Influence of manganese on apoptosis and glutathione content of cumulus cells during in vitro maturation in bovine oocytes.

We have investigated the effect of different Mn concentrations on (1) DNA integrity of cumulus cells by olive tail moment (OTM); (2) cumulus cells apoptosis by Annexin V staining assay; (3) intracellular total glutathione (GSH-GSSG) content; and (4) oocyte nuclear maturation and embryo cleavage after in vitro fertilisation (IVF). For this purpose, 0 (control), 2 (Mn1), 5 (Mn2) and 6 ng/mL (Mn3) Mn concentrations were added to IVM medium. Comet assay analysed by OTM was significantly higher in cumulus cells arising from COCs matured without Mn (control, P < 0.01) respect to cumulus cells obtained from COCs matured with Mn (control: 5.18 ± 2.3; Mn1: 2.93 ± 2.2; Mn2: 2.63 ± 2.4; Mn3: 2.92 ± 2.4). The frequency of apoptotic cells was higher in the control group (control: 6.63 ± 0.59; Mn1: 5.05 ± 0.5; Mn2: 4.61 ± 0.49; Mn3: 3.33 ± 0.42). Intracellular concentration of GSH-GSSG increased in oocytes and cumulus cells matured in the presence of Mn (P < 0.01). There were no differences in percentages of nuclear maturation when Mn was added to IVM medium at any concentration, but at 6 ng/mL Mn a higher cleavage rate was observed respect to the control group (P < 0.05). In conclusion, deficiency in Mn concentration during in vitro maturation increased the damage in the DNA molecule and the frequency of apoptotic cumulus cells. However, the addition of an adequate Mn concentration (6 ng/mL Mn) to IVM medium improved the health of cumulus-oocyte complexes and produced more cleaved embryos 48 h after IVF.

Association between dietary polyunsaturated fatty acids and their concentration in blood plasma, red blood cell, and semen of dogs.

Background: In dogs, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) affect the fatty acid (FA) profile of blood plasma, erythrocyte membrane (EM), and semen, but their correlation has not yet been investigated. Aim: In this study, we evaluated the association between dietary PUFA and their profile in blood plasma, EM, and semen of dogs, with the possibility to predict the semen profile using the values of the three first. Methods: Twelve male dogs received the same standard commercial diet for 4 weeks. The FA profile was analyzed by gas chromatography in paired diet, blood (plasma and EM determinations), and semen samples. Data were analyzed with SAS Proc Corr version 9.4. Pearson´s correlation coefficient (significant if p < 0.05) was used to assess the association of dietary FA profiles with those in blood plasma, EM, and semen. Results: There was a positive correlation between dietary eicosapentaenoic acid (EPA) and blood plasma (r = 0.97), EM (r = 0.94) and semen (r = 0.92) EPA, and between dietary docosahexaenoic acid (DHA) and arachidonic acid (ARA) and semen DHA (r = 0.93) and ARA (r = 0.92), respectively. There was a negative correlation between dihomo-gamma-linolenic acid (DGLA) in the diet and EM DGLA (r = -0.94). Conclusion: The dietary EPA is correlated with blood plasma, EM, and semen EPA concentrations, and dietary DHA and ARA are associated with semen DHA and ARA concentrations in dogs. These findings suggest that dietary EPA, DHA, and ARA concentrations could be helpful to predictive markers for such concentrations in the semen of dogs.

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.

Renal cortex copper concentration in acute copper poisoning in calves / Concentração de cobre no córtex renal na intoxicação aguda de cobre em bezerros

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 percent, 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 percent 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.

O objetivo deste trabalho foi estimar o valor diagnóstico de concentração de cobre (Cu) no córtex do rim em casos clínicos da intoxicação cobre aguda (ACP). Um total de 97 bezerros foi compilado em onze fazendas. Pelo menos, uma necropsia foi realizada em cada caso e foram colhidas amostras para análise complementar. O grau de autólise em cada necropsia foi avaliado. Os casos aparecem em criação extensiva e também em fazendas de confinamento intensivo. Os pesos dos animais variavam de 60 até 200 kg. Mortalidade variou entre 0,86 e 6,96 por cento, em todas as fazendas estudadas, o primeiro animal morto foi observado entre 6 e 72 horas após à administração parenteral de Cu. Surgirem discrepâncias em relação ao valor de referência a ser usado. O valor local (19. 9 partes por milhão) foi usado, confirmando o diagnóstico de intoxicação aguda de cobre em 93 por cento das amostras analisadas nos rins. Estes resultados confirmam o valor diagnóstico da concentração de Cu no rim córtex para o diagnóstico de ACP.

Diagnostic value of copper parameters to predict growth of suckling calves grazing native range in Argentina / Valor diagnóstico dos parâmetros de cobre para prever o crescimento de bezerros lactantes em pastagem nativas na 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.

Foi realizado um estudo para predisser o valor diagnóstico de diferentes parâmetros de cobre (Cu) como indicadores de ganho médio diário (ADG) na criação de bezerros. Também foram avaliados os efeitos da suplementação com Cu nas vacas no último terço da gestação. Cinco ensaios de suplementação, com um total de 300 bezerros, foram realizados. Dois grupos de 30 bezerros foram atribuídos aleatoriamente em cada proba, um grupo foi parenteralmente suple mentado (SG) e o outro não foi suplementado (NSG). Os ensaios começaram quando os bezerros tinham três meses de idade e terminou ao tempo do destete. Em cada ensaio os bezerros foram pesados e mostras do sangue foi tomada para determinar as concentrações Cu no plasma, sangue total (WB), eritrócitos (RC) e hematócrito (PCV). Amostras de fígado foram colhidas em seis animais de cada grupo tanto no início quanto no final do ensaio As mães do SG receberam suplementação de Cu no último terço da gestação em dois ensaios. Quatro dos cinco ensaios apresentavam baixa ADG nos NSGs. Nesses grupos, a concentração plasmática de Cu diminuiu rapidamente antes que a baixa do ADG fossei detectada, o que ocorreu com os valores abaixo 25 µg/d. A diminuição da concentração de RC Cu foi bastante lenta. WB mostrou uma posição intermédia. PCV nos SGs foi mais elevado do que nos NSGs em todos os ensaios. A suplementação das vacas foi insuficiente para gerar um armazenamento hepático capaz de durar até os 3 meses de idade dos bezerros. Estes dados poderiam ser úteis para prever o risco de baixa ADG em bezerros em pastagem.