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Influence of exogenous progesterone on early embryonic development in the mare
Theriogenology
INFLUENCE
38: 1055-I
063, 1992
OF EXOGENOUS PROGESTERONE ON EARLY EMBRYONIC DEVELOPMENT IN THE MARE B. A. Ball, P. G. Miller Department
Received
and P. F. Daels
of Clinical Sciences, Cornell Ithaca, New York 14853 for publication: Accepted:
University
March 24, 1992 September 28, 1992
ABSTRACT The influence of exogenous progesterone on the development of equine oviductal embryos was determined based upon the recovery of Day-7 uterine blastocysts from treated mares (n = 13) that were given 450 mg progesterone daily between Days 0 and 6 and from untreated control mares (n = 13). Daily administration of 450 mg progesterone in oil significantly (P < 0.02) increased There was no serum progesterone concentrations in the treated mares. significant difference in the recovery rate of Day-7 embryos between treated and control mares (8/l 3 versus 6/l 3, respectively). Embryonic development, assessed by morphologic evaluation, embryo diameter, and number of cell nuclei was not significantly different for embryos from treated and from control mares. The results of this study indicate that administration of progesterone beginning on the day of ovulation does not affect the embryo recovery rate or embryonic development, based on evaluation of uterine blastocysts recovered at Day 7 after ovulation. Key words:
equine,
embryo,
progesterone,
oviduct,
embryo
recovery
INTRODUCTION Synchrony between the developing embryo and the female reproductive tract, as influenced by hormonal stimulation, is critical for successful pregnancy
Acknowledgments We thank the Harry M. Zweig Memorial Fund for equine research and Thornbrook Farms for financial support; Dr. P.J. Burns for assistance in preparation of progesterone, Dr. J. Ellington for assistance with embryo staining; D. Waldow, and H. Das for technical assistance; and C. Collyer and the staff of the Equine Research Park for animal care. Portions of these results were presented at the 12th International Congress on Animal Reproduction, The Hague in August, 1992.
Copyright
Q 1992 Butterworth-Heinemann
1056
Theriogenology
in domestic animals. Synchrony between the embryo and the uterus appears to be more important for proper embryo-endometrial interactions, and coordination of development within the oviduct seems less critical (I 1. Steroids influence oviductal function, resulting in changes in fluid volume, electrolyte composition as well as in qualitative and quantitative changes in oviductal proteins (2). Therefore, alterations in the timing or the amount of progesterone present during the period of oviductal transit might alter oviductal transit or affect early embryonic development. Exogenous progesterone therapy is sometimes initiated during the immediate postovulatory period in mares. The equine embryo resides in the oviduct during the first 5 to 6 days after ovlation (31, and the equine embryo reaches a later stage of development prior to entry into the uterus than in other domestic animals (4,5). Therefore, the administration of progesterone to mares during the immediate postovulatory period might result in perturbations of the oviductal environment or oviductal transit. The effect of administration of progesterone following ovulation depends on the species under study and on the timing of progesterone administration relative to ovulation (2). In mice, progesterone appears to be important for normal development of cleavage-stage embryos and their transport through the oviduct (6-8). However, embryos from many domestic animals, including the horse, can develop in vitro from the zygote to blastocyst stage without the addition of progesterone to the co-culture (9-l 1). In addition, transferred rabbit zygotes undergo normal cleavage in the oviducts of ovariectomized rabbits (121, and the oviducts of anestrous ewes are frequently used as a surrogate host to support the development of heterologous embryos (13). These studies indicate that progesterone may be facilatory rather than obligatory in regulating development of oviductal embryos. Few investigators have examined the influence of exogenous progesterone on early embryonic development in the mare. Hinrichs and Watson examined the effect of administration of 250 mg progesterone at Days 0, 1 and 2 after ovulation on embryo collection from the uterus at Day 5 (14). They observed no difference in the embryo recovery rates between treated and control mares and concluded that administration of progesterone did not hasten transport of Weithenauer et al. (15) examined the effect of daily equine embryos. administration of either 150 mg or 450 mg of progesterone on the growth of the conceptus in utero between Days 10 and 18 and on the content of yolk sac fluid at Day 18. The concentration of proteins and glucose in yolk sac fluid was increased with the administration of 450 mg of progesterone, and there was a nonsignificant increase in growth of the conceptus as determined by ultrasonography.
Theriogenology
The objective of the current study was to determine the effect of administration of progesterone at Days 0 to 6 after ovulation on the development of the equine conceptus through Day 7. MATERIALS
AND METHODS
during estrus, and Pony mares fn = 26) were artificially inseminated ovulation was detected by daily transrectal ultrasonography (16). On the day that ovulation was detected (Day 01, mares were alternately assigned to either a treated or control group. Treated mares (n = 13) were given 450 mg progesterone (100 mg/ml progesterone dissolved in 20% benzyl alcohol in cottonseed oil; Sigma Chemical Co., St. Louis, MO) by intramuscular injection on a daily basis from Days 0 through Day 6. Control mares (n = 13) remained untreated. Jugular blood samples were taken daily beginning on Day 0 (prior to administration of progesterone) through Day 7 from treated and control mares. The blood was allowed to clot at ambient temperature, serum was separated by centrifugation and was stored at -20” C prior to radioimmunoassay for progesterone. Aliquots of serum (100 ,uI) were extracted in duplicate with petroleum ether and were assayed as described previously (17). The progesteroneantiserum usedcross-reacts 13.6% with 5/3_dihydroprogesterone, less than 6% with other progestins tested, and less than 0.01% with Sensitivity of the assay was 12 pglassay androgens and estrogens tested. tube or 0.12 ng/ml, and the intra- and inter-assay coefficients of variation were 9 and 6%, respectively. On Day 7 after ovulation, mares were restrained in stocks and were sedated Scientific; Shawnee, with 0.3 mg/lb xylazine HCI (Rompun @; Haver/Diamond KS) prior to embryo recovery. Embryos were recovered from treated and control mares by nonsurgical uterine lavage based on a modification of the technique of Hinrichs (16). The modification consisted of infusion of sufficient volume of lavage medium to distend the uterus followed by a 3-minute wait prior to recovery of the lavage effluent on only the first lavage. Thereafter, the lavage effluent was recovered immediately from the uterus after infusion. The uterus was lavaged 3 times with modified Dulbecco’s phosphate-buffered saline supplemented with 0.3% bovine serum albumin (Sigma). Lavage effluent was collected through an embryo filter (Veterinary Concepts, Inc., Spring Valley, WI) to isolate embryos. After isolation, the embryos were assessed morphologically with an inverted, phase-contrast microscope (60 to 460x; 19). Embryo diameter was determined with an ocular micrometer. The stage of embryonic development was assigned as 1 = early blastocyst; 2 = hatching blastocyst; or 3 = expanded, hatched blastocyst. Embryo quality scores were assigned as 1 = excellent; 2 = good; 3 = fair; or 4 = poor (19).
Theriogenology
1058
After the morpholgic evaluation, embryos were fixed in phosphate-buffered saline containing 1% formalin. The number of cell nuclei was subsequently determined by staining with Hoechst 33342 (Sigma) and examination with epifluroscence microscopy and a counting reticle (20). Embryo recovery rates were compared between groups based on Fisher’s exact test (21). Embryo quality and developmental scores were compared by a Kruskal-Wallis test (22). Embryo diameters and number of cell nuclei were analyzed via the General Linear Models procedure (GLM) of Minitab (221, with treatment, developmental score and treatment-by-development interactions. Serum progesterone concentrations were compared by GLM for repeated measures (231, with treatment, time, treatment-by-time interactions, and animal- within-treatment as the main effects. Data are expressed as mean + SEM. RESULTS There was no significant difference (P > 0.25) in the embryo recovery rate from the uterus at Day 7 after ovulation between treated and control mares (Table 1). Likewise, diameter, stage of development, quality score and cell number were not significantly different for embryos from treated or control mares (Table 1). However, there were significant effects of diameter (P < 0.01) and developmental score (P = 0.06) on the number of cell nuclei without a significant treatment effect on the the number of cell nuclei. Table
1.
Recovery embryos ovulation
Recovery Group
rate
rate, diameter, development, quality, and cell number for recovered from treated and control mares at Day 7 after
Embryo diameter
Embryonic developmenta
Quality scoreb
Cell nuclei
Control
6/l 3 (46%)
449 +_88 flrn
2.3 2 0.3
1.520.3
1108+374 (n = 6)
Treated
8113 (62%)
383 +_47 pm
2.3 +0.3
1.1 +O.l
1353 + 280 (n = 7)
&”
Embryonic development was scored as 1 = early blastocyst; 2 = hatching blastocyst; or 3 = expanded, hatched blastocyst. Embryo quality scores were assigned as 1 = excellent; 2 = good; 3 = fair; or 4 = poor.
The administration of 450 mg progesterone in cottonseed oil significantly (P < 0.02) increased serum progesterone concentrations in treated mares (Figure 1). There were also significant (P < 0.01) effects of day and a day-bygroup interaction on serum progesterone concentrations. Progesterone concentrations were significantly higher in treated than in control mares by Day 1 postovulation.
1059
Theriogenology
P
01.4. 0
1
I. 2
Days
I. 3
8. 4
I 5
*
8. 6
I 7
Postovulation
Serum progesterone concentrations (mean mares (0) and treated mares (WI between ovulation.
f SEMI for control Days 0 and 7 after
DISCUSSION Administration of 450 mg progesterone to pony mares daily between Days 0 and 6 after ovulation did not significantly affect embryonic development as determined by evaluation of embryos recovered from the uterus at Day 7 after ovulation. Embryo recovery rates were similar for control and treated pony mares and appeared similar to earlier studies that examined embryo recovery rates from the uterus at Day 7 (24,251. Embryonic development, assessed by morphologic evaluation, size and number of cell nuclei, was not different for embryos recovered from treated or control mares and also appeared similar to that reported for embryos recovered from the uterus at Day 7 (5,19,26). The similarity of embryo recovery rates from the uterus of treated and control mares at Day 7 suggests that treatment with progesterone did not affect embryo transport through the oviduct. However, because embryo recovery was conducted after the expected time of passage of the embryo into the uterus at Days 5 to 6 (31, this study did not critically test the effect of progesterone on oviductal transit time of embryos.
Theriogenology
1060
Based on the results of this study, there does not appear to be an effect of exogenous progesterone on development of the equine embryo within the oviductal environment. The requirement for progestational support of the oviductal environment in the horse is uncertain. Equine embryos can develop in vitro to the blastocyst stage in co-culture with oviductal epithelium without progesterone; however, development of embryos in this co-culture system is retarded compared to development in vivo (27). In cattle, Geisert et al. (28) reported that administration of progesterone to superovulated cows on Days 1 to 4 did not enhance embryonic development. They suggested that the alterations in embryo growth observed in progesterone-treated cows were related to effects of progesterone on the uterine environment after blastocyst hatching (28). It appears likely that any effect of exogenous progesterone on embryonic development in the mare would be manifested after the embryo reaches the uterus. It has been reported that administration of progesterone increases protein and uteroferrin concentrations in the uterus of mares (291, and the synthesis or secretion of a number of uterine proteins appears to be progesterone dependent (30,311. Weithenauer suggested that administration of progesterone during the first 18 days of pregnancy increased the size of the blastocyst in the mare (I 5,32). In the cow, accelerations in blastocyst growth occur after hatching and are apparent by Day 14 in progesterone-treated animals (28,331. The number of cell nuclei in Day 7 equine blastocysts has not been previously reported. Overall the mean cell number for Day 7 blastocysts in the current study was 1251 + 218; n = 12). For embryos from both groups of mares, cell numbers increased with developmental score and with increasing embryonic diameter. This difference is most likely due to the rapid expansion of the equine blastocyst after hatching from the zona pellucida at about Day 6 after ovulation (5,191. In summary, administration of progesterone to pony mares during the first 6 days after ovulation did not affect embryonic development based on evaluation of uterine blastocysts recovered at Day 7. Other studies suggest that any effect of progesterone on embryonic development would be related to the influence of progesterone on uterine secretions. Further studies are required to assess the possible influence of exogenous progesterone on development of the equine blastocyst in utero. REFERENCES 1.
Pope, W.F. Uterine asynchrony: =:999-1003 (1988).
A cause of embryonic
loss. Biol. Reprod.
Theriogenology
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2.
Hunter, R.H.F. The Fallopian Springer-Verlag, Heidelberg,
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Freeman, D.A., embryo transport (1991).
4.
Allen, W.R. Aspects of early embryonic development in farm animals. Proc. 10th Int. Cong. Anim. Reprod. Art. Insem. Iv:(Xll)l-9 (1984).
5.
Betteridge, K.J., Eaglesome, M.D., Mitchell, D., Flood, P.F. and Beriault, R. Development of horse embryos up to twenty-two days after ovulation: observation on fresh specimens. J. Anat. m: 191-209 (1982).
6.
Roblero, L. Effect of progesterone in vivo mouse embryos. J. Reprod. Fertil. =:153-l
7.
Roblero, L.S. and Garavagno, A.C. Effect of oestradiol-17b progesterone on oviductal transport and early embryo development mouse embryos. J. Reprod. Fertil. 57:91-95 (1979).
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Heap, R.B., Rider, V. and Feinstein, antibodies and early embryo development. Reprod. Art. Insem. Iv:(l984).
9.
Eyestone, W.H. and First, N.L. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned media. J. Reprod. Fertil. 85:715-720 (I 989).
IO.
Ellington, J.E., Carney, E.M., Farrell, P.B., Simkin, M.E. and Foote, R.H. Bovine I- to 2-cell embryo development using a semi-defined medium in three oviduct epithelial cell coculture systems. Biol. Reprod. a: 100-I 07 (1990).
11.
White, K.L., Hehnke, K.E., Rickorkds, L.F., Southern, L.L., Thompson, D.L. and Wood, T.C. Early embryonic development in vitro by coculture with oviductal epithelial cells in pigs. Biol. Reprod. &l:425-430 (1989).
12.
Stonen, S.L. and Hamner, oviduct on the development (1977).
13.
Prather, R.S., Sims, M.M. and First, N.L. Culture of porcine from the one- and two-cell stage to the blastocyst stage oviducts. Theriogenology =:I 147-I 151 (I 991).
Tubes: TheirRole 1988, pp. 30-74.
in Fertility
and Infertility.
Weber, J.A., Geary, R.T. and Woods, G.L. Time through the mare oviduct. Theriogenologys:823-830
upon the rate of cleavage 55 (1973).
A.
of
of
and of
Monoclonal progesterone Proc. 10th Int. Cong. Anim.
C.E. Hormonal and regional influences of the of rabbit embryos. Biol. Reprod. I&638-646
embryos in sheep
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14.
Hinrichs, K. and Watson, E.D. Effect of administration of phenylbutazone or progesterone on recovery of embryos from the uterus of mares 5 days after ovulation. Am. J. Vet. Res. 52:678-681 (1991).
15.
Weithenauer, J., Sharp, D.C. and Sherrin, P.C. The effects of steroids on nutrients in yolk sac fluid of Day 18 pony conceptuses. Equine Vet. J. jj (Suppl.):25-28 (1989).
16.
Ginther, O.J. Ultrasonic Imaging and Reproductive Events Equiservices, Cross Plains, WI, 1986, pp. 133-169.
17.
Beal, W.E., Milvae, R.A. and Hansel, W. Oestrous cycle length and plasma progesterone concentrations following administration of prostaglandin F-2a in the bovine oestrous cycle. J. Reprod. Fertil. s:393-396 (I 980).
18.
Hinrichs, recovery (1990).
19.
McKinnon, A.O. and Squires, E.L. Morphologic embryo. J. Am. Vet. Med. Assoc. N:401-406
20.
Ball, B.A., Altschul, M. and Ellington, J.E. In vitro development of Day-2 equine embryos co-cultured with oviductal explants or trophoblastic vesicles. Theriogenology =:669-682 (1991).
21.
Beyer, W.H. CRC Handbookof Tables for Probability Press, Inc., Boca Raton, FL 1966, pp. 266-281.
22.
Ryan, B.F., Joiner, Publishers, Boston,
23.
Gill, J.L. Repeated measurement: sensitive tests few animals. J. Anim. Sci. 6;1:943-954 (1986).
24.
Douglas, R.H. Some aspects (Suppl.):405-408 (1982).
25.
Woods, G.L., Baker, C.B., Hillman, R.B. and Schlafer, D.H. Recent studies relating to early embryonic death in the mare. Equine Vet. J. 3 (Suppl.):104-107 (1985).
26.
luliano, M.F., Squires, embryos and method &Q:258-263 (1985).
in the Mare.
K. A simple technique that may improve the rate of embryo on uterine flushing in mares. Theriogenology %:937-942
B.L. and Ryan, 1985.
T.A.,Jr.
of equine embryo
assessment (1988).
of the equine
and Statistics.
CRC
Handbook.
PWS
Minitab
for experiments
transfer.
J. Reprod.
with
Fertil.
E.L. and Cook, V.M. Effect of age of equine of transfer on pregnancy rate. J. Anim. Sci.
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27.
Ball, B.A. and Miller, P.G. Survival of equine embryos cocultured with equine oviductal epithelium from the 4-8-cell to blastocyst stage after transfer to synchronous recipient mares. Theriogenology 37:979-991, 1992.
28.
Geisert, R.D., Fox, T.C., Morgan, G.L., Wells, M.E., Wettemann, R.P. M.T. Survival of bovine embryos transferred to and Zavy, progesterone-treated asynchronous recipients. J. Reprod. Fertil. =:475-482 (1991).
29.
McDowell, K.J., Sharp, D.C. and Grubaugh, W. Comparison of progesterone and progesterone + oestrogen on total and specific uterine proteins in pony mares. J. Reprod. Fertil. a (Suppl.):335-342 (1987).
30.
Hinrichs, K. and Kenney, fluid related to duration mares used as embryo (1989).
31.
A.T. and Roberts, R.M. McDowell, K.J., Sharp, D.C., Fazleabas, Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares. J. Reprod. Fertil. @:107-l 15 (1990).
32.
Weithenauer, of exogenous pony mares.
33.
Garrett, J.E., Geisert, R.D., Zavy, M.T. and Morgan, G.L. Evidence for maternal regulation of early conceptus growth and development in beef cattle. J. Reprod. Fertil. &:437-446 (1988).
R.M. Differences in protein content of uterine of progesterone treatment in ovariectomised recipients. Equine Vet. J. 8 (Suppl.):49-55
J., McDowell, K.J., Davis, S.D. and Rothman, T.K. Effect progesterone and estrogen on early embryonic growth in Biol. Reprod. 34 (Suppl.) : abstr. #I80 (1986).
INFLUENCE
38: 1055-I
063, 1992
OF EXOGENOUS PROGESTERONE ON EARLY EMBRYONIC DEVELOPMENT IN THE MARE B. A. Ball, P. G. Miller Department
Received
and P. F. Daels
of Clinical Sciences, Cornell Ithaca, New York 14853 for publication: Accepted:
University
March 24, 1992 September 28, 1992
ABSTRACT The influence of exogenous progesterone on the development of equine oviductal embryos was determined based upon the recovery of Day-7 uterine blastocysts from treated mares (n = 13) that were given 450 mg progesterone daily between Days 0 and 6 and from untreated control mares (n = 13). Daily administration of 450 mg progesterone in oil significantly (P < 0.02) increased There was no serum progesterone concentrations in the treated mares. significant difference in the recovery rate of Day-7 embryos between treated and control mares (8/l 3 versus 6/l 3, respectively). Embryonic development, assessed by morphologic evaluation, embryo diameter, and number of cell nuclei was not significantly different for embryos from treated and from control mares. The results of this study indicate that administration of progesterone beginning on the day of ovulation does not affect the embryo recovery rate or embryonic development, based on evaluation of uterine blastocysts recovered at Day 7 after ovulation. Key words:
equine,
embryo,
progesterone,
oviduct,
embryo
recovery
INTRODUCTION Synchrony between the developing embryo and the female reproductive tract, as influenced by hormonal stimulation, is critical for successful pregnancy
Acknowledgments We thank the Harry M. Zweig Memorial Fund for equine research and Thornbrook Farms for financial support; Dr. P.J. Burns for assistance in preparation of progesterone, Dr. J. Ellington for assistance with embryo staining; D. Waldow, and H. Das for technical assistance; and C. Collyer and the staff of the Equine Research Park for animal care. Portions of these results were presented at the 12th International Congress on Animal Reproduction, The Hague in August, 1992.
Copyright
Q 1992 Butterworth-Heinemann
1056
Theriogenology
in domestic animals. Synchrony between the embryo and the uterus appears to be more important for proper embryo-endometrial interactions, and coordination of development within the oviduct seems less critical (I 1. Steroids influence oviductal function, resulting in changes in fluid volume, electrolyte composition as well as in qualitative and quantitative changes in oviductal proteins (2). Therefore, alterations in the timing or the amount of progesterone present during the period of oviductal transit might alter oviductal transit or affect early embryonic development. Exogenous progesterone therapy is sometimes initiated during the immediate postovulatory period in mares. The equine embryo resides in the oviduct during the first 5 to 6 days after ovlation (31, and the equine embryo reaches a later stage of development prior to entry into the uterus than in other domestic animals (4,5). Therefore, the administration of progesterone to mares during the immediate postovulatory period might result in perturbations of the oviductal environment or oviductal transit. The effect of administration of progesterone following ovulation depends on the species under study and on the timing of progesterone administration relative to ovulation (2). In mice, progesterone appears to be important for normal development of cleavage-stage embryos and their transport through the oviduct (6-8). However, embryos from many domestic animals, including the horse, can develop in vitro from the zygote to blastocyst stage without the addition of progesterone to the co-culture (9-l 1). In addition, transferred rabbit zygotes undergo normal cleavage in the oviducts of ovariectomized rabbits (121, and the oviducts of anestrous ewes are frequently used as a surrogate host to support the development of heterologous embryos (13). These studies indicate that progesterone may be facilatory rather than obligatory in regulating development of oviductal embryos. Few investigators have examined the influence of exogenous progesterone on early embryonic development in the mare. Hinrichs and Watson examined the effect of administration of 250 mg progesterone at Days 0, 1 and 2 after ovulation on embryo collection from the uterus at Day 5 (14). They observed no difference in the embryo recovery rates between treated and control mares and concluded that administration of progesterone did not hasten transport of Weithenauer et al. (15) examined the effect of daily equine embryos. administration of either 150 mg or 450 mg of progesterone on the growth of the conceptus in utero between Days 10 and 18 and on the content of yolk sac fluid at Day 18. The concentration of proteins and glucose in yolk sac fluid was increased with the administration of 450 mg of progesterone, and there was a nonsignificant increase in growth of the conceptus as determined by ultrasonography.
Theriogenology
The objective of the current study was to determine the effect of administration of progesterone at Days 0 to 6 after ovulation on the development of the equine conceptus through Day 7. MATERIALS
AND METHODS
during estrus, and Pony mares fn = 26) were artificially inseminated ovulation was detected by daily transrectal ultrasonography (16). On the day that ovulation was detected (Day 01, mares were alternately assigned to either a treated or control group. Treated mares (n = 13) were given 450 mg progesterone (100 mg/ml progesterone dissolved in 20% benzyl alcohol in cottonseed oil; Sigma Chemical Co., St. Louis, MO) by intramuscular injection on a daily basis from Days 0 through Day 6. Control mares (n = 13) remained untreated. Jugular blood samples were taken daily beginning on Day 0 (prior to administration of progesterone) through Day 7 from treated and control mares. The blood was allowed to clot at ambient temperature, serum was separated by centrifugation and was stored at -20” C prior to radioimmunoassay for progesterone. Aliquots of serum (100 ,uI) were extracted in duplicate with petroleum ether and were assayed as described previously (17). The progesteroneantiserum usedcross-reacts 13.6% with 5/3_dihydroprogesterone, less than 6% with other progestins tested, and less than 0.01% with Sensitivity of the assay was 12 pglassay androgens and estrogens tested. tube or 0.12 ng/ml, and the intra- and inter-assay coefficients of variation were 9 and 6%, respectively. On Day 7 after ovulation, mares were restrained in stocks and were sedated Scientific; Shawnee, with 0.3 mg/lb xylazine HCI (Rompun @; Haver/Diamond KS) prior to embryo recovery. Embryos were recovered from treated and control mares by nonsurgical uterine lavage based on a modification of the technique of Hinrichs (16). The modification consisted of infusion of sufficient volume of lavage medium to distend the uterus followed by a 3-minute wait prior to recovery of the lavage effluent on only the first lavage. Thereafter, the lavage effluent was recovered immediately from the uterus after infusion. The uterus was lavaged 3 times with modified Dulbecco’s phosphate-buffered saline supplemented with 0.3% bovine serum albumin (Sigma). Lavage effluent was collected through an embryo filter (Veterinary Concepts, Inc., Spring Valley, WI) to isolate embryos. After isolation, the embryos were assessed morphologically with an inverted, phase-contrast microscope (60 to 460x; 19). Embryo diameter was determined with an ocular micrometer. The stage of embryonic development was assigned as 1 = early blastocyst; 2 = hatching blastocyst; or 3 = expanded, hatched blastocyst. Embryo quality scores were assigned as 1 = excellent; 2 = good; 3 = fair; or 4 = poor (19).
Theriogenology
1058
After the morpholgic evaluation, embryos were fixed in phosphate-buffered saline containing 1% formalin. The number of cell nuclei was subsequently determined by staining with Hoechst 33342 (Sigma) and examination with epifluroscence microscopy and a counting reticle (20). Embryo recovery rates were compared between groups based on Fisher’s exact test (21). Embryo quality and developmental scores were compared by a Kruskal-Wallis test (22). Embryo diameters and number of cell nuclei were analyzed via the General Linear Models procedure (GLM) of Minitab (221, with treatment, developmental score and treatment-by-development interactions. Serum progesterone concentrations were compared by GLM for repeated measures (231, with treatment, time, treatment-by-time interactions, and animal- within-treatment as the main effects. Data are expressed as mean + SEM. RESULTS There was no significant difference (P > 0.25) in the embryo recovery rate from the uterus at Day 7 after ovulation between treated and control mares (Table 1). Likewise, diameter, stage of development, quality score and cell number were not significantly different for embryos from treated or control mares (Table 1). However, there were significant effects of diameter (P < 0.01) and developmental score (P = 0.06) on the number of cell nuclei without a significant treatment effect on the the number of cell nuclei. Table
1.
Recovery embryos ovulation
Recovery Group
rate
rate, diameter, development, quality, and cell number for recovered from treated and control mares at Day 7 after
Embryo diameter
Embryonic developmenta
Quality scoreb
Cell nuclei
Control
6/l 3 (46%)
449 +_88 flrn
2.3 2 0.3
1.520.3
1108+374 (n = 6)
Treated
8113 (62%)
383 +_47 pm
2.3 +0.3
1.1 +O.l
1353 + 280 (n = 7)
&”
Embryonic development was scored as 1 = early blastocyst; 2 = hatching blastocyst; or 3 = expanded, hatched blastocyst. Embryo quality scores were assigned as 1 = excellent; 2 = good; 3 = fair; or 4 = poor.
The administration of 450 mg progesterone in cottonseed oil significantly (P < 0.02) increased serum progesterone concentrations in treated mares (Figure 1). There were also significant (P < 0.01) effects of day and a day-bygroup interaction on serum progesterone concentrations. Progesterone concentrations were significantly higher in treated than in control mares by Day 1 postovulation.
1059
Theriogenology
P
01.4. 0
1
I. 2
Days
I. 3
8. 4
I 5
*
8. 6
I 7
Postovulation
Serum progesterone concentrations (mean mares (0) and treated mares (WI between ovulation.
f SEMI for control Days 0 and 7 after
DISCUSSION Administration of 450 mg progesterone to pony mares daily between Days 0 and 6 after ovulation did not significantly affect embryonic development as determined by evaluation of embryos recovered from the uterus at Day 7 after ovulation. Embryo recovery rates were similar for control and treated pony mares and appeared similar to earlier studies that examined embryo recovery rates from the uterus at Day 7 (24,251. Embryonic development, assessed by morphologic evaluation, size and number of cell nuclei, was not different for embryos recovered from treated or control mares and also appeared similar to that reported for embryos recovered from the uterus at Day 7 (5,19,26). The similarity of embryo recovery rates from the uterus of treated and control mares at Day 7 suggests that treatment with progesterone did not affect embryo transport through the oviduct. However, because embryo recovery was conducted after the expected time of passage of the embryo into the uterus at Days 5 to 6 (31, this study did not critically test the effect of progesterone on oviductal transit time of embryos.
Theriogenology
1060
Based on the results of this study, there does not appear to be an effect of exogenous progesterone on development of the equine embryo within the oviductal environment. The requirement for progestational support of the oviductal environment in the horse is uncertain. Equine embryos can develop in vitro to the blastocyst stage in co-culture with oviductal epithelium without progesterone; however, development of embryos in this co-culture system is retarded compared to development in vivo (27). In cattle, Geisert et al. (28) reported that administration of progesterone to superovulated cows on Days 1 to 4 did not enhance embryonic development. They suggested that the alterations in embryo growth observed in progesterone-treated cows were related to effects of progesterone on the uterine environment after blastocyst hatching (28). It appears likely that any effect of exogenous progesterone on embryonic development in the mare would be manifested after the embryo reaches the uterus. It has been reported that administration of progesterone increases protein and uteroferrin concentrations in the uterus of mares (291, and the synthesis or secretion of a number of uterine proteins appears to be progesterone dependent (30,311. Weithenauer suggested that administration of progesterone during the first 18 days of pregnancy increased the size of the blastocyst in the mare (I 5,32). In the cow, accelerations in blastocyst growth occur after hatching and are apparent by Day 14 in progesterone-treated animals (28,331. The number of cell nuclei in Day 7 equine blastocysts has not been previously reported. Overall the mean cell number for Day 7 blastocysts in the current study was 1251 + 218; n = 12). For embryos from both groups of mares, cell numbers increased with developmental score and with increasing embryonic diameter. This difference is most likely due to the rapid expansion of the equine blastocyst after hatching from the zona pellucida at about Day 6 after ovulation (5,191. In summary, administration of progesterone to pony mares during the first 6 days after ovulation did not affect embryonic development based on evaluation of uterine blastocysts recovered at Day 7. Other studies suggest that any effect of progesterone on embryonic development would be related to the influence of progesterone on uterine secretions. Further studies are required to assess the possible influence of exogenous progesterone on development of the equine blastocyst in utero. REFERENCES 1.
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