Guillermo Alberto Mattioli

Cattle hypocuprosis in Argentina associated with periodically flooded soils

Abstract Although it has been known for a long time that hypocuprosis affects cattle in the province of Buenos Aires (Argentine), mostly during the spring, there has been no attempt to estimate the occurrence of this deficiency. A survey involving more than 200 farms was carried out in five districts of the Salado river basin, where hypocuprosis is endemic. A total of 4750 plasma samples were obtained during 1993 to 1996, and the Cu content was analyzed by atomic absorption spectrophotometry. Fifty-six per cent of cattle population in the five districts was hypocupremic (plasma Cu −1 ), while 14% was severely hypocupremic ( −1 ). The highest incidence was during 1994, when 71% of cattle was hypocupremic and 26% was severely hypocupremic. During the spring a maximum of 37% of cattle was severely hypocupremic, which agrees with previous findings. The causes of hypocuprosis have been defined for the area, as low forage Cu with high S and Fe mainly during the autumn. A positive association between the occurrence of hypocuprosis and excesses of rainfall was found, that explain the seasonal pattern of hypocuprosis and the influence of floodings on hypocuprosis.

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes.

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ± 13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10(6) cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10(6) cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

THE OBJECTIVES WERE TO EVALUATE: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10(6) cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10(6) cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Characterization of cattle copper deficiency in the Magdalena district

Abstract A field survey involving 13 farms was carried out during the spring of 1994 in the Magdalena district to elucidate the causes of cattle hypocuprosis, that has been previously detected as a widespread condition in the area. From each farm, 30 blood samples and representative forage samples were obtained. Liver samples (N: 40) were obtained at a local slaughterhouse. Plasma and liver Cu, and forage Cu, Fe, Mo and S contents were analyzed. Plasma Cu concentrations were below 60 μg/dl in 78.6%, 37.6% and 61.9% of samples from calves, heifers and cows, respectively, in a total of 390 samples. Liver Cu concentration was below 75 ppm DM in 77.5% of the samples. The forage Cu concentration in 44 samples averaged 6.0 ppm DM, and was below the recommended level of 8 ppm in 84% of the samples. Molybdenum and S concentrations averaged 0.65 ppm DM and 0.28% DM, respectively. It is concluded that the widespread hypocuprosis develops as a simple deficiency, complicated by the high levels of S in the forages. This contrasts the Mo toxicity problems reported for other districts within the same basin.

A Single Administration of the GnRH Antagonist Acyline Inhibits Basal and GnRH‐Stimulated Serum Testosterone Concentrations in Male Dogs

The objective of this study was to describe testosterone (T) response to GnRH challenge in antagonist-treated dogs over a 30-day period. Eight mongrel dogs were randomly assigned to either the GnRH antagonist acyline 330 μg/kg sc (ACY; n = 4) or a placebo group (PLA; n = 4). The dogs were serially challenged with the GnRH agonist, buserelin 0.2 μg/kg sc on days -1, 1, 3, 7, 10, 14, 21 and 30. On these days, blood samples for T determinations were collected before (-30 min) and 60, 120 and 180 min after the agonist injection. Basal (-30 min) and post-GnRH agonist stimulation T values were compared by anova for repeated measures. Before treatments (day -1), there were no differences in basal T serum concentrations between groups (p > 0.1). After treatments, basal T showed a significant interaction between treatment and day (p < 0.05). Furthermore, when both groups were analysed independently, basal T varied in the ACY (p < 0.01) but not in the PLA group (p > 0.1). On day -1, before treatments, the stimulation tests had only a time effect (p = 0.05) although on days 1 (p < 0.01), 3 (p < 0.01), 7 (p < 0.01), 10 (p < 0.01) and 14 (p < 0.05), the response to the agonist differed between groups, becoming similar on days 21 (p > 0.05) and 30 (p > 0.05). It was concluded that, in dogs, a single administration of the GnRH antagonist prevented canine gonadal axis to physiologically respond to agonistic challenge during 14 days.

Electrocardiographic Changes in Normal and Abnormal Canine Pregnancy

The aim of this study was to describe the canine electrocardiographic changes in the course of normal and abnormal pregnancy. Twenty-three Brucellosis-negative pregnant bitches were retrospectively classified as normal (n = 12) or abnormal (n = 11). A control group of non-pregnant dioestrous bitches (n = 10) was also included. Normal pregnant females delivered healthy puppies at term while abnormal animals interrupted their pregnancy between days 52-60 (from estimated luteinizing hormone peak) or presented perinatal litter death higher than 60%. All the bitches were electrocardiographically evaluated every 10 days from day 0 to day 65 of the oestrous cycle, to parturition or abortion. Percentage heart rate change increased 31.3% from day 40 to 60 in normal gestation while it decreased -1.8% in dioestrous bitches, although it did not change in the abnormal group (p < 0.01). In the abnormal pregnant group but not in the others, percentage QRSa change fell to -34% on day 60 (p < 0.01). At the same time point, percentage QRSd change was 6.2% vs -4.9% in normal gestations and dioestrous animals, respectively (p < 0.05). Corrected QT interval augmented from day 40 onwards up to 9.9% and 4.3% in the normal pregnant and dioestrous groups, respectively, while it remained unchanged in abnormal gestations (p < 0.05). It is concluded that during normal canine pregnancy, some electrocardiographic parameters begin changing from day 40 onwards, and that pathological gestations differ from normality from day 30. The use of electrocardiography in canine obstetrics might contribute to identify abnormal outcomes before they become clinically evident.

Analysis of apoptosis and DNA damage in bovine cumulus cells after exposure in vitro to different zinc concentrations.

The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dose‐dependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P=0.014 for Zn1; P<0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P=0.0308), control and Zn2 (P=0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P=0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations ‘protect’ the integrity of DNA molecule and diminish the percentage of apoptotic CC.

Diagnostic value of copper parameters to predict growth of suckling calves grazing native range in Argentina

A study was conducted to evaluate the predictive diagnostic value of different copper (Cu) parameters as indicators of average daily gain (ADG) in growing calves. The effects in calves of cow Cu supplementation in the last one-third gestation period were also evaluated. Five supplementation trials, with a total of 300 calves, were carried out. Two groups of 30 calves were randomly assigned to each trial, one group was parenterally supplemented (SG) and the other was not supplemented (NSG). Trials began when calves were three-month-old and ended at weaning time. At each sampling calves were weighed and blood was taken to determine Cu concentrations in plasma, Whole Blood (WB), Red Cells (RC) and Packed Cell Volume (PCV). Liver samples from six animals of each group were taken both at the beginning and at the end of the trial. In two trials the mothers of the SG received Cu supplementation at the last one- third gestation period. Four of the five trials exhibited low ADG in the NSGs. In these groups, plasma Cu concentration decreased rapidly before low ADG was detected, which occurred with values remaining below 25µg/dl. The decrease of RC Cu concentration was considerably slow. WB showed an intermediate position. PCV in the SGs was higher than in the NSGs in all trials. Cow supplementation was insufficient to generate a liver storage able to last after calves reached the 3 months of age. These data could be useful to predict the risk of low ADG in grazing calves.

Renal cortex copper concentration in acute copper poisoning in calves

The aim of this study was to estimate the diagnostic value of renal cortex copper (Cu) concentration in clinical cases of acute copper poisoning (ACP). A total of 97 calves that died due to subcutaneous copper administration were compiled in eleven farms. At least, one necropsy was conducted on each farm and samples for complementary analysis were taken. The degree of autolysis in each necropsy was evaluated. The cases appeared on extensive grazing calf breeding and intensive feedlot farms, in calves of 60 to 200 kg body weight. Mortality varied from 0.86 to 6.96 %, on the farms studied. The first succumbed calf was found on the farms between 6 and 72 hours after the susbcutaneous Cu administration. As discrepancies regarding the reference value arose, the local value (19.9 parts per million) was used, confirming the diagnosis of acute copper poisoning in 93% of the analyzed kidney samples. These results confirm the value of analysis of the cortical kidney Cu concentration for the diagnosis of acute copper poisoning.

Analyses of apoptosis and DNA damage in bovine cumulus cells after in vitro maturation with different copper concentrations: consequences on early embryo development

The aim of this study was to investigate the influence of copper (Cu) during in vitro maturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu, P < 0.01) with respect to cells treated with Cu for cumulus-oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. After in vitro fertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.