Mitochondrial function and sperm viability were quantified in samples of cryopreserved bovine spermatozoa from 12 bulls using fluorometric techniques. The active mitochondria of the spermatozoa were fluorescently stained using three different fluorophores: rhodamine 123 (R123), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) or MitoTracker Green FM (MITO). The stained spermatozoa, and companion aliquots that had been stained with SYBR-14 (a living-cell nucleic acid stain) and propidium iodide to assess viability, were quantified using flow cytometry. The resulting fluorescent measurements of mitochondrial function were compared with microscopic assessments of progressive sperm motility immediately after thawing, with motility after 3-h incubation at 37 degrees C, and with the fluorescent assessment of sperm viability. Staining with either R123 or MITO resulted in a single green population. In contrast, the JC-1 staining of mitochondria produced both green and red-orange populations of spermatozoa and sometimes a progressive gradient between the two populations. The ability of JC-1 to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provided a more rigorous estimate of metabolic function than the other two fluorescent stains. Overall, the three fluorometric measurements of mitochondrial function were highly correlated with each other, with the SYBR-14 assessment of viability, and with the microscopic estimates of motility.

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Flow cytometry was used to compare the functional status of fluorescently stained sperm organelles from 12 Holstein bulls after storage for 24 h at 5 degrees C and after cryopreservation. The organelle-specific stains, SYBR-14 and LysoTracker Green DND-26, identified spermatozoa with intact plasmalemma and those with intact acrosomes, respectively. The mitochondria-specific stain, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1), identified two populations of spermatozoa. One population stained red-orange because the JC-1 accumulated in the mitochondria as aggregates (characteristic of cells exhibiting a high membrane potential); a second population stained green because of JC-1 monomers within the mitochondria (characteristic of cells exhibiting a lower membrane potential). Analysis of variance revealed that within bulls, the properties of sperm viability, intact acrosomes, and mitochondrial status differed in spermatozoa stored for 24 h (p < 0.001) but not in cryopreserved spermatozoa (p > 0.11). Linear regression analyses resulted in significant models in which the proportions of stained spermatozoa stored for 24 h were indicative of those proportions observed in the cryopreserved fractions. These findings suggest that the plasmalemma, the acrosome, and the mitochondria of unfrozen spermatozoa varied as to their functional status. The cryopreservation process, however, resulted in a more uniform status of sperm organelles.

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Ejaculates were collected by artificial vagina from 3 Holstein sires and sorted to 90% purity for X-chromosome-bearing spermatozoa (range 88 to 93%) using flow cytometry. Sorted sperm were diluted to 2.1, 3.5, or 5.0 x 10(6) sperm per dose in an egg yolk (20%), Tris, glycerol (7%) extender. Collections were repeated until >600 straws per sperm dose per sire were obtained. Each sperm dose was loaded into color-coded 0.25-mL French straws, with alternate colors used to define treatments across sires. Within sires, straws were packaged at 9 per cane (3 of each color) and strategically allocated to 75 Holstein herds with targets for 50% use in heifers and 50% in lactating cows. Straw color was recorded in the on-farm record-keeping system at the time of insemination. Data were analyzed separately for cows and heifers. Among heifers, a total of 2,125 usable records were retrieved from 51 herds (238 +/- 5.5 services/ sperm dose per sire, range: 218 to 263). Conception rates in heifers were influenced by the sire x sperm dosage interaction. Within sire A, conception rates of heifers were greater for the 5 x 10(6) (59.5%) than for the 2.1 x 10(6) (46.4%) sperm dose and intermediate for the 3.5 x 10(6) sperm dose (52.2%). However, across sires, sperm dosage had no effect on heifer conception rates (46.7, 51.2, and 52.5% for the 2.1, 3.5, and 5.0 x 10(6) sperm dosages, respectively). Among cows, a total of 2,369 services were retrieved from 56 herds (263 +/- 8.8 services/sperm dose per sire, range: 233 to 303). Conception rates of cows (29.4%) were not affected by sire or sperm dosage (27.0, 29.1, and 30.3% for the 2.1, 3.5, and 5.0 x 10(6) sperm dosages, respectively). In conclusion, these data indicate that an increased sperm dosage may enhance virgin heifer conception rates for some (but not all) sires, whereas neither sire nor sexed-sperm dosage affected conception rates of lactating cows. Additional studies of sexed-sperm dosage across a larger sampling of bulls are warranted to determine whether and how such a practice can be implemented cost effectively for the benefit of the dairy industry.