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.

This study was conducted to determine the osmotic properties of bull spermatozoa, including the effects of osmotic stress and cryoprotectant agent (CPA) addition and removal, on sperm motility. Semen from beef bulls was collected by electroejaculation and extended 1:3 in TL-Hepes containing 100 micro g/ml pyruvate and 6 mg/ml BSA. In solutions of 150-1200 mOsmolal (mOsm), bull spermatozoa behaved as linear osmometers (r(2) = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 micro m(3). Motility was determined after exposure to anisosmotic solutions ranging from 35 to 2400 mOsm and after return to isosmotic conditions. Retention of at least 90% of isosmotic motility could be maintained only between 270-360 mOsm. Bull spermatozoa were calculated to retain 90% of their isosmotic motility at 92-103% of their isosmotic cell volume. Motility following a one-step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31%, 90%, and 6%, respectively, compared with CPA addition only. These data indicate that, during bull spermatozoa cryopreservation, osmotically driven cell volume excursions must be limited by exposure to a very narrow range that may be facilitated by the use of ethylene glycol as a CPA.

The physiological regulation of follicular atresia was investigated during the early luteal phase after ovulation and during altrenogest-synchronized preovulatory maturation in pigs (gilts). Apoptosis in dispersed granulosa cells was determined by flow cytometry. Apoptotic (A0) cells contain low, subdiploid amounts of DNA fluorescence. Follicles were classified biochemically as atretic or nonatretic based on the percentage of A0 (% A0) cells, atretic with > or = 10%, and nonatretic with < 10% A0 granulosa cells. The % A0 granulosa cells/follicle ranged from .02 to 89. Follicles containing debris in their isolated granulosa cells were classified as morphologically atretic. The morphological and biochemical criteria of atresia were in agreement for 224 of 248 follicles. Internucleosomal DNA cleavage, the hallmark of apoptosis, was determined by autoradiographic analysis of [32P]3'-end labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]3'-end labeled DNA fragments in the .18 to 20 kbp size range was correlated with the % A0 cells (R > .9, n = 22, P < .001). During altrenogest-synchronized preovulatory maturation, < 5% of large (> 6 mm in diameter) follicles were atretic. Among medium-sized follicles (3 to 6 mm) on d 1 and 3 of preovulatory maturation, only 17% were atretic, in contrast with d 5 when 87% were atretic. During the early luteal phase, atretic follicles/pig increased from 6% on d 5 to 50% on d 7 after estrus. Follicular fluid estradiol-17 beta concentration was greater (P < .001) in nonatretic than in atretic follicles on d 5 and 6 after estrus, but by d 7 estradiol-17 beta had decreased to a mean < 1 ng/mL in nonatretic and atretic follicles. The increase in apoptosis in granulosa cells and loss of estradiol-17 beta production in vivo indicated a high incidence of atresia among the first group of follicles grown after ovulation in pigs. These results indicate that apoptotic cell death was involved in degeneration of granulosa cells and atresia during two different stages of follicular development.

The incidence of atresia in the first group of follicles grown after ovulation was investigated in the pig. At slaughter, 113 follicles 3-6 mm in diameter were dissected from the ovaries of four pregnant pigs per day on Days 5, 6, and 7 after the onset of estrus. Granulosa cells were isolated from each follicle. The percentage of granulosa cells containing sub-diploid amounts of DNA (%Ao cells), a measure of apoptosis, was determined for each follicle by DNA fluorescence flow cytometry of propidium iodide (PI) stained nuclei of ethanol-fixed cells. Granulosa cell DNA condition was used to classify follicles. Follicles with > or = 10% Ao cells (n = 33) were designated biochemically atretic (BA), and follicles with < 10% Ao cells (n = 80) were designated biochemically healthy (BH). Internucleosomal cleavage, also indicative of apoptosis, was determined by autoradiographic analysis of [32P]-3'-end-labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]-3'-end-labeled DNA fragments in the 0.18-20-kb size range was correlated with the %Ao cells (R > or = 0.90, N = 22, p < 0.001). The incidence of pigs with BA follicles was 2 of 4, 3 of 4, and 4 of 4 on Days 5, 6, and 7, respectively. The %BA follicles per pig (mean +/- SEM) increased (p < or = 0.01) between Days 5 and 7; values were 6.2 +/- 3.6, 28.1 +/- 13.5, and 50.0 +/- 7.1, respectively, on Days 5, 6, and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

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