Effect of Heat-Stress on Bovine Embryo Development In Vitroli2 D. P. Ryan*v3,4, E. G. Blakewood*, J. W. Lynn+, L. Munyakazi", and R. A. Godke* Departments of *Animal Science and +Zoology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge 70803
ABSTRACT: Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43°C (acute)has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6"C (TI),4OoC (T2),38.6OC after a prior pulse treatment (20 m i d at 43°C with 5 % C02 (T31, or 38.6"C after a prior pulse treatment (20 min) at 43°C with 100% C 0 2 (T4). During incubation, embryos cocultured a t 40" C had a greater P e .05) mean embryo development score at 36 h than embryos cocultured at 38.6"C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6"C than for those cocultured at 40°C. The number of embryos
hatched at 60 h was similar after coculture at 38.6"C (TI)or a prior pulse treatment with 5 % COz and 43°C (T31, but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embyros in T4 had greater (P c .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4)resulted in a greater P e .05)number of hatched embryos at 60 h than TI, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching. However, a n acute rise in temperature at the morula stage increased the rate of embryo development. This may be associated with the production of heat-shock proteins that enabled embyros to tolerate the in vitro stress of the culture environment.
Key Words: Bovidae, Heat Stress, Embryonic Development, Prostaglandins
J. h i m . Sci. 1992. 70:3490-3497
Introduction Fertility is reduced in dairy cows exposed to heat stress (Gwazdauskas et al., 1973; Badinga et al., 1985). Dunlap and Vincent (1971) reported that
'Approved for publication by the Director of the Louisiana Agric. Exp. Sta. as manuscript no. 91-11-5465. This study was supported, in part, by funds from the Louisiana m c . Exp. Sta., LSUAC, and the Federal Southern Regional Project 5-137. 2Mention of trademark or a proprietary product does not constitute a guarantee or a warranty of the product by Louisiana State Univ. and does not imply its approval to the exclusion of other products that also may be suitable. 3Present address: Dept. of Anim. Husbandry, Teagasc, Moorepark, Fermoy, County Cork, Ireland. 4To whom correspondence should be addressed. Received January 14, 1992 Accepted June 24, 1992
when rectal temperatures of cattle increased from 38.5 to 40°C for 72 h postinsemination, pregnancy rates decreased from 48 to 0%. When dairy cows were exposed to heat stress, it was concluded that the majority of embryo mortality occurred either before d 7 of gestation (Monty and Racowsky, 1987; Putney et al., 1988a) or continued through d 14 of gestation (Ryan et al., 1992). Induction of heatshock proteins (HSPI may enable embryos to tolerate the stress of an abnormal uterine environment aindquist, 1986) by inducing thermotolerance and, possibly, the tolerance to additional forms of cellular stress. Harper et al. (1981) concluded that prostaglandin E2 (PGE21 production by rabbit embryos (d 4 to 6) was important in the developmental process and that the embryo may initiate this development. Furthermore, Hwang et al. (1988) have suggested
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AND BOVINE EMBRYO DEVELOPMENT
that PGE2 produced by bovine embryos before d 12 of gestation may be involved in luteal maintenance and, possibly, in the maternal recognition of pregnancy in cattle. Therefore, PGE2 production by bovine embryos may be an assessment of viability. The objectives of the present study were 1) to evaluate in vitro embryo development after exposure of bovine morulae to different heat-stress treatments and 21 to determine the relationship between the heat-stress treatments and subsequent PGE2 production by bovine embryos.
Materials and Methods Preparation of Oviduct Epithelial Cell Monolayer Caprine oviductal monolayers were prepared using a modified procedure similar to that previously reported (Ouhibi et al., 1989; Thibodeaux et al., 1991). In a preliminary experiment, we found that caprine oviduct cells were equal to and often superior to bovine oviduct cells for culturing early morula-stage bovine embryos in vitro. The reproductive tract was surgically removed from a mature, luteal-phase doe and placed in .9% saline. Oviducts were aseptically removed and washed three times in tissue culture medium-199 [TCM-199; Gibco, Grand Island, NYI containing 100 pg of penicillin, 100 pg of streptomycin, and .25 pg of amphotericin-B (PSA; Gibco) per milliliter of medium. Each oviduct was flushed with a .25% trypsin solution (TCM-199)using a 20-gauge needle attached to a sterile 1-mL plastic syringe. The oviducts were then filled with the .25% trypsin solution, and the distal end of the oviduct was clamped with a pair of hemostats and incubated a t 3 7 O C in an atmosphere of 5 % C02 in air for 10 min. Medium was then released from the oviducts into a centrifuge tube containing 5 mL of TCM-199with PSA and centrifuged (200 x gl for 15 min, and the supernatant was discarded. The resulting cell pellet was resuspended in TCM-199 containing 10% fetal bovine serum (FBS; Gibco) and PSA, transferred to a 60-mL cell culture flask (Corning Glassware, Corning, NY), and incubated at 3 7 O C with 5% C02 in an atmosphere of air. After reaching confluence, oviduct cells were detached with a .25% trypsin solution (TCM-199)and were counted using a hemocytometer. Aliquots of approximately 200,000 cells were subsequently transferred into additional 60-mL cell culture flasks. At 48 h before embryo collection, 100,000 cells were placed in wells of a 24-well cell culture cluster dish (Gibcoware@,Gibco). Then 1 mL of Menezo’s Bz medium (API System, Montailieu Vercieu, France) and 1 mL of TCM-199 were added Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
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to each culture well. Medium was changed in each of the wells 12 h before collecting the embryos. Before embryo coculture, each well with a confluent monolayer was washed twice with 4 mL of Dulbecco’s phosphate-buffered saline (DPBS; Gibco) to remove dead cells and FBS, which could subsequently interfere with the radioimmunoassay for PGE2.
Superovulation and Embryo Recovery Superovulation and embryo recovery were performed on a group of crossbred beef donor cows (n = 17). Cows were between d 8 and 14 of an estrous cycle at the onset of gonadotropin treatment. A 5-MHz probe attached to an ultrasound unit (LS1000@, Tokyo Keiki, Japan) was used to verify luteal tissue. Follicle-stimulating hormone (FSHP@, Schering, Kenilworth, NJ) was administered twice daily (0800 and 1800) to each donor cow in a descending dose schedule of 5 , 4 , 3 , and 2 mg. Each cow received a 25-mg dose of PGFza (Lutalyse@, Upjohn, Kalamazoo, MI) in the morning of the 3rd d of FSH injections. At the onset of standing estrus, donor cows were mated with two fertile beef bulls. Each donor was then artificially inseminated 24 h after the onset of estrus with two units of frozenthawed semen. On d 6 to 6.5 after the onset of standing estrus (estrus = d 01, embryos were nonsurgically collected from donors as previously described [Looney et al., 1981). Dulbecco’s PBS, supplemented with 1YO FBS and 1%O PSA, was used as the flushing medium. Each uterine horn was flushed with 200 to 250 mL of DPBS flushing medium. Flushing medium recovered was passed through a 70-pm screen of a filter apparatus [Emcon*, Veterinary Concepts, Spring Valley, WI) to collect the embryos. Flushing medium retained in the filter apparatus (25 to 30 mL1 was examined for embryos using a Zeiss stereomicroscope (20x1.
Allocation of Embryos to Culture Treatments Recovered embryos were placed in modified DPBS containing 10% FBS, 1 mg of glucose, .036 mg of pyruvate, . 1YO phenol red, and .05% PSA per milliliter of medium. Embryos were maintained at room temperature until they were assigned an embryo quality grade (Lindner and Wright, 1983) ranging from 1 to 4 (1 = excellent, 2 = good, 3 = fair, and 4 = poor). Embryos with quality grades 1, 2, and 3 were randomly allotted by donor and quality grade to treatment groups (Table 1). In Treatment 1 (TI), embryos were cocultured at 38.8OC for 60 h in 5% C02,5% 0 2 , and 90% N in B2 medium on a caprine oviduct monolayer. The rationale for TI was to mimic the normal internal core body temperature of the cow. Embryos in
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Table 1. Design and experimental conditions for allocation of bovine embryos across temperature treatments Treatment Item Embryos per group Culture, h Temperature, C (0 to 20 m i d c02, Yo (0 to 20 min) Temperature, C (20 min to 60 hl c02, Yo (20 min to 60 hl O
T1
T2
27 60
27 60
38.6
T3
T4 27
40
27 60 43
5
5
100
38.6
40
38.6
38.6
5
5
5
5
5
Treatment 2 (T$ were cocultured at 4OoC for 60 h in 5 % C02, 5 % 0 2 , and 90% N in B2 medium on a caprine oviduct epithelial cell monolayer (COECM). The rationale for T2 was to mimic a chronic heat stress. Embryos in Treatment 3 (Tal were incubated at 43°C with 5% C02, 5 % 02,and 90% N for 20 min before coculture at 383°C in 5 % C02, 5% 0 2 and 90% N in B2 medium on a COECM for a total incubation period of 60 h. In Treatment 4 (T41, the embryos were incubated a t 43O C with 100% C02 for 20 min before coculture at 38.6"C in 5 % C02, 5 % 0 2 , and 90% N in B2 medium on a COECM for a total incubation period of 60 h. The rationale for T3 and T4 was to mimic the effects of embryo exposure to an acute stress. At each of the coculture incubation temperatures (38.6 and 40" C1, supplemental culture wells contained either .5 mL of B2 medium (T5)or .5 mL of B2 medium with a COECM (Ts). The rationale for T5 and T6 was to identify whether there was a n additive effect of embryos in the coculture system on the production of PGE2. Embryos were placed in .5 mL of B2 medium (43"C) in groups of four per well of a four-well plate (Nunc@,Roskilde, Denmark) to apply the "acute" temperature stress (43OC) in T3 and Tq. Embryos were then placed in a modular incubator chamber (Billups-Rothering, Del Mar, CAI and sealed after introducing either 5 or 100% C02. The modular incubator containing the embryos was placed in a larger temperature-controlled incubator for 20 min at 43°C. Embryos were placed either directly (TI and T2) or immediately after the acute stress (T3 and T4) into wells of a 24-well cell culture cluster dish containing .5 mL of B2 medium on a caprine oviduct cell monolayer. Fetal bovine serum was not included in the coculture system for the first 24 h of coculture. The B2 medium was replaced in each well for all treatments at 4-h intervals during the first 24 h of incubation. The B2 medium recovered was frozen for subsequent PGE2 determination by RIA (Hwang, 1985). Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
60 43
After a preliminary assay to determine the concentration of PGE2 in B2 samples, the samples were diluted by normalizing a .l-mL aliquot of B2 medium from the test sample with .9 mL of PBS. 1YO gelatin. A double-antibody technique was used to separate unbound antigen from bound antigen and circumvent the problems associated with charcoal stripping of the antigen bound to the antibody. Sensitivity of the assay was .5 pg/mL. Within- (n = 2) and between- (n = 3) assay CV for PGE2 were 5.4 and 8.8%, respectively. All embryos were placed in agar cylinders before coculture as previously described (Blakewood and Godke, 1989; Blakewood et al., 1989). Lowmeltingpoint, electrophoresis-grade agarose (Bethesda Research Laboratories, Gaithersburg, MD) was added to 1 mL of DPBS at a concentration of 1.5%. Embryos were embedded in agarose solution at 37"C, and agarose was allowed to solidify at 20 to 23OC. Agarose cylinders (four embryos per cylinder) were used to reduce the risk of embryo damage during the first 24 h of culture when media were changed at 4-h intervals. Embryos were removed from agar cylinders after 24 h of coculture and placed back into the coculture system. During h 24 to 60 of coculture, 1.5 mL of B2 medium was placed in each well. Embryo morphology, stage of development, and embryo quality were assessed within each stage a t 12-h intervals from 24 to 60 h of coculture. Each embryo was assigned a quality grade score from 1 to 4 at each 12-h evaluation interval. Each embryo was also assigned a Ryan embryo development (RED) score based on a combination of stage of development and grade at that stage (Table 2). The RED score system for potentially viable embryos can be described using the additive mathematical formula Yij = p + $i + (P, where Yij = the RED score stage of development and for a n embryo at the receiving the jth grade, p = the overall mean RED score for viable embryos, $i = Yi - pi and $j = Yj Pi*
dh
HEAT STRESS AND BOVINE
Measurement of pH of the Medium During the Incubation Period The B2 medium was heated to 37OC in a water bath. The 24-well culture dishes were preheated to the incubation test temperature for 1 h. This procedure was conducted before incubation of 2 mL of B2 medium in each of 12 wells at 38.6, 40, and 43OC with 5 % C02, 5% 0 2 , and 90% N or 43OC with 100% C02 for 20 min. The pH was measured in each of the 12 wells immediately after the incubation period.
Statistical Analysis Analysis of variance for repeated measures (Gill and Hafs, 1971) was used to examine the effect of treatment and time on RED score as follows: Yijk = + ai + Pj + apij + eijk, where Yijk represented the assigned score, p was the overall mean, ai was the ith treatment effect, pj was the jth time of measurement effect, apij was the interaction effect between time and treatment, and eijk was the residual subplot effect. The pH of the B2 medium was analyzed as a completely randomized design that involved the unique effect of treatment (ai). Prostaglandin E2 production was analyzed for the effects of group, temperature, group x temperature, treatment within group x temperature, time of sample measurement, and its interaction with group and temperature. In this analysis, there were three group levels identified by 1) PGE2 levels in the presence of embryos in the coculture system, 2) no embryo in the coculture system, and 3) PGE2 levels in the presence of B2 medium alone. The temperature effect examined pertained to the two incubation temperatures used from the onset of coculture (38.6 and 40' C). Chi-square analysis (Parker, 1976) was used to detect differences between treatments for the number of embryos hatched at 60 h after the onset of coculture. The SAS (1985) software was used to perform these procedures.
Results Short-term culture of embryos in B2 medium a t 43°C with 5 % or 100% C02 was not detrimental to the viability of embryos at 60 h after the onset of coculture compared with control embryos cocultured continuously at 38.6"C during the same interval. After embryo culture at 43OC with 100% C02 for 20 min in B2 medium and subsequent coculture in B2 medium on caprine oviduct epithelial cells a t 38.6"C with 5% C02 (T& 78% (21/27) of the embryos hatched by 60 h. This hatching rate was greater (P < .05) than the 33% (9127) of the embryos that hatched by 60 h with the coculture system maintained at 38.6"C. In T3 Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
EMBRYO DEVELOPMENT
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(pulse treated at 43OC with 5% C02),48% (13/27) of the embryos hatched by 60 h, which was similar to TI, T2 (13/27), and T4. In contrast, only 11% (3/27) of the embryos in T2 seemed to be viable after 60 h of sustained coculture at 40°C, which was less (P c .05) than any of the other treatment groups, in which all hatched embryos were viable a t 60 h. There was a significant effect of time in coculture and treatment on resulting RED scores. Mean RED scores for embryos in coculture at different time periods are presented in Table 3. At 24 h after the onset of coculture, there was no evidence of differences in RED scores among treatments. Embryos in T1, however, tended to lag in morphological development with time in culture. Embryos in T2, T3, and T4 had greater (P c .05) mean RED scores at 36 h than embryos in TI.Although a difference in mean RED score among T2, T3, and T4 was not evident at 48 h, T3 and T4 had greater (P < .05)mean RED scores than did embryos in T1. After 60 h in culture, the mean RED score for embryos in T2 decreased (P c .05) from the previous RED score a t 48 h and was the cause of the statistical interaction noted between treatment and time (P < .001). This marked decline in the RED score resulted from the death of embryos just before, during, or shortly after hatching; 48.1% of embryos (13/27) in T2 hatched during coculture at 4OoC, but only 11.1% (3/27) were viable at 60 h of incubation. Although the number of embryos hatched at 60 h was not significantly different between T1 (9/27) and T3 (13/27), the RED score for T3 was greater (P c .05) than that for TI. This pattern resulted from embryo death, which was 30% in TI and 4 % in T3 at 60 h of coculture. Mean pH of B2 medium after incubation at 43" C and 100% C02 for 20 min (T4) was 6.3 and was more acidic (P < .001) than the pH of 7.4 for B2 medium after culture at 38.6 (TI),40 (T$, and 43OC for 20 min with 5% C02 (T31, respectively. There was no evidence of a n effect of incubation temperature on the production of PGE2; however, there was a n effect (P c .001) of time in culture (Figure 11. Mean PGE2 productions during the first (20.8 f .8 ng/mL) and second (7.0 k .8 ng/mL) 4-h periods of incubation were different (P c .05), and both were greater (P c .05) than PGE2 concentrations a t subsequent 4-h intervals. The concentration of PGE2 in medium recovered from oviduct cells alone followed a trend a t each time period similar to that of PGE2 concentrations of embryos in coculture with oviduct cells. There was, however, some evidence (P = .06) of a n additive effect of embryos in the coculture system. Prostaglandin E2 production was 7.9 k .4 ng/mL in the presence of oviduct cells and embryos and was greater (P c .05) than the 5.9 .8 ng/mL determined when no embryos were present in the culture system.
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Table 2. The Ryan embryo development score system developed for evaluating bovine embryos in culture ~
~~
State of development
Embryo quality grade
Morula
Blastocyst ~
1 2 3 4
Expanded blastocyst
Hatching blastocyst
Hatched blastocyst
~
1 .oo .66 .33
2.00 1.60 1.33
3.00 2.66 2.33
4.00 3.66 3.33
5.00 4.86 4.33
.oo
.oo
.oo
.oo
.oo
Discussion In this study, chronic elevation of temperature above the normal culture temperature resulted in a high incidence of embryo death that was evident shortly after embryo hatching. These findings are in agreement with recent field studies that reported the stages of embryo mortality in dairy cows during the hot and cool months in Saudi Arabia (Ryan et al., 1992). In that study, embryo mortality in vivo was increased from 4 1% a t d 8 and 7 to 73% at d 13 and 14 of gestation during the hot season. However, there was no increase in embryo mortality between d 6 and 14 of gestation during the cool season. In the present study, embryos cocultured at an elevated temperature of 40°C (T2) had a greater mean development score at 38 h than did embryos cocultured at 38.6" C (T1). However, between 48 and 80 h of coculture at 4OoC,there was a n increase in embryo mortality resulting from death at the hatched blastocyst stage. It had previously been noted that small elevations in incubation temperature in vitro ffrom 37 to 39" Cl were associated with an increase in the mitotic cycle and the cellular metabolic rate of somatic cells (Sisken et al., 1985). The cleavage rate of morula-stage mouse embryos has also been shown to increase by elevating the incubation temperature from 37 to 39OC (Lavy et al., 1988). Findings reported herein suggest that at elevated temperatures bovine embryos may develop at a faster rate to the hatched blastocyst stage and then begin to degenerate rapidly.
There is evidence that an acute rise in C02 concentration may have increased subsequent rate of embryo development in this study. An atmosphere of 5 % C02 is generally used for in vitro culture of mammalian embryos, but this has not been proven to be optimal for embryo development in all species. Carney and Bavister (1987) have shown that development of 8-cell hamster embryos in vitro was improved by using a 10% C02 gas atmosphere compared with a 5 YO C02 gas atmosphere. These workers hypothesized that COz was acting as a weak acid to maintain a lower and possibly a more favorable pH for the developing embryos. When they substituted the nonmetabolizable, weak acid 2,4-dimethyloxazolidinedione (DMO) for the elevated C02 concentration in their study, embryos placed in culture medium with 5 % C02 and DMO developed faster than control embryos incubated in medium with 5 O/O C 0 2 alone. A possible explanation for the increased embryo viability in vitro associated with either an acute elevation in temperature or this in combination with a reduced pH may be the induction of HSP, thus altering gene expression in the embryonic cells. When d-6 rabbit blastocysts were heatshocked at 43OC for 2 h, the incorporation of labeled amino acid was reduced by 0 to 50%, but the labeling of a 70-kDa HSP was increased in the embryos (Heikkila et al., 1985). More recently, Putney et al. (1988b) reported that d-17 bovine conceptuses produced HSP after cows were exposed to an induced heat stress. It has been
Table 3. Mean Ryan embryo development for bovine embryos by treatment across time in coculture Time in culture, h Treatment
24
TI (Controll
Tz (40°C) T, (43OC with Td (43°C with
5 % COZI 100% COZ)
1.43' 1.70' 1.81& 1.63'
36
1.70' 2.30b 2.8sb 2.75b
48
2.83' 3.10ab 3.30b 3.00b
60 3.00& .65b 4.12' 4.55c
*b*cMeanswith different superscripts in the same column indicate means that differ (P e ,051. Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
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HEAT STRESS AND BOVINE EMBRYO DEVELOPMENT
proposed that the cellular production of HSP protects the developing embryo against the toxic effects of elevated temperature and other types of cellular stress (Lindquist, 19881. Lindquist (19881 noted that the ubiquitin form of HSP was induced by heat shock in both yeast and chicken and proposed that a function of this protein was to remove denatured protein from the cell through its protease activity. In addition, HSP were proposed to prevent the production of protein aggregates or to disaggregate them after formation. Therefore, production of HSP may have removed toxic breakdown products in cells of harvested bovine embryos, thus allowing cellular components to remain metabolically active in vitro. Embryos could then proceed at a faster rate to the hatched blastocyst stage after the heatshock episode. However, synthesis and function of
HSP remains unclear and warrants further investigation with bovine embryos. Further research is required to identify whether HSP are produced after reducing the pH of the culture medium or whether the improved rate of development was associated with a reduction of intracellular pH in the embryo. One possible technique would be to measure the internal pH using fluorescent probes (Heiple and Taylor, 1982; Whitaker et al., 19881. Induction of HSP and thermotolerance in embryos before transfer to a recipient may increase tolerance to an abnormal environment and, therefore, increase embryo transfer pregnancy rates. This may be particularly beneficial in countries in which pregnancy rates in cows are seasonally reduced due to environmental heat stress (Monty and Racowsky, 1987; Putney et al., 1988a; Ryan et al., 1992). If an acute stress were
: T1 T2 E
T3
E
T5
T4 T6 h
4
. E
20
M
v
N
w
15
0
a 10
5
0
i
4
8
12
16
20
24
Time in C u l t u r e ( h )
Figure 1. Mean prostaglandin E2 (PGE2)production for bovine embryos by treatment across time in coculture. In T1, embryos were cocultured at 38.6'C in 5% C02,5% 0 2 , and 90% N in B2 medium on a caprine oviduct epithelial cell monolayer (COECM);in T2, embryos were cocultured at 40'C for 60 h in 5% C02, 5% 0 2 , and 90% N in B2 medium on a COECM; in T3, embryos were incubated at 43'C with 5% C02, 5% 0 2 , and 90% N for 20 min before coculture at 38.6'C in 5% COz, 5% 0 2 , and 90% N in B2 medium on a COECM; in T4, embryos were incubated at 43°C with 100% C02 for 20 min before coculture at 38.6"C in 5 % COz, 5% 0 2 , and 90% N in B2 medium on a COECM; in T5 culture wells contained B2 medium; and in T6 contained Bz medium with a COECM. Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
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applied to bovine embryos before transfer to recipients, it could reduce the seasonal decline in pregnancy rates due to heat stress. It was not possible to assess embryo viability from the production of P G E 2 in this study. Prostaglandin E2 synthesis from early-stage bovine embryos during in vitro culture has been previously used in this laboratory to identify viable embryos after culture (Hwang et al., 1988). More recently, it has been suggested that PGE2 secretion by d-5 equine embryos may initiate oviductal transport (Weber et al., 1991). Secretion of PGE2 by d-10 bovine embryos in a perifusion system was only detectable during the last 8 h of a 24-h perifusion period (Wilson et al., 1992). Detection of PGE2 production by d-8 to -8.5 embryos in this study may be related to co-incubation of embryos with oviduct epithelial cells. Co-incubation of embryos and endometrial tissue shifted prostaglandin secretion away from PGF2, toward PGE2 (Lewis and Waterman, 1983; Lewis, 1088). Patterns of PGE2 production by all treatments during the first 24 h of coculture were not expected. Reduction in P G E 2 production after the first 4 h of oviduct cell culture in the presence or absence of embryos may be explained by the removal of fetal calf serum from the culture medium. Fetal calf serum may have provided the precursors for the production of PGE2.
Implications A chronic increase in culture temperature for bovine embryos increases the rate of development to the hatched blastocyst stage but rapid degeneration occurs after hatching. However, when bovine embryos are exposed to an acute stress (e.g., heat pulse) before embryo culture, the rate of development and viability at the hatched blastocyst stage is increased compared with embryos cultured at body temperature without prior exposure to a n acute stress. Exposure of bovine embryos to a heat pulse may increase thermotolerance when the embryos are placed into recipient cows living under a heat-stress environment.
Literature Cited Badinga, L., R. J. Collier, W. W. Thatcher, and C. J. Wilcox. 1985. Effects of climatic and management factors on conception rate of dairy cattle in subtropical environment. J. Dairy Sci. 88:78. Blakewood, E. G., and R. A. Godke. 1989. A method using the chick embryo amnion for mammalian embryo culture. J. Tissue Cult. Methods 12:73. Blakewood, E. G., J. M. Jaynes, W. A. Johnson, and R. A. Godke. 1989. Using the amniotic cavity of the developing chick embryo for the in vivo culture of early-stage mammalian Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
embryos. Poult. Sci. 88:1895. Carney, E. W., and B. D. Bavister. 1987. Regulation of hamster embryo development in vitro in carbon dioxide. Biol. Reprod. 36:1155. Dunlap, S. E., and C. K. Vincent. 1971. Influence of postbreeding thermal stress on conception rate in beef cattle. J. h i m . Sci. 32:1216. Gill, J. L., and H. D. Hafs. 1971. Analysis of repeated measurements of animals. J. h i m . Sci. 33:331. Gwazdauskas, F. C., W. W. Thatcher, and C. J. Wilcox. 1973. Physiological environmental, and hormonal factors at insemination which may affect conception. J. Dairy Sci. 50: 873.
Harper, M.J.K., C. J. Norris, and K. Rajkumar. 1981. Prostaglandins released by zygotes and endometria of pregnant rabbits. Biol. Reprod. 28:350. Heikkila, J. J., J.G.O. Miller, G. A. Schultz, M. Kloc, and L. W. Browder. 1985. Heat shock gene expression during early animal development. In:B. G. Atkinson and D. B. Walden (Ed.) Changes in Eukaryotic Gene Expression in Response to Environmental Stress. p 135. Academic Press, New York. Heiple, J. M., and D. M. Taylor. 1982. An optical technique for measurement of intracellular pH in single-living cells. I n R. Nuccitelli and D. W. Deamer (Ed.) Intracellular pH. Its Measurement, Regulation and Utilization in Cellular Functions. p 21. Alan R. Liss, New York. Hwang, D. H. 1985. Radioimmunoassay for eicosanoids. I n G. L. Longenecker and S.W.W. Schaffer (Ed.) Prostaglandin: Research and Clinical Update. p 303. Southeastern Pharmacology Society, Burgess Publishing, Minneapolis, MN. Hwang, D. H., S. H. Pool, R. W. Rorie, M. Boudreau, and R. A. Godke. 1888. Transitional changes in arachidonic acid metabolism by bovine embryos at different developmental stages. Prostaglandins 35:387. Lavy, G., M. P. Diamond, A. Pellicer, W. K. Vaughn, and A. H. Decherney. 1988. The effect of the incubation temperature on the cleavage rate of mouse embryos in vitro. J. In Vitro Fertil. Embryo Transfer 5:187. Lewis, G. S. 1986. Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast. Prostaglandins 32:275. Lewis, G. S., and R. A. Waterman. 1983. Effects of endometrium on metabolism of arachidonic acid by bovine blastocysts in vitro. Prostaglandins 25:881. Lindner, G. M., and R. W. Wright, Jr. 1083. Bovine embryo morphology and evaluation. Theriogenology 20:407. Lindquist, S. 1986. The heat-shock response. Annu. Rev. Biochem. 55:1151. Looney, C. R., B. W. Boutte, L. F. Archibald, and R. A. Godke. 1981. Comparison of once daily and twice daily FSH injections for superovulating beef cattle. Theriogenology 15:13. Monty, D. E., and C. Racowsky. 1987. In vitro evaluation of early embryo viability and development in summer heatstressed, superovulated dairy cows. Theriogenology 28:451. Ouhibi, N., Y.Menezo, G. Benet, and B. Nicollet. 1989. Culture of epithelial cells derived from the oviduct of different spscies. Human Reprod. 4:229. Parker, R. E. 1978. Use of X2:Single classifications. In: R. E. Parker (Ed.) Introductory Statistics for Biology. p 35. Edward Arnold, London. Putney, D. J., M. Drost, and W. W. Thatcher. 1988a. Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between days 1 to 7 postinsemination. Theriogenology 30:195. Putney, D. J., J. R. Malayer, T. S. Gross, W. W. Thatcher, P. J. Hansen, and M. Drost. 1988b. Heat stress-induced alterations in the synthesis and secretion of proteins and prostaglandins by cultured bovine conceptuses and uterine endometrium. Biol. Reprod. 39:717. Ryan, D. P., J. F. Prichard, E. Kopel, and R. A. Godke. 1992.
HEAT STRESS AND BOVINE EMBRYO DEVELOPMENT Comparing early embryo mortality in dairy cows during hot and cool seasons of the year. Theriogenology 37:Un press). SAS.1985. SAS User's Guide: Statistics. SAS Inst. Inc., Cary, NC. Sisken, J. E., L. Morasca, and S.Kibby. 1965. Effects of temperature on the kinetics of the mitotic cycle of mammalian cells in culture. Exp. Cell Res. 39:103. Thibodeaux, J. K., L. L. Goodeaux, J. D. Roussel, Y.MefiBzo, G. F. Amborski, J. D. Moreau, and R. A. Godke. 19Ql.Effects of the bovine oestrous cycle on invitro characteristics of uter-
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ine and oviductal epithelial cells. Human Reprod. 6:751. Weber, J. A., D. A. Freeman, D. K. Vandewall, and G. L. Woods. 1991. Prostaglandin E2 secretion by oviductal transport-stage equine embryos. Biol Reprod. 45540. Whitaker, J. E., R. P. Haugland, and F. G. Prendergast. 1988. Seminaphto-fluoresceins and -rhodafluors: Dual fluoresence pH indicators. Biophys. J. 53: 197a (Abstr.1. Wilson, J. M., D. D. Zalesky, C. R. Looney, K. R. Bondioli, and R. R. Magness. 1992. Hormone secretion by preimplantation embryos in a dynamic in vitro culture system. Biol. Reprod. 46:295.
ABSTRACT: Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43°C (acute)has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6"C (TI),4OoC (T2),38.6OC after a prior pulse treatment (20 m i d at 43°C with 5 % C02 (T31, or 38.6"C after a prior pulse treatment (20 min) at 43°C with 100% C 0 2 (T4). During incubation, embryos cocultured a t 40" C had a greater P e .05) mean embryo development score at 36 h than embryos cocultured at 38.6"C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6"C than for those cocultured at 40°C. The number of embryos
hatched at 60 h was similar after coculture at 38.6"C (TI)or a prior pulse treatment with 5 % COz and 43°C (T31, but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embyros in T4 had greater (P c .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4)resulted in a greater P e .05)number of hatched embryos at 60 h than TI, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching. However, a n acute rise in temperature at the morula stage increased the rate of embryo development. This may be associated with the production of heat-shock proteins that enabled embyros to tolerate the in vitro stress of the culture environment.
Key Words: Bovidae, Heat Stress, Embryonic Development, Prostaglandins
J. h i m . Sci. 1992. 70:3490-3497
Introduction Fertility is reduced in dairy cows exposed to heat stress (Gwazdauskas et al., 1973; Badinga et al., 1985). Dunlap and Vincent (1971) reported that
'Approved for publication by the Director of the Louisiana Agric. Exp. Sta. as manuscript no. 91-11-5465. This study was supported, in part, by funds from the Louisiana m c . Exp. Sta., LSUAC, and the Federal Southern Regional Project 5-137. 2Mention of trademark or a proprietary product does not constitute a guarantee or a warranty of the product by Louisiana State Univ. and does not imply its approval to the exclusion of other products that also may be suitable. 3Present address: Dept. of Anim. Husbandry, Teagasc, Moorepark, Fermoy, County Cork, Ireland. 4To whom correspondence should be addressed. Received January 14, 1992 Accepted June 24, 1992
when rectal temperatures of cattle increased from 38.5 to 40°C for 72 h postinsemination, pregnancy rates decreased from 48 to 0%. When dairy cows were exposed to heat stress, it was concluded that the majority of embryo mortality occurred either before d 7 of gestation (Monty and Racowsky, 1987; Putney et al., 1988a) or continued through d 14 of gestation (Ryan et al., 1992). Induction of heatshock proteins (HSPI may enable embryos to tolerate the stress of an abnormal uterine environment aindquist, 1986) by inducing thermotolerance and, possibly, the tolerance to additional forms of cellular stress. Harper et al. (1981) concluded that prostaglandin E2 (PGE21 production by rabbit embryos (d 4 to 6) was important in the developmental process and that the embryo may initiate this development. Furthermore, Hwang et al. (1988) have suggested
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AND BOVINE EMBRYO DEVELOPMENT
that PGE2 produced by bovine embryos before d 12 of gestation may be involved in luteal maintenance and, possibly, in the maternal recognition of pregnancy in cattle. Therefore, PGE2 production by bovine embryos may be an assessment of viability. The objectives of the present study were 1) to evaluate in vitro embryo development after exposure of bovine morulae to different heat-stress treatments and 21 to determine the relationship between the heat-stress treatments and subsequent PGE2 production by bovine embryos.
Materials and Methods Preparation of Oviduct Epithelial Cell Monolayer Caprine oviductal monolayers were prepared using a modified procedure similar to that previously reported (Ouhibi et al., 1989; Thibodeaux et al., 1991). In a preliminary experiment, we found that caprine oviduct cells were equal to and often superior to bovine oviduct cells for culturing early morula-stage bovine embryos in vitro. The reproductive tract was surgically removed from a mature, luteal-phase doe and placed in .9% saline. Oviducts were aseptically removed and washed three times in tissue culture medium-199 [TCM-199; Gibco, Grand Island, NYI containing 100 pg of penicillin, 100 pg of streptomycin, and .25 pg of amphotericin-B (PSA; Gibco) per milliliter of medium. Each oviduct was flushed with a .25% trypsin solution (TCM-199)using a 20-gauge needle attached to a sterile 1-mL plastic syringe. The oviducts were then filled with the .25% trypsin solution, and the distal end of the oviduct was clamped with a pair of hemostats and incubated a t 3 7 O C in an atmosphere of 5 % C02 in air for 10 min. Medium was then released from the oviducts into a centrifuge tube containing 5 mL of TCM-199with PSA and centrifuged (200 x gl for 15 min, and the supernatant was discarded. The resulting cell pellet was resuspended in TCM-199 containing 10% fetal bovine serum (FBS; Gibco) and PSA, transferred to a 60-mL cell culture flask (Corning Glassware, Corning, NY), and incubated at 3 7 O C with 5% C02 in an atmosphere of air. After reaching confluence, oviduct cells were detached with a .25% trypsin solution (TCM-199)and were counted using a hemocytometer. Aliquots of approximately 200,000 cells were subsequently transferred into additional 60-mL cell culture flasks. At 48 h before embryo collection, 100,000 cells were placed in wells of a 24-well cell culture cluster dish (Gibcoware@,Gibco). Then 1 mL of Menezo’s Bz medium (API System, Montailieu Vercieu, France) and 1 mL of TCM-199 were added Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
349 1
to each culture well. Medium was changed in each of the wells 12 h before collecting the embryos. Before embryo coculture, each well with a confluent monolayer was washed twice with 4 mL of Dulbecco’s phosphate-buffered saline (DPBS; Gibco) to remove dead cells and FBS, which could subsequently interfere with the radioimmunoassay for PGE2.
Superovulation and Embryo Recovery Superovulation and embryo recovery were performed on a group of crossbred beef donor cows (n = 17). Cows were between d 8 and 14 of an estrous cycle at the onset of gonadotropin treatment. A 5-MHz probe attached to an ultrasound unit (LS1000@, Tokyo Keiki, Japan) was used to verify luteal tissue. Follicle-stimulating hormone (FSHP@, Schering, Kenilworth, NJ) was administered twice daily (0800 and 1800) to each donor cow in a descending dose schedule of 5 , 4 , 3 , and 2 mg. Each cow received a 25-mg dose of PGFza (Lutalyse@, Upjohn, Kalamazoo, MI) in the morning of the 3rd d of FSH injections. At the onset of standing estrus, donor cows were mated with two fertile beef bulls. Each donor was then artificially inseminated 24 h after the onset of estrus with two units of frozenthawed semen. On d 6 to 6.5 after the onset of standing estrus (estrus = d 01, embryos were nonsurgically collected from donors as previously described [Looney et al., 1981). Dulbecco’s PBS, supplemented with 1YO FBS and 1%O PSA, was used as the flushing medium. Each uterine horn was flushed with 200 to 250 mL of DPBS flushing medium. Flushing medium recovered was passed through a 70-pm screen of a filter apparatus [Emcon*, Veterinary Concepts, Spring Valley, WI) to collect the embryos. Flushing medium retained in the filter apparatus (25 to 30 mL1 was examined for embryos using a Zeiss stereomicroscope (20x1.
Allocation of Embryos to Culture Treatments Recovered embryos were placed in modified DPBS containing 10% FBS, 1 mg of glucose, .036 mg of pyruvate, . 1YO phenol red, and .05% PSA per milliliter of medium. Embryos were maintained at room temperature until they were assigned an embryo quality grade (Lindner and Wright, 1983) ranging from 1 to 4 (1 = excellent, 2 = good, 3 = fair, and 4 = poor). Embryos with quality grades 1, 2, and 3 were randomly allotted by donor and quality grade to treatment groups (Table 1). In Treatment 1 (TI), embryos were cocultured at 38.8OC for 60 h in 5% C02,5% 0 2 , and 90% N in B2 medium on a caprine oviduct monolayer. The rationale for TI was to mimic the normal internal core body temperature of the cow. Embryos in
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RYAN ET AL.
Table 1. Design and experimental conditions for allocation of bovine embryos across temperature treatments Treatment Item Embryos per group Culture, h Temperature, C (0 to 20 m i d c02, Yo (0 to 20 min) Temperature, C (20 min to 60 hl c02, Yo (20 min to 60 hl O
T1
T2
27 60
27 60
38.6
T3
T4 27
40
27 60 43
5
5
100
38.6
40
38.6
38.6
5
5
5
5
5
Treatment 2 (T$ were cocultured at 4OoC for 60 h in 5 % C02, 5 % 0 2 , and 90% N in B2 medium on a caprine oviduct epithelial cell monolayer (COECM). The rationale for T2 was to mimic a chronic heat stress. Embryos in Treatment 3 (Tal were incubated at 43°C with 5% C02, 5 % 02,and 90% N for 20 min before coculture at 383°C in 5 % C02, 5% 0 2 and 90% N in B2 medium on a COECM for a total incubation period of 60 h. In Treatment 4 (T41, the embryos were incubated a t 43O C with 100% C02 for 20 min before coculture at 38.6"C in 5 % C02, 5 % 0 2 , and 90% N in B2 medium on a COECM for a total incubation period of 60 h. The rationale for T3 and T4 was to mimic the effects of embryo exposure to an acute stress. At each of the coculture incubation temperatures (38.6 and 40" C1, supplemental culture wells contained either .5 mL of B2 medium (T5)or .5 mL of B2 medium with a COECM (Ts). The rationale for T5 and T6 was to identify whether there was a n additive effect of embryos in the coculture system on the production of PGE2. Embryos were placed in .5 mL of B2 medium (43"C) in groups of four per well of a four-well plate (Nunc@,Roskilde, Denmark) to apply the "acute" temperature stress (43OC) in T3 and Tq. Embryos were then placed in a modular incubator chamber (Billups-Rothering, Del Mar, CAI and sealed after introducing either 5 or 100% C02. The modular incubator containing the embryos was placed in a larger temperature-controlled incubator for 20 min at 43°C. Embryos were placed either directly (TI and T2) or immediately after the acute stress (T3 and T4) into wells of a 24-well cell culture cluster dish containing .5 mL of B2 medium on a caprine oviduct cell monolayer. Fetal bovine serum was not included in the coculture system for the first 24 h of coculture. The B2 medium was replaced in each well for all treatments at 4-h intervals during the first 24 h of incubation. The B2 medium recovered was frozen for subsequent PGE2 determination by RIA (Hwang, 1985). Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
60 43
After a preliminary assay to determine the concentration of PGE2 in B2 samples, the samples were diluted by normalizing a .l-mL aliquot of B2 medium from the test sample with .9 mL of PBS. 1YO gelatin. A double-antibody technique was used to separate unbound antigen from bound antigen and circumvent the problems associated with charcoal stripping of the antigen bound to the antibody. Sensitivity of the assay was .5 pg/mL. Within- (n = 2) and between- (n = 3) assay CV for PGE2 were 5.4 and 8.8%, respectively. All embryos were placed in agar cylinders before coculture as previously described (Blakewood and Godke, 1989; Blakewood et al., 1989). Lowmeltingpoint, electrophoresis-grade agarose (Bethesda Research Laboratories, Gaithersburg, MD) was added to 1 mL of DPBS at a concentration of 1.5%. Embryos were embedded in agarose solution at 37"C, and agarose was allowed to solidify at 20 to 23OC. Agarose cylinders (four embryos per cylinder) were used to reduce the risk of embryo damage during the first 24 h of culture when media were changed at 4-h intervals. Embryos were removed from agar cylinders after 24 h of coculture and placed back into the coculture system. During h 24 to 60 of coculture, 1.5 mL of B2 medium was placed in each well. Embryo morphology, stage of development, and embryo quality were assessed within each stage a t 12-h intervals from 24 to 60 h of coculture. Each embryo was assigned a quality grade score from 1 to 4 at each 12-h evaluation interval. Each embryo was also assigned a Ryan embryo development (RED) score based on a combination of stage of development and grade at that stage (Table 2). The RED score system for potentially viable embryos can be described using the additive mathematical formula Yij = p + $i + (P, where Yij = the RED score stage of development and for a n embryo at the receiving the jth grade, p = the overall mean RED score for viable embryos, $i = Yi - pi and $j = Yj Pi*
dh
HEAT STRESS AND BOVINE
Measurement of pH of the Medium During the Incubation Period The B2 medium was heated to 37OC in a water bath. The 24-well culture dishes were preheated to the incubation test temperature for 1 h. This procedure was conducted before incubation of 2 mL of B2 medium in each of 12 wells at 38.6, 40, and 43OC with 5 % C02, 5% 0 2 , and 90% N or 43OC with 100% C02 for 20 min. The pH was measured in each of the 12 wells immediately after the incubation period.
Statistical Analysis Analysis of variance for repeated measures (Gill and Hafs, 1971) was used to examine the effect of treatment and time on RED score as follows: Yijk = + ai + Pj + apij + eijk, where Yijk represented the assigned score, p was the overall mean, ai was the ith treatment effect, pj was the jth time of measurement effect, apij was the interaction effect between time and treatment, and eijk was the residual subplot effect. The pH of the B2 medium was analyzed as a completely randomized design that involved the unique effect of treatment (ai). Prostaglandin E2 production was analyzed for the effects of group, temperature, group x temperature, treatment within group x temperature, time of sample measurement, and its interaction with group and temperature. In this analysis, there were three group levels identified by 1) PGE2 levels in the presence of embryos in the coculture system, 2) no embryo in the coculture system, and 3) PGE2 levels in the presence of B2 medium alone. The temperature effect examined pertained to the two incubation temperatures used from the onset of coculture (38.6 and 40' C). Chi-square analysis (Parker, 1976) was used to detect differences between treatments for the number of embryos hatched at 60 h after the onset of coculture. The SAS (1985) software was used to perform these procedures.
Results Short-term culture of embryos in B2 medium a t 43°C with 5 % or 100% C02 was not detrimental to the viability of embryos at 60 h after the onset of coculture compared with control embryos cocultured continuously at 38.6"C during the same interval. After embryo culture at 43OC with 100% C02 for 20 min in B2 medium and subsequent coculture in B2 medium on caprine oviduct epithelial cells a t 38.6"C with 5% C02 (T& 78% (21/27) of the embryos hatched by 60 h. This hatching rate was greater (P < .05) than the 33% (9127) of the embryos that hatched by 60 h with the coculture system maintained at 38.6"C. In T3 Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
EMBRYO DEVELOPMENT
3493
(pulse treated at 43OC with 5% C02),48% (13/27) of the embryos hatched by 60 h, which was similar to TI, T2 (13/27), and T4. In contrast, only 11% (3/27) of the embryos in T2 seemed to be viable after 60 h of sustained coculture at 40°C, which was less (P c .05) than any of the other treatment groups, in which all hatched embryos were viable a t 60 h. There was a significant effect of time in coculture and treatment on resulting RED scores. Mean RED scores for embryos in coculture at different time periods are presented in Table 3. At 24 h after the onset of coculture, there was no evidence of differences in RED scores among treatments. Embryos in T1, however, tended to lag in morphological development with time in culture. Embryos in T2, T3, and T4 had greater (P c .05) mean RED scores at 36 h than embryos in TI.Although a difference in mean RED score among T2, T3, and T4 was not evident at 48 h, T3 and T4 had greater (P < .05)mean RED scores than did embryos in T1. After 60 h in culture, the mean RED score for embryos in T2 decreased (P c .05) from the previous RED score a t 48 h and was the cause of the statistical interaction noted between treatment and time (P < .001). This marked decline in the RED score resulted from the death of embryos just before, during, or shortly after hatching; 48.1% of embryos (13/27) in T2 hatched during coculture at 4OoC, but only 11.1% (3/27) were viable at 60 h of incubation. Although the number of embryos hatched at 60 h was not significantly different between T1 (9/27) and T3 (13/27), the RED score for T3 was greater (P c .05) than that for TI. This pattern resulted from embryo death, which was 30% in TI and 4 % in T3 at 60 h of coculture. Mean pH of B2 medium after incubation at 43" C and 100% C02 for 20 min (T4) was 6.3 and was more acidic (P < .001) than the pH of 7.4 for B2 medium after culture at 38.6 (TI),40 (T$, and 43OC for 20 min with 5% C02 (T31, respectively. There was no evidence of a n effect of incubation temperature on the production of PGE2; however, there was a n effect (P c .001) of time in culture (Figure 11. Mean PGE2 productions during the first (20.8 f .8 ng/mL) and second (7.0 k .8 ng/mL) 4-h periods of incubation were different (P c .05), and both were greater (P c .05) than PGE2 concentrations a t subsequent 4-h intervals. The concentration of PGE2 in medium recovered from oviduct cells alone followed a trend a t each time period similar to that of PGE2 concentrations of embryos in coculture with oviduct cells. There was, however, some evidence (P = .06) of a n additive effect of embryos in the coculture system. Prostaglandin E2 production was 7.9 k .4 ng/mL in the presence of oviduct cells and embryos and was greater (P c .05) than the 5.9 .8 ng/mL determined when no embryos were present in the culture system.
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RYAN ET AL.
Table 2. The Ryan embryo development score system developed for evaluating bovine embryos in culture ~
~~
State of development
Embryo quality grade
Morula
Blastocyst ~
1 2 3 4
Expanded blastocyst
Hatching blastocyst
Hatched blastocyst
~
1 .oo .66 .33
2.00 1.60 1.33
3.00 2.66 2.33
4.00 3.66 3.33
5.00 4.86 4.33
.oo
.oo
.oo
.oo
.oo
Discussion In this study, chronic elevation of temperature above the normal culture temperature resulted in a high incidence of embryo death that was evident shortly after embryo hatching. These findings are in agreement with recent field studies that reported the stages of embryo mortality in dairy cows during the hot and cool months in Saudi Arabia (Ryan et al., 1992). In that study, embryo mortality in vivo was increased from 4 1% a t d 8 and 7 to 73% at d 13 and 14 of gestation during the hot season. However, there was no increase in embryo mortality between d 6 and 14 of gestation during the cool season. In the present study, embryos cocultured at an elevated temperature of 40°C (T2) had a greater mean development score at 38 h than did embryos cocultured at 38.6" C (T1). However, between 48 and 80 h of coculture at 4OoC,there was a n increase in embryo mortality resulting from death at the hatched blastocyst stage. It had previously been noted that small elevations in incubation temperature in vitro ffrom 37 to 39" Cl were associated with an increase in the mitotic cycle and the cellular metabolic rate of somatic cells (Sisken et al., 1985). The cleavage rate of morula-stage mouse embryos has also been shown to increase by elevating the incubation temperature from 37 to 39OC (Lavy et al., 1988). Findings reported herein suggest that at elevated temperatures bovine embryos may develop at a faster rate to the hatched blastocyst stage and then begin to degenerate rapidly.
There is evidence that an acute rise in C02 concentration may have increased subsequent rate of embryo development in this study. An atmosphere of 5 % C02 is generally used for in vitro culture of mammalian embryos, but this has not been proven to be optimal for embryo development in all species. Carney and Bavister (1987) have shown that development of 8-cell hamster embryos in vitro was improved by using a 10% C02 gas atmosphere compared with a 5 YO C02 gas atmosphere. These workers hypothesized that COz was acting as a weak acid to maintain a lower and possibly a more favorable pH for the developing embryos. When they substituted the nonmetabolizable, weak acid 2,4-dimethyloxazolidinedione (DMO) for the elevated C02 concentration in their study, embryos placed in culture medium with 5 % C02 and DMO developed faster than control embryos incubated in medium with 5 O/O C 0 2 alone. A possible explanation for the increased embryo viability in vitro associated with either an acute elevation in temperature or this in combination with a reduced pH may be the induction of HSP, thus altering gene expression in the embryonic cells. When d-6 rabbit blastocysts were heatshocked at 43OC for 2 h, the incorporation of labeled amino acid was reduced by 0 to 50%, but the labeling of a 70-kDa HSP was increased in the embryos (Heikkila et al., 1985). More recently, Putney et al. (1988b) reported that d-17 bovine conceptuses produced HSP after cows were exposed to an induced heat stress. It has been
Table 3. Mean Ryan embryo development for bovine embryos by treatment across time in coculture Time in culture, h Treatment
24
TI (Controll
Tz (40°C) T, (43OC with Td (43°C with
5 % COZI 100% COZ)
1.43' 1.70' 1.81& 1.63'
36
1.70' 2.30b 2.8sb 2.75b
48
2.83' 3.10ab 3.30b 3.00b
60 3.00& .65b 4.12' 4.55c
*b*cMeanswith different superscripts in the same column indicate means that differ (P e ,051. Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
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HEAT STRESS AND BOVINE EMBRYO DEVELOPMENT
proposed that the cellular production of HSP protects the developing embryo against the toxic effects of elevated temperature and other types of cellular stress (Lindquist, 19881. Lindquist (19881 noted that the ubiquitin form of HSP was induced by heat shock in both yeast and chicken and proposed that a function of this protein was to remove denatured protein from the cell through its protease activity. In addition, HSP were proposed to prevent the production of protein aggregates or to disaggregate them after formation. Therefore, production of HSP may have removed toxic breakdown products in cells of harvested bovine embryos, thus allowing cellular components to remain metabolically active in vitro. Embryos could then proceed at a faster rate to the hatched blastocyst stage after the heatshock episode. However, synthesis and function of
HSP remains unclear and warrants further investigation with bovine embryos. Further research is required to identify whether HSP are produced after reducing the pH of the culture medium or whether the improved rate of development was associated with a reduction of intracellular pH in the embryo. One possible technique would be to measure the internal pH using fluorescent probes (Heiple and Taylor, 1982; Whitaker et al., 19881. Induction of HSP and thermotolerance in embryos before transfer to a recipient may increase tolerance to an abnormal environment and, therefore, increase embryo transfer pregnancy rates. This may be particularly beneficial in countries in which pregnancy rates in cows are seasonally reduced due to environmental heat stress (Monty and Racowsky, 1987; Putney et al., 1988a; Ryan et al., 1992). If an acute stress were
: T1 T2 E
T3
E
T5
T4 T6 h
4
. E
20
M
v
N
w
15
0
a 10
5
0
i
4
8
12
16
20
24
Time in C u l t u r e ( h )
Figure 1. Mean prostaglandin E2 (PGE2)production for bovine embryos by treatment across time in coculture. In T1, embryos were cocultured at 38.6'C in 5% C02,5% 0 2 , and 90% N in B2 medium on a caprine oviduct epithelial cell monolayer (COECM);in T2, embryos were cocultured at 40'C for 60 h in 5% C02, 5% 0 2 , and 90% N in B2 medium on a COECM; in T3, embryos were incubated at 43'C with 5% C02, 5% 0 2 , and 90% N for 20 min before coculture at 38.6'C in 5% COz, 5% 0 2 , and 90% N in B2 medium on a COECM; in T4, embryos were incubated at 43°C with 100% C02 for 20 min before coculture at 38.6"C in 5 % COz, 5% 0 2 , and 90% N in B2 medium on a COECM; in T5 culture wells contained B2 medium; and in T6 contained Bz medium with a COECM. Downloaded from https://academic.oup.com/jas/article-abstract/70/11/3490/4705790 by guest on 24 February 2018
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applied to bovine embryos before transfer to recipients, it could reduce the seasonal decline in pregnancy rates due to heat stress. It was not possible to assess embryo viability from the production of P G E 2 in this study. Prostaglandin E2 synthesis from early-stage bovine embryos during in vitro culture has been previously used in this laboratory to identify viable embryos after culture (Hwang et al., 1988). More recently, it has been suggested that PGE2 secretion by d-5 equine embryos may initiate oviductal transport (Weber et al., 1991). Secretion of PGE2 by d-10 bovine embryos in a perifusion system was only detectable during the last 8 h of a 24-h perifusion period (Wilson et al., 1992). Detection of PGE2 production by d-8 to -8.5 embryos in this study may be related to co-incubation of embryos with oviduct epithelial cells. Co-incubation of embryos and endometrial tissue shifted prostaglandin secretion away from PGF2, toward PGE2 (Lewis and Waterman, 1983; Lewis, 1088). Patterns of PGE2 production by all treatments during the first 24 h of coculture were not expected. Reduction in P G E 2 production after the first 4 h of oviduct cell culture in the presence or absence of embryos may be explained by the removal of fetal calf serum from the culture medium. Fetal calf serum may have provided the precursors for the production of PGE2.
Implications A chronic increase in culture temperature for bovine embryos increases the rate of development to the hatched blastocyst stage but rapid degeneration occurs after hatching. However, when bovine embryos are exposed to an acute stress (e.g., heat pulse) before embryo culture, the rate of development and viability at the hatched blastocyst stage is increased compared with embryos cultured at body temperature without prior exposure to a n acute stress. Exposure of bovine embryos to a heat pulse may increase thermotolerance when the embryos are placed into recipient cows living under a heat-stress environment.
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