Use of Hoechst 33342 stain to evaluate live fresh and frozen bull sperm by computer-assisted analysis.

The objective of this research was to investigate possible procedures for evaluating living bull sperm stained with Hoechst 33342 while in a simple medium and in commonly used complex egg yolk-glycerol-Tris (EYGT) and whole milk-glycerol (WMG) extenders. The two semen extenders provide good cryoprotection, but the latter one virtually obscures the sperm. To evaluate sperm motion characteristics when static nonsperm particles are present, a new Hamilton Thorne epifluorescent optical system (UV) with a strobe light was developed for potential use with DNA-stained sperm. This system permitted examination for the first time of sperm motion characteristics in milk. In Experiment 1 (four bull semen replicates with five dye concentrations and three incubation times), 2.5 microg/ml of Hoechst 33342 stained live and dead sperm sufficiently in a modified Tyrode's solution to measure all sperm characteristics without depressing motility, which was validated by using phase-contrast to analyze stained and unstained controls. In Experiments 2a and 2b, each using semen from four bulls with a 5 x 5 factorial arrangement, it was determined that 40 to 60 microg/ml of dye in EYGT or WMG, with UV illumination for 20 minutes, was optimal. There was no detrimental effect on sperm motility. In Experiment 3, analyses of two ejaculates, from each of eight bulls, confirmed that motion characteristics of sperm in EYGT and WMG were not depressed when the sperm were stained with Hoechst 33342. These experiments demonstrate that the dye concentrations and exposure times developed for use with the new epifluorescent optics facilitate evaluating bull sperm frozen in particle-filled whole milk and should be useful for sperm evaluation of a variety of species when nonsperm particulate matter may otherwise interfere.

Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility.

Two experiments were conducted to evaluate semen quality of bulls housed under controlled conditions at a large AI facility and relate results to fertility. In Experiment 1 semen was collected from six 6-yr-old bulls twice daily at 3- to 4-d intervals for 3 d. In Experiment 2 eleven 6- to 11-yr-old bulls were used. Extensive breeding information was available and semen was collected as in Experiment 1 but replicated 4 times. Standard semen analysis and computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, model 10 unit, were performed on 36 first and second ejaculates in Experiment 1 and on 44 first ejaculates in Experiment 2. Sixteen fields (2 chambers with 8 fields per chamber) were examined per sample. In Experiment 1 the correlation between estimated sperm concentration by spectrophotometry and CASA was 0.91 (P < 0.01). Among bulls the range in the percentage of motile spermatozoa was 52 to 82 for CASA versus 62 to 69 for subjective measurements made by highly experienced technicians. Thus, CASA, with high repeatability, provided a more discriminating estimate of the percentage of motile sperm cells than did the subjective procedure. Bull effect was much greater than any other variable in the experiments. Chamber differences were small and so the results for the 2 chambers with 8 fields each were combined. One to five CASA values were correlated with bull fertility, defined as 59-day nonreturn rates corrected for cow and herd effects. The percentage of motile spermatozoa accounted for a small fraction of the total variation in fertility (r2 = 0.34). However higher r2 values (0.68 to 0.98) were obtained for 2 to 5 variables used in the multiple regression equations. The results are promising, and further testing will determine more precisely which of these CASA variables are most useful in estimating bull fertility potential.

Computer-assisted sperm analysis for assessing initial semen quality and changes during storage at 5 degrees C.

Computer-assisted sperm analysis equipment was used to evaluate bull sperm initially in a modified Tyrode's solution, in Cornell University extender, and in egg yolk-glycerol-Tris extender (following cooling and storage in the latter two extenders). Two ejaculates of semen were collected from each of eight bulls. Semen was divided into aliquots using a factorial arrangement. The semen, diluted to approximately 20 x 10(6) sperm/ml, was loaded into two 20-micron chambers, and six microscope fields from each chamber were videotaped for each treatment of each ejaculate of semen. Eight sperm characteristics analyzed with the Hamilton Thorne integrated visual optical system (Hamilton Thorne, Beverly, MA) were reported, and some of these characteristics differed significantly among bulls. The initial values of motile sperm in modified Tyrode's solution, Cornell University extender, and egg yolk-glycerol-Tris extender were 87, 79, and 66%; little change followed cooling and storage at 5 degrees C in the latter two extenders. Also, there was a small but significant decline in sperm velocity during 3 d of storage. Hyperactive sperm increased slightly during storage. The procedures used can rapidly and accurately measure many sperm characteristics in fresh semen and in semen stored in egg yolk extenders, and differences among bulls can be detected.

Computerized imaging and scanning electron microscope (SEM) analysis of co-cultured fresh and frozen bovine sperm.

Fresh and frozen-thawed bull sperm were incubated with bovine oviductal epithelial cells and segments from the oviducts to examine the usefulness of these culture systems to model sperm changes in vivo. Changes in sperm motion characteristics [computer-assisted sperm analysis (CASA)] and surface morphology [scanning electron microscopy (SEM)] were evaluated. In Experiment 1, fresh and frozen sperm were suspended in the Brackett and Oliphant medium or modified Tyrodes medium (mTALP) and incubated for 0, 3, 6, and 9 hours in direct contact with bovine oviductal-epithelial cell (BOEC) monolayers prepared from oviducts of cows in the periovulatory phase of estrus. The percentage of motile sperm decreased gradually in mTALP, but decreased rapidly in Brackett's defined medium after 3 hours of incubation, with overall averages of 55 and 32%, respectively. The percentage of motile fresh sperm exceeded frozen-thawed sperm under all conditions. In Experiment 2, sperm suspended with mTALP were incubated in dishes without monolayers (control), with monolayers, and within the segments of the oviduct for 0, 3, and 6 hours. In the epithelial cell monolayers, the percentage of motile sperm was similar to the controls throughout incubation, but after 3 hours in the oviductal segments, a decrease, partly associated with more rapid rupture of acrosomal membranes occurred. Sperm velocity was higher (100 microns/second) in fresh sperm than in frozen sperm (85 microns/second). Acrosomal changes, discernible with SEM after 3 hours of incubation, increased with time and were always found more often in frozen than in fresh sperm. The BOEC-monolayer system provided a useful in vitro model to study pre-fertilization changes in sperm.

Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342.

OBJECTIVE: To develop methods for using a DNA-specific dye to discriminate between motile and nonmotile sperm and static particulate matter in fresh and diluted semen, using computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, TOX version (Hamilton-Thorne Research, Beverly, MA). DESIGN: Donor semen was divided for treatment as fixed stained sperm (Hoechst 33342 stain; Sigma Chemical Company, St. Louis, MO), fresh motile and nonmotile stained sperm, and unstained control sperm. SETTING: Normal human volunteers in an academic research and medical environment. PATIENTS: Selected healthy student volunteers. INTERVENTIONS: Delivered semen to the laboratory within 1 hour of collection. MAIN OUTCOME MEASURE: Semen quality measured by CASA. RESULTS: Fixed or fresh human sperm stained with Hoechst 33342 dye should be diluted to < or = 50 x 10(6) sperm/mL to count sperm accurately. Motile and nonmotile sperm were stained suitably with 5 to 10 micrograms/mL of dye when diluted with a simple diluent, but the dye concentration should be increased to 40 micrograms/mL when egg yolk is in the diluent. CONCLUSIONS: The DNA-specific dye, Hoechst 33342, can be used to discriminate between motile and nonmotile sperm and other particulate matter when evaluated by CASA with instrumentation equipped with suitable optics.

Media and dilution procedures tested to minimize handling effects on human, rabbit, and bull sperm for computer-assisted sperm analysis (CASA).

Proper handling of semen prior to computer-assisted sperm analysis (CASA) is critical if the analysis is to be representative of the fresh sample. The effects of diluting medium or dilution and holding time before CASA on multiple sperm characteristics were studied. Four replicates of unselected semen samples from each of eight human donors were diluted with phosphate-buffered saline (PBS)-glucose plus bovine serum albumin (BSA), with Tyrode's albumen lactate pyruvate (TALP), and with high-potassium TALP (K-TALP) to a concentration of approximately 25 x 10(6) sperm/ml. The diluted semen was held for 0, 1, and 2 hours at approximately 30 degrees C before CASA, with little difference between the three diluents in all 12 variables measured. There was a decline of 3-6% in the proportion of motile sperm over a 2-hour period (P < 0.05). Donors were the largest source of differences (P < 0.05). Rabbit sperm (five bucks, four ejaculates per buck) were processed in a manner similar to that of the human sperm. There was a major effect of media. The average percentages of motile sperm over 2 hours in TALP, K-TALP, and PBS were 76, 42, and 29%, respectively (P < 0.05), with a decline of only 3% in TALP during the 2 hours. Hyperactivity and other characteristics were affected by treatment. Donors were a large source of variation. Bull semen (10 bulls, two ejaculates per bull) either was not diluted or diluted with TALP 2x or 4x and held for 0, 1, and 2 hours at 30 degrees C. It was then diluted to 25 x 10(6) sperm/ml with TALP. There was little change in most sperm characteristics in any treatment during the first hour, although many of the changes were statistically significant. The percentage of motile sperm in undiluted semen declined from 87% to 82% over 2 hours. Modified TALP was a suitable medium for sperm from all three species, and a simple PBS-glucose-BSA medium can be used for human sperm.

Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits.

Male Dutch rabbits were weighed and randomly assigned within each weight group to five groups of six animals each (plus one more in the highest dose group). They received 0, 12.5, 25.0, 37.5, or 50.0 mg of ethylene glycol monomethyl ether (EGME) per kg of body weight in the drinking water 5 d/week for 12 weeks. Feed and water consumption were monitored daily and body weight weekly. All animals consumed the water and feed, maintained body weight, and were in good health throughout the experiment. Semen was collected twice weekly for 12 weeks, and 96% of the ejaculates were obtained. By weeks 6 and 9, most males in groups receiving 50.0 or 37.5 mg of EGME per kg were oligospermic. Only minor changes in other characteristics of sperm obtained from treated animals were found, as measured by computer-assisted sperm analysis. Fertility of the males still producing sufficient sperm during week 12 to use for insemination was tested with 96 does producing 2839 oocytes, and fertility of treated males (41%) was not lower (P > 0.05) than 47% in controls. At necropsy, all vital organs were grossly normal, with no notable histopathology. However, the groups of animals receiving 37.5 and 50 mg of EGME per kg of body weight produced fewer sperm and had smaller testes than controls (P < 0.05). Although all rabbits appeared grossly normal, there was a marked disruption of spermatogenesis as ingestion of EGME increased above 25 mg/kg of body weight. Rabbit testes appear to be more sensitive to EGME than testes of rats or mice.

Beneficial effects of culturing rabbit zygotes to blastocysts in 5% oxygen and 10% carbon dioxide.

Zygotes were collected from superovulated Dutch-belted rabbits 19 h after injection of LH and insemination. Oocytes that appeared to be unfertilized were discarded. The zygotes were distributed equally within each donor female across all culture treatments. Culture dishes contained 500 microliters of macromolecule-free RD medium consisting of equal parts of RPMI 1640 and low glucose Dulbecco's modified Eagle's medium. Embryos were cultured at 39 degrees C in several gas combinations of N2 plus the following: (1) 1% O2:10% CO2; (2) 5% O2:10% CO2; and (3) 20% O2:10% CO2. The control (4) was 95% air:5% CO2. The experiment was replicated with embryos from 11 donors providing 295 usable zygotes. After 84 h of culture, the percentages of blastocysts formed in treatments 1 to 4, respectively, were 13, 86, 82 and 59 (P < 0.01). The corresponding mean cell counts, including all cleaved embryos cultured (but not degenerate ones), were 55, 183, 118 and 68 (P < 0.01). These results indicate that 10% CO2 combined with 5% O2 is a more effective gas phase for culturing rabbit zygotes in a synthetic medium than is the commonly used 5% CO2, and that 5% O2 is superior to either 1% or 20% O2.

Relationship of semen quality, number of sperm inseminated, and fertility in rabbits.

The relationship between the total number of sperm inseminated, semen quality, and fertility in rabbits was investigated, using fractionated or unfractionated semen and different diluting fluids. Semen was from Dutch-belted males collected twice weekly with an artificial vagina. All does were superovulated except in Experiment 3. In Experiment 1, sperm were fractionated on discontinuous 4% and 10% bovine serum albumin columns. Sperm from each portion of the gradient, along with unfractionated controls, were diluted to give 0.25 x 10(6), 0.5 x 10(6), 1.0 x 10(6), and 2.0 x 10(6) total sperm per insemination. In Experiment 2, sperm were diluted with Dulbecco's phosphate-buffered saline to provide 0.10 x 10(6), 0.50 x 10(6), and 1.0 x 10(6) total sperm per insemination, with minimal processing time. In Experiment 3, does were allowed to kindle after inseminating 0.1 x 10(6) or 1.0 x 10(6) sperm. In Experiment 4, sperm were diluted with TALP buffer: seminal plasma 1:1 to 0.025 x 10(6), 0.05 x 10(6), and 0.10 x 10(6) total sperm per insemination. Over 2,800 embryos or unfertilized oocytes were obtained either 24 or 48 hours after insemination to measure fertility. Sperm numbers required for normal fertility were 0.50 x 10(6) in Experiment 1 and only 0.05 x 10(6) in Experiment 4. This reduction presumably was due primarily to reduced processing time and diluent change. Litter size was normal with 0.1 x 10(6) sperm (Experiment 3). In Experiment 4, computer-assisted sperm analysis (HTM 2030 system; Beverly, Massachusetts) was adapted to successfully screen out some of the "interfering" granules in rabbit semen.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of six-day bovine embryo development in uterine tube (oviduct) epithelial cell co-culture versus in vivo development in the cow.

A study was designed to evaluate and compare the appearance of embryos recovered from donor cows on Day 6 to embryos from in vivo fertilized cow zygotes developed to Day 6 on uterine tube (oviduct) epithelial cell co-culture using serum-free CZB medium. Embryo stage of development and quality score were assessed. Hoechst 33342 DNA stain was then used to determine the total number of blastomeres, the number of poor nuclei and the number of nuclei in mitosis. Mean cell counts did not differ for the 70 embryos evaluated in each group (65 cells in vivo, 61 cells in vitro). The percentage of transferable emryos (excellent, good or fair quality), in each group also did not differ (57% in vivo, 56% in vitro). There were no significant differences in any of the measured parameters. Our findings suggest that co-culture of in vivo produced cow zygotes can result in embryos comparable in developmental stage and quality to embryos developed in vivo in the cow for 6 d.

Method for obtaining bovine zygotes produced in vivo.

A superovulatory and surgical protocol was developed for recovery of bovine zygotes. Holstein cows and heifers were given follicle-stimulating hormone and cloprostenol to induce superovulation. Surgical cannulation and lavage of the uterine tube was performed 40 to 48 hours after the start of standing estrus. In general, cows had more corpora hemorrhagica than did heifers, but a higher percentage (P less than 0.05) of ova recovered from cows were infertile. Several heifers were subjected to the procedure twice, and embryo recovery rates were equivalent both times.

Bovine 1-2-cell embryo development using a simple medium in three oviduct epithelial cell coculture systems.

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells.

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.