High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows.

Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season.

Effects of dexamethasone administration to diestrus cows on systemic progesterone, estrogen and uterine cyclooxygenase production.

Parenteral administration of dexamethasone to diestrus cattle can extend the length of the natural estrous cycle. In mice, dexamethasone has been shown to inhibit production of the second isozyme of the cyclooxygenase (COX) enzyme (a rate limiting enzyme in prostaglandin formation). Therefore, the purpose of this study was to determine the effect of dexamethasone on estrous cycle length and COX-1 and -2 production by the uterine endometrium of cyclic cattle. Nine crossbred beef cows that exhibited two previous normal estrous cycles were randomly assigned to two treatments; a control group administered intramuscular injections of vehicle, and a dexamethasone group administered 8 mg of dexamethasone (Azium, Schering Corp., Kenilworth, NJ). Both groups received twice daily injections on day 13-22 of the treatment cycle. Uterine endometrial biopsies were collected on days 16, 19 and 22 of the treatment cycle. Blood samples were collected daily on day 13-22 of the treatment cycle for plasma progesterone and estradiol concentrations.

Effect of media substitutes on bovine granulosa cell function and proliferation during in vitro culture.

The development of a serum-free culture system for bovine granulosa cells that would allow for cellular proliferation without induction of steroidogenesis would provide researchers with an important in vitro tool for determining differentiation mechanisms during folliculogenesis. The objective of the present study was to determine the effect of a commercially prepared serum substitute and a medium supplement on proliferation and progesterone production by bovine granulosa cells. Granulosa cells were obtained by aspirating the follicular fluid of follicles 2 to 8 mm in diameter. For each experiment, growth curves to determine the proliferative and steroidogenic response of granulosa cells to several different medium additions were constructed. Cells were counted on d 1, 2, 4, 6, and 8 of culture to determine cell concentration and the media harvested to determine progesterone content. In Exp. 1, granulosa cells were seeded at an initial rate of 5.0 x 10(5) for 48 h in serum-supplemented medium then allotted to one of five treatments including medium alone or medium containing fetal bovine serum (FBS; 1%), Gibco BRL media supplement-x (GMS-X; 1%), fatty acid-free bovine serum albumin (FAF-BSA; 4 mg/mL), or SerXtend (5%). For Exp. 2 and 3, granulosa cells were plated in serum-supplemented medium for either 48 or 24 h and seeded at either 5.0 x 10(5) or 2.5 x 10(5) cells/flask, respectively, and cultured in different concentrations of SerXtend. All treatment media supported granulosa cell proliferation to some extent; the SerXtend treatment provided the highest proliferation rate at all concentrations above .3125%. Also, during the proliferative stage of the growth curve, cells in the SerXtend treatment produced lower amounts of progesterone compared with cells in the other treatments. In summary, granulosa cells may be propagated in vitro in a serum-free environment without inducing progesterone production.

Transvaginal aspiration of oocytes from hormone-treated pregnant beef cattle for in vitro fertilization.

The ability to produce oocytes from genetically valuable, pregnant donors in a safe, repeatable manner would broaden the application of in vitro fertilization (IVF) procedures for beef and dairy cattle. The objectives of this study were to evaluate two gonadotropin treatment schedules for follicle stimulation of pregnant donor cattle and to determine the efficacy and safety of the repeated oocyte aspiration procedure from pregnant cattle. In Exp. 1, pregnant donors at 60 to 90 d of gestation were randomly allotted to three treatment groups. Cows in Treatment A received a total dose of 40 mg of FSH. Cows in Treatment B were administered a total of 20 mg of FSH, and females in Treatment C served as pregnant vehicle-treated controls. A group of luteal phase cows received a total of 40 mg of FSH and served as nonpregnant controls (Treatment D). Ultrasound-guided transvaginal oocyte aspiration was performed 12 h following the last FSH or saline injection. Following follicle aspiration, oocytes were matured for 24 h and then entered a standard bovine IVF procedure. Experiment 2 was conducted to determine the repeatability of this procedure on first trimester cows. Cows in Exp. 2 were selected (after a 20-d recovery period) from each of the three pregnant treatment groups in Exp. 1 and each given 40 mg of FSH before a second oocyte aspiration procedure. The number of follicles aspirated per cow in treatment groups receiving the high FSH dose treatment (40 mg of FSH total dose) was not different (Treatment A, Treatment D, and cows in Exp. 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Established cell lines and their conditioned media support bovine embryo development during in-vitro culture.

These experiments were conducted to evaluate the ability of different somatic-cell monolayers or conditioned medium from somatic cells for supporting bovine embryo development in vitro. In the first experiment, bovine embryos (2- to 4-cells) were allocated randomly to a control (medium 199 with 10% fetal bovine serum and antibiotics) group or co-cultured with bovine oviduct epithelial (BOEC), buffalo rat liver (BRL), Madin Darby bovine kidney (MDBK) or African green monkey kidney (Vero) cells. In the second experiment, bovine embryos (1-cell) were allocated randomly to the following groups: control medium or conditioned medium from BOEC, BRL, MDBK and Vero monolayers. In both experiments, development to the blastocyst stage was assessed after 8 days of incubation at 39 degrees C and 5% CO2. In Experiment 1, coculture improved development to the blastocyst stage compared with control medium alone, and the highest development was observed after co-culture with BOEC. In Experiment 2, conditioned medium enhanced development to morulae and blastocysts compared with the control medium; however, no differences were detected among different cell supports. These results indicate that both co-culture and conditioned medium from different cell monolayers supported development to the blastocyst stage at a higher efficiency than control medium alone.

Stimulation of progesterone production in bovine luteal cells by co-incubation with bovine blastocyst-stage embryos or trophoblastic vesicles.

A study was conducted to determine whether bovine blastocyst-stage embryos and trophoblastic vesicles stimulate the production of progesterone in bovine luteal cells during incubation in vitro. The effects of co-incubation of these embryos and vesicles with uterine endometrial tissue on progesterone production was also investigated. Bovine small and large luteal cells were obtained on day 12 of the oestrous cycle, dispersed by unit gravity sedimentation and recombined to provide preparations free of accessory cells. Blastocyst-stage embryos were obtained on day 7 and trophoblastic vesicles were obtained from bovine embryos on day 12. A uterine endometrial tissue sample was obtained from the same cow from which the corpus luteum was taken. Treatment groups were arranged in 24-well plates as follows: luteal cells alone; luteal cells and one trophoblastic vesicle; luteal cells and one blastocyst embryo; luteal cells and a 10 mg uterine endometrial sample; luteal cells, one trophoblastic vesicle and a uterine endometrial sample; and luteal cells, one blastocyst embryo and a uterine endometrial sample. All treatment groups were incubated (at 37 degrees C under 5% CO2) in Ham's F-12 medium supplemented with antibiotics (100 micrograms penicillin ml-1 and 100 U streptomycin ml-1, L-glutamine (0.29 mg ml-1), insulin (5 micrograms ml-1), transferrin (5 micrograms ml-1) and selenium (5 ng ml-1) for 12 h. Samples of the medium were harvested 10 min (basal concentration) and 2, 6 and 12 h after incubation to determine the concentrations of progesterone and prostaglandin.(ABSTRACT TRUNCATED AT 250 WORDS)

Frozen-thawed cumulus-granulosa cells support bovine embryo development during coculture.

OBJECTIVE: To evaluate the ability of bovine cumulus-granulosa cells to survive cryopreservation and subsequently support bovine embryo development during coculture. DESIGN: In vitro-matured and -fertilized bovine embryos (two- to four-cell) were allotted randomly to one of three treatment groups: [1] control medium alone consisting of Medium 199 containing 10% fetal bovine serum and antibiotics, [2] cocultured on fresh bovine cumulus-granulosa cells in control medium, or [3] cocultured on frozen-thawed cumulus-granulosa cells in control medium. Embryo development was assessed on days 7 and 8 after IVF. RESULTS: Coculture improved embryo development on days 7 and 8 compared with the control group. However, embryo development on days 7 and 8 did not differ among coculture groups. CONCLUSIONS: Frozen-thawed cumulus-granulosa cells enhance embryo development similar to fresh cells during in vitro coculture.

Stimulation of development of in vitro-matured and in vitro-fertilized bovine embryos by platelets.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.

The effects of different types of syringes on equine spermatozoa.

Control extender was incubated at 4 degrees C for 24 hours. Rubber or plastic syringe plungers were separately incubated in semen extender for 24 hours at 4 degrees C. Following incubation, the extender was stored at -20 degrees C until the time of semen collection. The treatments consisted of the following: Group A = equine semen plus control extender; Group B=equine semen plus extender incubated with rubber plungers and Group C=equine semen plus extender incubated in plastic plungers; Group D=equine semen plus control extended in rubber plunger syringes and Group E=equine semen plus control extender in plastic plunger syringer. Each group contained a 5-ml volume of semen and extender at a concentration of 1.0 x 10(8) sperm/ml. The number of live spermatozoa, percentage of progressively motile spermatozoa and rate of progressive motility were taken following collection and every 15 minutes for 1 hour following application of treatments. In experiment 2, treatments were allowed to incubate with semen for 45 minutes, then the extender was removed and was replaced with fresh extender. The rate of progressive motility and the percentage of progressively motile spermatozoa were taken immediately, at 45 minutes, and then every 15 minutes for 1 hour. In experiment 1, the number of live spermatozoa was not affected among the 5 groups. However, there was a decrease (P<0.01) in the rate of progressive motility and in the percentage of progressively motile spermatozoa in Group B compared with the remaining 4 treatment groups at 30, 45 and 60 minutes, with no differences noted when semen was held in syringes with a rubber or a plastic plunger. In experiment 2, the percentage of progressively motile spermatozoa increased after the addition of the control extender.

The use of image analysis to evaluate the development of uterine and oviduct epithelial cells during in vitro culture. A potential quality assurance procedure for in vitro laboratories.

The objective of this study was to establish selection criteria for morphologic assessment of cell quality using a computer image-analysis system. Uterine and oviduct epithelial cells were isolated from the reproductive tracts of cyclic cows using a trypsin solution. Harvested cells were cultured in Tissue Culture Medium-199 with 10% fetal bovine serum and maintained at 37 degrees C with 5% carbon dioxide in air. Subcultures of different confluent monolayers were established using a weak trypsin solution. Morphologic assessment of cell integrity and viability were made visually, with electron microscopy and image analysis. After morphologic assessment, cells were assigned quality scores. Evaluations were conducted on cells during primary culture and following the first and third subpassages. Image-analysis evaluation was conducted on cells using a silicon-intensified target camera and computer-based software. Gray-level density was determined on cells from each flask. There was no difference (P greater than .05) between sample replicates, indicating repeatable measurements were obtained with the image-analysis system. In addition, there was no significant difference in parameters measured between uterine and oviduct cells; therefore, data from both cell types were combined for further statistical comparisons. Gray-level density values for combined uterine and oviduct cells during primary culture and following the first and third subpassages were as follows: 139.7, 154.5, and 173.3, respectively. There was an increase (P less than .05) in gray-level density values with each subpassage of the cell populations. Furthermore, gray level was influenced (P less than .01) by cell quality for combined uterine and oviduct cell populations. These results indicate that epithelial cell quality during in vitro culture may be effectively determined using image analysis. This approach should not be overlooked in establishing quality control measures for uterine and oviduct cells before mammalian embryos are cocultured in vitro.

Evaluating an in vitro culture system of bovine uterine and oviduct epithelial cells for subsequent embryo co-culture.

Three experiments were conducted to evaluate the effects of culture medium and incubation temperature on bovine uterine and oviduct epithelial cell growth, so that the most efficient combination could then be used to develop a co-culture system for bovine embryos. In the first experiment, uterine and oviduct epithelial cells at either the second or third subpassage were incubated for 8 days at 37 degrees C with 5% CO2 in Tissue Culture Medium-199, CMRL-1066, Minimal Essential Medium, Ménézo's B2 or Ham's F-12 medium. In addition to plotting growth curves of cell populations, the cell cycle was monitored for 8 days by flow cytometry. Uterine and oviduct epithelial cells incubated in CMRL-1066 exhibited the highest growth rates during the 8-day culture period. However, there were no differences in cell cycle analysis among treatment groups during the incubation period. In the second experiment, CMRL-1066 medium was used to evaluate growth and proliferation of uterine and oviduct epithelial cells incubated at 37 degrees C or 39 degrees C; temperature had no significant effect on growth rates or proliferation rates for either uterine or oviduct cells during the 8-day incubation. In the third experiment, the more promising culture media for epithelial cell culture studies were chosen for in vitro maturation and subsequent in vitro fertilization (IVF) of bovine oocytes. Early cleavage-stage embryos produced by IVF procedures were subsequently cultured in vitro for 7 days in medium alone or with oviduct epithelial cells. In this study, the culture medium did not influence fertilization or cleavage rates. However, more embryos co-cultured with oviduct epithelial cells were considered viable after 7 days of incubation compared with embryos incubated in medium alone. These results indicate that various incubation conditions can influence the growth of bovine uterine and oviduct epithelial cells in vitro. However, in spite of changes in cell growth patterns, there does not appear to be a change in their embryotropic capabilities in vitro.