The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.

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Daily, weekly, and within-day variations in copper, iron, and zinc contents of human milk were investigated in order to determine whether one sample from an individual is representative of these elements. Total solids, fat, and protein contents were also measured. Fifty women in their 6th to 12th week of lactation each provided seven milk samples consisting of five consecutive daily samples and two additional samples collected either within a single day or at weekly intervals. Fat varied the most of all constituents and total milk solids reflected this variability. Values ranged from 0.2 to 10.4 g/100 ml for fat and from 8.58 to 17.49 g/100 ml for total solids. Protein varied from 0.76 to 2.04 g/100 ml among individuals, with little variation within an individual. Copper content varied considerably among women and within the same woman. With a large proportion of low values, the range was 0.09 to 0.63 mug/ml. Iron content was also found to vary within women as well as among women. Values ranged from less than 0.1 to 1.6 mug/ml with a preponderance of low values. Zinc content was more evenly distributed over the range of 0.14 to 3.95 mug/ml,and within an individual it did not vary widely. A representative estimate of copper and iron contents would therefore require multiple samples, whereas only one sample may provide a representative estimate of zinc content. Comparison of morning, midday, and evening values showed that copper and zinc are higher in the morning and iron is lower at this time. Increased amounts of copper, iron, and zinc were found in multiparous women whether or not they had previously lactated. Milk from older women had lower iron and higher copper and zinc contents than that from younger women. No differences were found in milk of women receiving dietary mineral and vitamin supplements. Calculations indicated that fully breast fed infants under 3 months of age receive approximately 0.35 mg/kg per day of zinc and 0.05 mg/kg per day of both copper and iron.