Effects of heat stress and dietary zinc source on performance and mammary epithelial integrity of lactating dairy cows.

Dietary Zn and heat stress alter gut integrity in monogastric animals. However, effects of Zn on mammary epithelial integrity in heat-stressed lactating dairy cows have not been studied. Multiparous lactating Holstein cows (n = 72) were randomly assigned to 1 of 4 treatments with a 2 × 2 factorial arrangement to study the effects of environment and Zn source on performance and mammary epithelial integrity. Treatments included 2 environments [cooled (CL) or not cooled (NC)] and 2 Zn sources [75 mg/kg of supplemental Zn as Zn hydroxychloride (IOZ) or 35 mg/kg of Zn hydroxychloride + 40 mg/kg of Zn-Met complex (ZMC)]. The experiment was divided into baseline and environmental challenge phases of 84 d each. All cows were cooled during the baseline phase (temperature-humidity index = 72.5), whereas NC cows were not cooled during environmental challenge (temperature-humidity index = 77.7). Mammary biopsies were collected on d 7 and 56 relative to the onset of environmental challenge to analyze gene expression of claudin 1, 4, and 8, zonula occludens 1, 2, and 3, occludin, and E-cadherin and protein expression of occludin and E-cadherin. Deprivation of cooling increased respiration rate (64.8 vs. 73.9 breaths/min) and vaginal temperature (39.03 vs. 39.94°C) and decreased dry matter intake (26.7 vs. 21.6 kg/d). Energy-corrected milk yield decreased for NC cows relative to CL cows (24.5 vs. 34.1 kg/d). An interaction between environment and Zn source occurred for milk fat content as CL cows fed ZMC had lower milk fat percentage than other groups. Relative to CL cows, NC cows had lower concentrations of lactose (4.69 vs. 4.56%) and solids-not-fat (8.46 vs. 8.32%) but a higher concentration of milk urea nitrogen (9.07 vs. 11.02 mg/mL). Compared with IOZ, cows fed ZMC had lower plasma lactose concentration during baseline and tended to have lower plasma lactose concentration during environmental challenge. Plasma lactose concentration tended to increase at 3, 5, and 41 d after the onset of environmental challenge in NC cows relative to CL cows. Treatment had no effect on milk BSA concentration. Cows fed ZMC tended to have higher gene expression of E-cadherin relative to IOZ. Compared with CL, NC cows had increased gene expression of occludin and E-cadherin and tended to have increased claudin 1 and zonula occludens 1 and 2 gene expression in the mammary gland. Protein expression of occludin and E-cadherin was unchanged. In conclusion, removing active cooling impairs lactation performance and affects gene expression of proteins involved in the mammary epithelial barrier, and feeding a portion of dietary zinc as ZMC improves the integrity of the mammary epithelium.

Short communication: Effect of heat stress on markers of autophagy in the mammary gland during the dry period.

Heat stress (HT) during the dry period compromises mammary gland (MG) growth, thus negatively affecting subsequent milk yield. Cooling during the late dry period, when mammary tissue proliferates, is a common management practice. However, it neglects MG involution during the early dry period, a process that is accomplished by both apoptosis and autophagy. Our objective was to evaluate the effect of HT on MG autophagy during the early dry period. Holstein cows were dried off ~45d before expected calving and randomly assigned to 1 of 2 treatments: HT or cooling (CL). All cows were housed in the same free stall barn during the dry period, but only the stall area for CL cows was equipped with soakers and fans. Rectal temperature and respiration rate were measured daily during the dry period. Mammary gland biopsies were collected from each cow 3d before dry-off and on d 3, 7, 14, and 22±2 after dry-off. Autophagy in the MG was determined by measuring protein expression of 2 autophagic markers, autophagy-related protein 7 and microtubule-associated protein light chain 3 (LC3). The average temperature-humidity index during the dry period was 77.7, which indicated that HT and CL cows were exposed to significant heat stress. However, the cooling system effectively alleviated heat strain in CL cows by decreasing the rectal temperature (39.0 vs. 39.4°C) and respiration rate (47.3 vs. 71.2 breaths per minute) relative to HT cows. Protein expression of autophagy-related protein 7, a marker for early autophagosome formation, did not change within or between groups. In contrast, protein expression of LC3-II, a marker of autophagosomes, and its precursor LC3-I showed a dynamic expression pattern in MG from CL cows during the early dry period. Relative to HT cows, MG from CL cows displayed higher expression of LC3-I and LC3-II on d 7 and lower expression of LC3-II on d 14 and 22 after dry-off. Collectively, our data provide a possible mechanistic explanation for the impairment of MG capacity in HT dairy cows. Heat stress-related perturbations of autophagic activity may compromise the regenerative MG involution that is necessary for optimal cell proliferation.

Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.

Ventilatory and metabolic responses to hypoxia and sulphide in the lugworm Arenicola marina (L.).

We examined the effects of hypoxia and sulphide levels on the ventilatory activity of Arenicola marina and determined whether ventilation compensates for oxygen deficiency and affects the mode of energy provision. A. marina ventilated intermittently, irrespective of ambient P(O2) and sulphide concentration. The ventilation rate was 28.5+/-16 ml h(-1) g(-1) wet mass during normoxia, but increased to 175+/-60% of this value during moderate hypoxia, during which aerobic energy metabolism was maintained. Below a P(O2) of 6.2 kPa, A. marina reduced the ventilated volume to 54+/-16% of the normoxic value and became anaerobic, as indicated by the accumulation of succinate and strombine. Incubation with 27 micromol l(-1) ambient sulphide had no effect on the normoxic and hypoxic ventilation rates or on the P(O2) below which anaerobiosis started (P(cM)). Increased sulphide concentrations reduced the ventilation rate and shifted the P(cM) towards a higher P(O2) below 10.7 kPa. Sulphide diffused into the body and was at least partially detoxified to thiosulphate when oxygen was present. Under normoxia, sulphide accumulated in the body wall tissue and coelomic fluid when ambient sulphide levels exceeded 117 micromol l(-1) and 216 micromol l(-1), respectively. A decrease in P(O2) in the presence of 27 or 117 micromol l(-1) ambient sulphide had no significant effect on sulphide accumulation.