 therapy reduced these effects (P < 0.05). Specifically, PEG–pGLP-2 infusion significantly decreased (P < 0.05) the mRNA expression levels of interleukin (IL)-8 and TNF-α in the duodenum and jejunum, IL-10 in the duodenum, and IFN-γ in the jejunum compared with the LPS treatment. LPS treatment increased the caspase-3 activity of the ileum mucosal (P < 0.05), and this effect was significantly reduced by PEG–pGLP-2 treatment. These results indicate that PEG–pGLP-2 infusion alleviates the severity of intestinal injury in weaning piglets by reducing the secretion of inflammatory cytokines and the caspase-3 activity, and increasing the villus height/crypt depth ratio.     

The influence of drinker location and colour on drinking behaviour and water intake of newborn pigs under hot environments

The effect of drinker location within the farrowing crate in relation to its microenvironment and the effect of dispenser colour on drinking behaviour and water intake of newborn pigs for the first two days of their lives were studied. In the first trial, 16 sows were randomly assigned to four treatment groups with the piglet's water dispenser placed in the front right (FR), front left (FL), back right (BR) or back left (BL) corner of the farrowing pen, respectively. In the second trial nine sows were randomly assigned to three treatment groups with the piglets’ water dispenser, placed in the front left corner of the farrowing pen, and coloured as red (CR), green (CG) or blue (CB), respectively. Trials commenced between 08:00 and 10:00 h on the first morning after the litters were born and the piglets’ age was considered to be zero (“age”) by that time.The average age of the piglets at their first visit to water dispensers was 15.9 ± 1.8 h. The newborn pigs visited the drinker more frequently (P < 0.001) during daytime than during night, with two peaks soon after sow feeding time. The duration of drinking time was long at the time of higher (noon) and shorter at the time of lowest (early morning) temperatures (P < 0.01) in the farrowing house, respectively. The greatest attention at the drinkers occurred soon after suckling (P < 0.001). The longest time spends at the drinker during the first visits and decreased as the pigs visited dispensers repeatedly. The profile of visit frequency and duration differed between piglet sexes (P < 0.001). The pigs that did not visit the drinker grew slower and were lighter at 48 h of age than the pigs visited the drinker. First visit and overall mean visit age were significantly shorter for BL than for FR and BR groups. Piglets visited the FL drinker for shorter time than the others. Water consumption per pig/24 h was lower for FL and higher for BL groups (P < 0.05). Water consumption was higher for CR and CB groups than for CG group. The overall mean visit age was shorter for CG than for CB group. Significant interactions were observed between sex and colour groups for the number of visits (P < 0.05). The results of the present study revealed that water dispenser use by newborn piglets is characterised by diurnal and between two successive milk consumptions distributions. Water consumption by piglets from birth to 48 h of their lives is strongly influenced by water dispenser location in the farrowing pen and the dispenser colour. It also appears that males and females behave differently to the three colour dispensers used in this study.     

The effect of providing shredded paper or ropes to piglets in farrowing crates on their behaviour and health and the behaviour and health of their dams

The objective of this study was to evaluate the effect of providing paper or rope, alternative enriching substrates to straw, to piglets in farrowing crates on piglet and sow welfare.Sixty multiparous sows and their litters were housed in crates that were either barren (BARREN), enriched with shredded paper (PAPER) or natural fibre rope (ROPE). Enriching substrates were introduced when piglets were 10 days old. The proportion of sows with udder and teat lesions before parturition and at weaning was recorded. Piglet facial lesions were scored according to severity on days 11, 18 and 27. Sow and piglet behaviour was recorded using scan sampling on days 14, 18, 22 and 26. Furthermore, the behaviour of one male and one female focal piglet per litter was recorded continuously for 10 min twice per day on days 14, 18, 22 and 26. On day 27 post-partum, focal piglets were observed for 5 min in a novel arena and for a further 5 min after a novel object was introduced.On day 27, there was a tendency for more BARREN sows to have teat lesions (P = 0.07). PAPER litters tended to have a smaller proportion of piglets with facial lesions (P = 0.06). ROPE piglets were active in the enriched area of the crate in more observations than BARREN and PAPER piglets (P < 0.01). PAPER piglets spent less time inactive (P < 0.01), less time exploring the pen-fittings (P < 0.01) and more time interacting with the enriching substrate (P < 0.001) than piglets in the other two treatments. In the first 5 min in the novel arena BARREN piglets froze more than PAPER and ROPE piglets (P = 0.05).In conclusion, shredded paper improved piglet welfare and was easily incorporated into the farrowing crates.     

Study of porcine hepatocyte-entrapped bioartificial liver in surgery-induced fulminant hepatic failure rabbits

ICP is an important cause of mortality in fulminant hepatic failure. The aim of this study was to prolonged survival in surgery-induced FHF rabbit with BAL. Four hours after induction of FHF, rabbits were connected to a 4-h whole blood perfusion through the BAL containing 1.2 × 10⁹ fresh isolated porcine hepatocytes (estimated 30% of liver volume of rabbit) via an arterial-venous shunt. Survival times in control group, sham-BAL group and BAL group were14.4 ± 4.4 h, 12.6 ± 2.6 h and 22.6 ± 5.2 h, respectively (BAL vs. control, p = 0.026; BAL vs. sham-BAL, p = 0.006). BAL group remain ICP level less than 10 mmHg for near 13 h after FHF induction while 19 and 15 mmHg were observed in control and sham-BAL groups, respectively. BAL containing porcine hepatocytes significantly prolonged survival time by delayed increase of intracranial pressure compared with the control and sham-BAL groups.     

Correlations between ovarian follicular blood flow and superovulatory responses in ewes

The primary goal of this study was to employ ultrasonography to examine the ovaries of ewes undergoing superovulatory treatment for correlations between antral follicular blood flow and ovarian responses/embryo yields. Five Santa Inês ewes were subjected to a short- (Days 0–6, Group 1) and five to a long-term progesterone-based protocol (Days 0–12, Group 2) to synchronize estrus and ovulations after the superovulatory treatment. Porcine FSH (pFSH, 200 mg) was administered in 8 decreasing doses over 4 days, starting on Days 4 and 10 in Groups 1 and 2, respectively. After CIDR removal, all ewes were bred by a ram and embryos were recovered surgically 7 days later. Transrectal ovarian ultrasonography was performed the day before and on all 4 days of the superovulatory treatment. Both an arbitrary-scale [(0) non-detectable; (1) small; (2) moderate; (3) intense blood flow] and quantitative analysis of the blood flow area were used to assess the follicular blood flow in color Doppler images. There were no significant correlations between the arbitrary blood flow scores and superovulatory responses in the ewes of the present study. However, there was a positive correlation between the quantitative estimates of follicular blood flow on the final day of the superovulatory treatment, and the number (DA: r = 0.68, P < 0.05; DA/TA × 100%: r = 0.85, P < 0.05) and percentage (DA: r = 0.65, P < 0.05; DA/TA × 100%: r = 0.91, P < 0.001) of unfertilized eggs (DA: Doppler area, TA: total area of the largest ovarian cross section). This experiment presents a commercially practical tool for predicting superovulatory outcomes in ewes and evidence for the existence of follicular blood flow threshold that may impinge negatively on oocyte quality when surpassed during hormonal ovarian superstimulation.     

Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein

This study was performed to develop a system for porcine somatic cell nuclear transfer (SCNT) and to produce human erythropoietin (hEPO)-transgenic cloned piglets. Porcine fetal fibroblasts were transfected with an expression plasmid (phEPO-GFP). In Experiment 1, the effect of transfection of phEPO-GFP transgene on development of porcine SCNT embryos was investigated. Three fetal fibroblast cell lines (two male and one female) with or without transfected with phEPO-GFP trasngene were used as donor cells for SCNT. Lower fusion rates were observed in two lines of transfected cells as compared to those of the control cells. In Experiment 2, the effect was examined of elevated Ca2+ concentration in the fusion/activation medium on development of transfected SCNT embryos. The rates of fusion and blastocyst formation were significantly increased by supplementing 1.0 mM of CaCl2 (versus 0.1 mM) into the fusion/activation medium. In Experiment 3, the effect was studied of a chemical treatment (cytochalasin B) after electric fusion/activation (F/A) on porcine transgenic SCNT embryo development. The electric F/A + cytochalasin B treatment increased total cell number in blastocysts as compared to that of electric F/A treatment alone. In Experiment 4, transgenic cloned embryos were transferred to surrogate mothers and a total of six cloned piglets were born. Transgenic cloned piglets were confirmed by polymerase chain reaction and Southern blot analysis. From a single surrogate mother, female and male transgenic cloned piglets were produced by transferring pooled SCNT embryos derived from female and male transfected donor cells. In conclusion, a system for porcine SCNT was developed and led to the successful production of hEPO transgenic cloned piglets.     

Chemical and acidic composition of Longissimus dorsi muscle of Comisana lambs fed with Trifolium subterraneum and Lolium multiflorum

Aim of this study was to evaluate the effects of grazing on Trifolium subterraneum and Lolium multiflorum, as pure or associated crops, on the chemical composition and on the fatty acid profile of the intramuscular lipids of the meat of lambs. Forty Comisana male lambs, on average weighing 13.75 ± 1.90 kg, were divided into four homogenous groups of ten and called, in relation to the diet: group T those grazing on T. subterraneum; Group L on L. multiflorum; Group TL on adjacent monocultures of T. subterraneum and L. multiflorum (66.6 and 33.3% of surface, respectively); Group LT on adjacent monocultures of T. subterraneum and L. multiflorum (33.3 and 66.6% of surface, respectively). Every 10 days, samples of forage species ingested by grazing lambs were collected and analysed. At 90 days of age, with an average live weight of 25.44, 23.44, 24.69 and 24.75 kg for T, L, TL and LT group, respectively, all lambs were slaughtered and a sample of Longissimus dorsi muscle for each animal was collected to study the chemical and acidic composition. No significant differences among the groups were observed for the growth performance and for the chemical composition of the meat. As regards the fatty acid classes, significant differences (P < 0.05) were observed for the monounsaturated fatty acids, which were lower in the group T (35.46%) than those of the groups L (38.24%), TL (38.63%) and LT (38.59%), whereas, significant higher values for the group T were observed for the polyunsaturated fatty acids of the n-3 (4.49%) and n-6 (8.26%) series than those of the n-6 series for group L (6.79%; P < 0.05) and than those of both series for group LT (n-3 = 3.64%; P < 0.05 and n-6 = 6.43%; P < 0.05). The fatty acids that have significantly determined the modifications of the acidic classes were: oleic acid, which showed significant (P < 0.05) lower values in the group T (26.70%) than the levels observed in the groups L (30.33%), TL (30.39%) and LT (30.63%) and the linoleic, linolenic and rumenic acids which were significantly (P < 0.05) higher in the groups T (linoleic = 5.13%; linolenic = 1.97%; rumenic = 0.46%) and TL (linoleic = 4.75%; linolenic = 1.82%; rumenic = 0.41%) than those of the groups L (linoleic = 4.10%; linolenic = 1.52%; rumenic = 0.26%) and LT (linoleic = 3.95%; linolenic = 1.42%; rumenic = 0.33%). These differences could be due to the different dynamic activity of the cellulolytic bacteria in the rumen, related to the different levels of fibrous fractions of the diets. No significant difference was observed for saturated fatty acid, unsaturated/saturated fatty acids ratio and Atherogenic and Thrombogenic indices among the groups, whereas, PUFA/SFA ratio showed significant (P < 0.05) higher value in group T than that in the group LT.T. subterraneum monoculture grazed as monoculture (T) and in mixture with L. multiflorum (66/33, TL) increased the linoleic, linolenic and rumenic acids improving the dietetic-nutritional characteristics of the lamb meat.     

Protection against X-ray radiation-induced cellular damage of human peripheral blood lymphocytes by an aminothiazole derivative of dendrodoine

The present study was aimed to evaluate the radioprotective efficacy of dendrodoine analog (DA), an aminothiazole derivative against X-ray radiation-induced cellular damage in cultured human peripheral blood lymphocytes. Different concentrations of DA (2, 4, 6, 8, 10 μg/ml or 6.15, 12.29, 18.44, 24.59, 30.73 μM) were pre-incubated with lymphocytes for 30 min prior to irradiation [4 Gy] and the micronuclei (MN) scoring and comet assay were performed to fix the effective concentration of DA against 4 Gy irradiation-induced cellular damage. The results indicated that among all the concentrations, 6 μg/ml concentration of DA showed optimum protection by effectively decreasing the MN frequencies and comet attributes. Based on the above results, 6 μg/ml concentration of DA was fixed as the effective dose to further investigate its radioprotective efficacy. This was carried out by pre-incubating the lymphocytes with 6 μg/ml concentration of DA followed by exposure of the lymphocytes to different doses (1, 2, 3 and 4 Gy) of radiation and investigating the radiation-induced genetic damage (MN, comet assay, DNA fragmentation assay) and biochemical changes (changes in the level of enzymic and non-enzymic antioxidants, lipid peroxidation). The results indicated a dose-dependent increase in both genetic damage and thiobarbituric acid reactive substances (TBARS), accompanied by a significant decrease in the antioxidant status in the irradiated groups compared to DA treated groups which modulated the toxic effects through its antioxidant potential. Thus the current study shows DA to be an effective radioprotector against X-ray radiation induced in vitro cellular damage in lymphocytes.     

Analogs of zanamivir with modified C4-substituents as the inhibitors against the group-1 neuraminidases of influenza viruses

Unlike the group-2 neuraminidase, the group-1 neuraminidase of influenza virus possesses a flexible loop (the 150-loop) and a cavity (the 150-cavity) adjacent to the active site, and renders a conformational change from the ‘open’ form to the ‘closed’ form on binding with substrate (sialo-glycoprotein) or inhibitor (e.g., zanamivir). Zanamivir derivative 8a having an extended (piperazinocarbonyl)propyl substituent at the internal N-position of the guanidino group is designed as a possible inhibitor on the basis of computer docking to the open form of N1 subtype neuraminidase. Indeed, compound 8a exhibits strong neuraminidase inhibition and good anti-influenza activity against H1N1 virus with IC₅₀ = 2.15 μM and EC₅₀ = 0.77 μM, respectively. This study may provide a clue to future design of better group-1 neuraminidase inhibitors.     

The effect of inspiratory and expiratory loads on abdominal muscle activity during breathing in subjects “at risk” for the development of chronic obstructive pulmonary disease and healthy

The abdominal muscle activity has been shown to be variable in subjects with chronic obstructive pulmonary disease (COPD) when respiratory demand increases and their recruitment pattern may change the mechanics, as well as the work and cost of breathing. The scientific evidence in subjects “at risk” for the development of COPD may be important to understand the natural history of this disease. This study aims to evaluate the effect of inspiratory and expiratory loads on the abdominal muscle activity during breathing in subjects “at risk” for the development of COPD and healthy. Thirty-one volunteers, divided in “At Risk” for COPD (n = 17; 47.71 ± 5.11 years) and Healthy (n = 14; 48.21 ± 6.87 years) groups, breathed at the same rhythm without load and with 10% of the maximal inspiratory or expiratory pressures, in standing. Surface electromyography was performed to assess the activation intensity of rectus abdominis (RA), external oblique and transversus abdominis/internal oblique (TrA/IO) muscles, during inspiration and expiration. During inspiration, in “At Risk” for COPD group, RA muscle activation was higher with loaded expiration (p = 0.016); however, in Healthy group it was observed a higher activation of external oblique and TrA/IO muscles (p < 0.050). During expiration, while in “At Risk” for COPD group, RA muscle activation was higher with loaded inspiration (p = 0.009), in Healthy group TrA/IO muscle showed a higher activation (p = 0.025). Subjects “at risk” for the development of COPD seemed to have a specific recruitment of the superficial layer of ventrolateral abdominal wall for the mechanics of breathing.     

Nephrotoxicity assessments of acetaminophen during zebrafish embryogenesis

We used a green fluorescent kidney line, Tg(wt1b:GFP), as a model to access the acetaminophen (AAP)-induced nephrotoxicity dynamically. Zebrafish (Danio rerio) embryos at different developmental stages (12–60 hpf) were treated with different dosages of AAP (0–45 mM) for different time courses (12–60 h). Results showed that zebrafish embryos exhibited no evident differences in survival rates and morphological changes between the mock-treated control (0 mM) and 2.25 mM AAP-exposure (12–72 hpf) groups. In contrast, after higher doses (22.5 and 45 mM) of exposure, embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tube, pronephric duct, and a cystic and atrophic glomerulus. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AAP increased. Interestingly, under the same exposure time course (12 h) and dose (22.5 mM), embryos displayed higher percentages of severe defects at earlier developmental stage of exposure (12–24 hpf), whereas embryos displayed higher percentages of mild defects at later exposure (60–72 hpf). With an exposure time course less than 24 h of 45 mM AAP, no embryo survived by the developmental stage of 72 hpf. These results indicated that AAP-induced nephrotoxicity depended on the exposure dose, time course and developmental stages. Immunohistochemical experiments showed that the cells' morphologies of the pronephric tube, pronephric duct and glomerulus were disrupted by AAP, and consequently caused cell death. Real-time RT-PCR revealed embryos after AAP treatment decreased the expression of cox2 and bcl2, but increased p53 expression. In conclusion, AAP-induced defects on glomerulus, pronephric tube and pronephric duct could be easily and dynamically observed in vivo during kidney development in this present model.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Growth and domoic acid content of Pseudo-nitzschia australis isolated from northwestern Baja California,Mexico, cultured under batch conditions at different temperatures and two Si:NO3 ratios

Domoic acid (DA) poisoning in the southern part of the California Current System has been associated typically with blooms of Pseudo-nitzschia australis. The environmental variables that promote growth and DA production in the Mexican part of this system have not been identified. The present study investigated the effect of temperature and two nutrient ratios on the growth characteristics and DA content of two (BTS-1, BTS-2) P. australis strains isolated from the Pacific coast of northern Baja California peninsula, México. Of the different temperatures assayed (10, 12, 14, 15, 18 and 20 °C), the maximum cell abundance was detected at 12 °C for BTS-2 and 14 °C for BTS-1. The highest maximum specific growth rate (1.69 day⁻¹) was measured at 15 °C for BTS-2. With the exception of cells maintained at 15 °C, growth characteristics were similar in P. australis cultured in a high Si:NO₃ (2.5) or low Si:NO₃ (0.5) ratio at each temperature. Dissolved (dDA) and cellular (cDA) DA content measured at the stationary phase of growth was similar in cells cultivated at the different temperatures. No difference in cDA (between 0.11 and 1.87 pg DA cell⁻¹) was observed in cells cultivated at the two nutrient ratios. To evaluate if P. australis accumulates DA (cDA + dDA) at different stages of the culture and not only during the stationary phase of growth, the BTS-1 strain was cultivated at 14 °C and the content of this toxin was measured during culture development. The cultures were maintained at high (HL; 200 μmol quanta m⁻² s⁻¹) and low light (LL; 30 μmol quanta m⁻² s⁻¹) and in the two nutrient ratios to evaluate the effect of these variables on DA content. The photosynthetic performance and pigment concentration were measured as indicators of the physiological condition of the cells. cDA was detected in all culture conditions and during the different stages of growth. The highest DA content was measured during the lag phase of growth and it was present mainly in the medium (dDA = 70.83 pg DA cell⁻¹). Cells cultivated at HL produced more DA than LL cultured cells. P. australis cultured in HL presented lower photosynthetic rates than LL cells and had similar concentrations of photoprotective pigments and the highest maximum photosynthetic rates were detected during the lag phase of growth in all culture conditions. The results demonstrate that P. australis from northern Baja California peninsula presents a narrow temperature range for optimal growth under batch culture conditions. P. australis produce DA at different stages of growth, and DA content was related to the light intensity at which the cells were cultivated.     

Effect of artificial vs. natural rearing on milk yield,kid growth and cost in Payoya autochthonous dairy goats

Seventy full lactations of Payoya dairy goats were used to study the effect of two different kid-rearing systems, natural or artificial, on milk yield, composition, hygiene-sanitary quality, kid growth and rearing cost. Two animal groups were established, one with goats under natural rearing (NS) and the other under artificial rearing (AR). In the NS group, the kids had free access to goat milk 18–20 h a day and were suckled up to 5 weeks of age and then the dams were milked twice daily. Dams in the AR were separated from their kids at 48 h post-partum; then, kids were reared artificially, and the dams were milked twice daily. The number of animals used in each type of rearing system was 35 (23 goats giving birth to twins and 12 goats giving birth to a single kid). Each week during suckling phase the volume of milk produced was measured, and individual samples were taken. From 5th week and until 210 days lactation, test-day yields recorded at intervals of 30 ± 3 days were obtained. The chemical composition of the milk, the bacteriology, and the somatic cell count was analyzed. The kids of both sexes were assigned to two groups, natural suckling (NS, n = 58) and ad libitum artificial rearing (AR, n = 58). Within each treatment, two groups of kids were formed depending on prolificacy: first group with kids from single birth (n = 12) and the second with twin kids (n = 46). Birth weight and weight every week upto the 4th week of life were recorded. During the 5 weeks of lactation the total milk yield per goat was higher for the NS group (140.2 L vs. 95.4 L; P < 0.001), although the total amount of marketable milk was greater for the AR group, with a difference of some 21 L (P < 0.05). Throughout the entire lactation the milk yield was higher in the group of natural rearing (total yield of 508 L vs. 400 L; P < 0.05). Although a significant effect of prolificacy was found during suckling phase (P < 0.001), during 30 weeks of lactation this factor did not affect milk yield (P > 0.05). For the milk composition and hygiene-sanitary quality there were no effects on the type of rearing system or the type of birth (P > 0.05). No significant effect was observed either for the feeding system or the sex or the prolificacy on the live weight of the kids at 28 days and the postnatal growth rate from birth to 28 days (P > 0.05). Natural rearing system had higher cost per kid comparing with artificial rearing system (€18.63/kid vs. €14.70/kid, respectively). However, when comparing total incomes during a full lactation, goats with natural rearing system had a higher income because of increment of total milk production (€29.95/kid).