Summary Introduction

E F F E C T OF P R O G E S T E R O N E INDUCED I N C R E A S E IN U T E R I N E S E C R E T O R Y A C T I V I T Y ON D E V E L O P M E N T O F T H E P O R C I N E CONCEPTUS 1,2 James W. Knight, Fuller W. Bazer and H. D. Wallace University o f Florida, Gainesville 32611

Summary

.05) heavier empty uterine weight, greater Twelve crossbred gilts were randomly allantoic fluid volume and longer placentae. assigned to be either sham-operated or bi- Total allantoic fluid protein was not differlaterally ovariectomized on day 4 after onset ent in the SO-CO vs. SO-lIP ~ t s , but was of estrus and treated as follows: (1) sham- significantly (P < .01) greater in the OVX-HP operated gilts received either corn oil alone vs. OVX-LP gilts. In general, progesterone (SO-CO) or 3.3 mg progesterone (P) and therapy had no significant affect on embry0.55 ug estradiol (E)/kg/day (SO-HI') and (2) onic survival or embryonic development, but bilaterally ovariectomized gilts received either it did affect development of the placentae 1.1 mg P and 0.55 ug E/kg/day (OVX-LP) and allantoic fluid volume at day 40 of or 3.3 mg P and 0.55 ug E/kg/day (OVX- pregnancy. HP). These gilts were treated from day 4 to Introduction day 15 after onset of estrus when uterine flushings were obtained and total recoverable Conflicting results have been reported as to uterine protein determined. Total protein the effects of progesterone and estrogen recovered was 87.7, 133.4, 86.1 and 133.5 therapy on embryonic survival in gilts. mg for the SO-CO, SO-HP, OVX-LP and Reddy, Lasley and Mayer (1958) and Day et OVX-I-IP groups, respectively. The quantity al. (1959) presented evidence that certain of uterine protein recovered was significantly dosages of exogenous progesterone and estra( P < .01) greater for the SO-HP and OVX- diol may enhance embryonic survival, whereHP groups. as, Haines, Warnick and Wallace (1958) and Thirteen contemporary gilts of the same Spies et at (1959) reported that proage and weight were randomly assigned to gesterone-estrogen therapy had no effect on the same treatments after breeding, and were embryonic survival. In contrast to these also either sham-operated or bilaterally ova- results, Sammelwitz, Dziuk and Nalbandov riectomized on day 4 after onset of estrus. (1956) indicated that certain levels of exHowever, in these gilts, treatment was carried ogenous progesterone may be detrimental to out from day 4 to day 40 of gestation when embryonic survival. the gilts were hysterectomized. There were Knight, Bazer and Wallace (1973a) demonno significant treatment effects on concep- strated a positive relationship between quantion rate, number of corpora lutea, percent tity of luteal tissue and quantity of recoverembryo survival, number of embryos or dry able uterine protein on day 15 of the weight of the embryos. The SO-HP and estrous cycle. They also detected a positive OVX-HP treated gilts had significantly ( P < correlation between level of progesterone administered between days 4 and 15 after 1Department of Animal Science, Florida Agri- onset of estrus and quantity of uterine cultural Experiment Station Journal Series No. 5244. protein recovered (Knight et al., 1973b). 2This research was supported by U.S.D.A. Grant Therefore, this study was designed to evaluNO. 12-14-100-9962(44). ate the effects of two different levels of 743 JOURNAL OF ANIMAL SCIENCE, vol. 39, no. 4, 1974

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progesterone therapy at a constant level of estradiol on recoverable uterine protein and development of the porcine conceptus to day 40 of gestation. Materials and Methods E x p e r i m e n t One. Twelve crossbred gilts which had exhibited at least one estrous cycle were randomly assigned to be 'either sham-operated or bilaterally ovariectomized on day 4 after onset ofestrus(day ofestrus = day 0). Sham-operated gilts received either corn oil (4 ml) daily (SO-CO) or 3.3 mg progesterone (P) and 0.55 ug estradiol (E)/kg/day (SO-HP). Bilaterally ovariectomized gifts received either 1.1 mg P and 0.55 ug (E)/kg/day (OVX-LP) or 3.3 mg P and 0.55 /ag (E)/kg/day (OVX-HP). Treatment was initiated on day 4 and continued until day 1 5 after onset of estrus when uterine flushings were obtained to determine total recoverable uterine protein secretions as previously described by Knight et al. (1973a). The hormones were dissolved in corn oil and absolute ethanol (90:10) and administered subcutaneously in the neck region each day. Previous studies indicated that ovariectomized gilts treated with corn oil alone had little recoverable uterine protein (Knight et al. 1973a). E x p e r i m e n t Two. Thirteen contemporary crossbred gilts of the same age and weight as the females used for Experiment One, were mated at 12 and at 24 hr. after onset of estrus. They were randomly assigned to be either sham-operated or bilaterally ovariectomized on day 4 of pregnancy and treated as SO-CO, SO-HP, OVX-LP or OVXHP groups from day 4 to day 40 of gestation. On day 40 of gestation they were hysterectomized so that the uterus and its contents could be examined. Data were coUected so that treatment comparisons could be made with respect to number of corpora lutea (CL), number of live embryos, percent embryo survival, embryo wet weight and dry weight, embryo crown-rump length, placental length, placental wet weight, aUantoic fluid volume, allantoic fluid protein concentration (using the method of Lowry et al., 1951), allantoic fluid total protein, total allantoic fluid protein per litter and empty uterine weight. Embryo dry weight was determined by drying each embryo to a constant weight, generally for 72 hr., at 100C in a drying oven. Weights were determined using an analytical balance. Fluid

volumes were measured directly in a graduated cylinder and measures of length were determined with a metric rule. Analysis of variance and orthogonal comparisons were the statistical techniques utilized to evaluate the data (Steel and Torrie, 1960). Results and Discussion E x p e r i m e n t One. The quantity (X + SE) of recoverable uterine protein recovered from the SO-CO, SO-HP, OVX-LP and OVX-HP treated gilts was 87.66 +_ 6.54, 133.44 + 26.16, 86.13 -+ 15.38 and 133.54 + 27.06 mg, respectively. Difference between the SO-CO vs. OVX-LP and SO-HP vs. OVX-HP were not significantly ( P < .05) different. However, the quantity of recoverable protein for the SO-HP and OVX-HP was significantly ( P < .01) greater than that obtained from the SO-CO and OVX-LP treated gilts. On the basis of these data, it was assumed that secretion of uterine proteins would be increased in a similar manner for pregnant gilts studied in Experiment Two. E x p e r i m e n t Two. Results of the experiment are summarized in table 1. Treatment had no effect on pregnancy rate, number of live embryos or percent embryo survival at day 40 of gestation. These findings are consistent with previous reports (Haines et al., 1958; Reddy et al., 1958; Day et al., 1959; Spies et al., 1959; Webel et al., 1972) which indicated that exogenous progesterone therapy did not result in a significant (P < .05) improvement in either litter size or percent embryo survival. There was no evidence of a detrimental effect of the high progesterone level on litter size or percent embryo survival in the SO-HP and OVX-HP groups compared with the SO-CO and OVX-LP groups. In this respect, these data are not in agreement with data reported by Sammelwitz et al. (1956). Embryo wet weight and crown-rump length were significantly ( P < .05) greater for the SO-CO group as compared with embryos from gilts in the other treatment groups. However, there was no significant ( P < .05) treatment effect on the dry weight of the embryos. This indicates that the embryos from the SO-CO group contained more moisture and may have been longer because of distension resulting from water accumulation or, stated in another way, embryos from the other three treatments were relatively dehydrated. Placental length was significantly (P < .05)

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DEVELOPMENT OF THE PORCINE CONCEPTUS TABLE 1. E F F E C T OF P R O G E S T E R O N E T H E R A P Y ON DEVELOPMENT OF THE PORCINE CONCEPTUS TO DAY 49 OF GESTATION

Treatments, a,b,c Item

SO-CO

No. of females 3 Females pregnant 3 No. c o r p o r a l u t e a , avg 12.0-+ 0 No. live embryos, avg 10.3 -+ 0.7 Embryo survival, avg % 86.1 +- 2.0 Empty uterine wt, avg, g 1496.4 + 30.2a Fetal wet wt, avg, g 10.6 +- 0.2 a Fetal d r y w t , avg, g 2.0 + 0.04 Crown-rump length, avg, mm 50.2 -+ 0.3a Placental length, avg, cm 55.1 -+ 2.0a Placental wt, avg, g 61.0 +- 2.4a Allantoic fluid vol, avg, ml 93.9 +- 11.5 a Allantoic fluid protein concentration avg, mg/ml 5.9 -+ 0.5a Allantoic fluid total protein, avg, g 441.0 +_ 22.1a Total allantoic fluid protein/litter, avg, g 4.4 + 0.03a

SO-HP

OVX-LP

OVX-HP

3 3 11.7 -+ 0.7 9.7 +- 0.3 82.9 +- 2.9

4 4 13.3-+ 0.5 11.8 + 0.9 88.7 + 3.3

1903.1 +- 59.2b 9.5 +- 0.3b 1.9 +- 0.03

1217.3 +- 34.4 c 9.2-+ 0.2b 1.9 +- 0.04

1879.0 +- 64.1b 9.3 +- 0.2 b 1.9 -+ 0.02

48.2 + 0.5b

47.8 + 0.6 b

47.5 -+ 0.4b

62.4 +- 3.5 b 68.5 -+ 3.8b

57.4 + 2.8 a 53.8 -+ 3.6 c

65.2 -+ 3.0 b 54.3 +- 2.8c

147.5 -+ 14.3 b

94.4 + 13.9a

177.4 +- 20.4b

3.9 -+ 0.4b

5.2 + 0.7 a

3.2 +- 0.2b

430.3 + 21.5 a

308.4 +- 21.2b

426.4 +- 24.8a

3 3 14.7-+ 1.7 13.0 -+ 1.2 88.6 -+ 3.8

5.0 +- 0.02a

2.9 -+ 0.02 b

4.4 + 0.02a

a V a l u e s represent means +- S E . bMearLs in t h e same row w i t h different superscript letters are significantly (P < .05) different f r o m each other. CSee t e x t for e x p l a n a t i o n of treatments.

increased by about 7 cm in the SO-HP and OVX-HP treated gilts compared with the SO-CO and OVX-LP treated gilts. Allantoic fluid volume was also significantly greater (P < .05) in the SO-HP vs. SO-CO (147.49 vs. 93.90 ml) and in the OVX-HP vs. OVX-LP (177.44 vs. 94.36 ml) groups. The combination of those two factors, i.e., increased placental length and allantoic fluid volume, are critical factors in determining the placental surface area which achieves contact with the maternal endometrium. Brambell (1933) found that the number of placental areolae is related to placental surface area and becomes fixed at about day 35 of gestation. The number of areolae may be critical since they represent the placental sites of absorption o f nutrients from the uterine glands. Chen, Bazer and Roberts (1973) have reported the presence of the purple porcine Fraction IV uterine protein in allantoic fluid after day 30 o f gestation which indicates that proteins from the uterine glands may be transported into the placenta and fetal fluids via the areolae.

Placental weights were significantly ( P < .05) greater in the sham-operated compared to the bilaterally ovariectomized females and the SO-HP treated gilts had significantly (P < .05) heavier placentae than those from gilts in the other three treatment groups. Since an increase in placental length precedes the increase in placental weight in normal development o f the porcine placentae (Warwick, 1928), this experiment may have been terminated too early to fully evaluate the effect of treatment qn placental weight. In contrast to these results, passive immunization of gilts with antisera against one of the pig uterine proteins (Fraction IV) resulted in a significant reduction in placental weight and length (Chen and Bazer, 1973). Allantoic fluid total protein per conceptus and per litter was significantly (P < .05) less for the OVX-LP group compared with the other three treatment groups. This suggests that the combination of exogenous and endogenous progesterone and estrogen used in this study did not significantly ( P < .05) affect the

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accumulation of protein in the allantoic fluid of the sham-operated gilts. However, the secretory activity of the uterine glands and/or the availability of protein from other sources, e.g., maternal blood, was significantly ( P < .05) decreased in the OVX-LP compared with the OVX-HP treated gilts. Collectively, these data suggest that increased uterine secretory activity, which resuited from the high progesterone level, enhanced placental development. This may be accomplished by an increase in allantoic fluid volume which leads to an increase in the placental surface area which is in contact with the maternal endometrium, particularly the placental areolae surface area which may serve as the area for nutrient and water absorption (Brambell, 1933; Chen et al., 1973). The establishment of maximum placental surface area early in gestation may be of critical importance with respect to fetal growth and survival as pregnancy progresses towards term. Further experiments are necessary to determine whether or n o t we must evaluate the development of the placental membranes, and not simply the number of embryos, in order to fully understand the problem of prenatal mortality in swine.

Literature Cited Brambell, Charles E. 1933. Atlantochorionic differentiations of the pig studied morphologically and histochemically. Amer. J. Anat. 52: 397.

Chen, T. T. and Fuller W. Bazer. 1973. Effect of antiserum to porcine fraction IV uterine protein on the conceptus. J. Anim. Sci. 37:304. (Abstr.). Chen, T. T., Fuller W. Bazer and R. M. Roberts. 1973. A study of porcine lavender protein fraction IV. J. Anim. Sci. 37:304. (Abstr.). Day, B. N., L. L. Anderson, M. A. Emmerson, L. N. Hazel and R. M. Melampy. 1959. Effect of estrogen and progesterone on early embryonic mortality in ovariectomized gilts. J. Anim. Sci. 18:607. Haines, C. E., A. C. Warnick and H. D. Wallace. 1958. The effect of exogenous progesterone and level of feeding on prenatal survival in gilts. J. Anim. Sci. 17:879. Knight, J. W., Fuller W. Bazer and H. D. Wallace. 1973a. Effects of superovulation and unilateral ovariectomy-hysterectomy on porcine uterine secretions. J. Anim. Sci. 36:61. Knight, J. W., Fuller W. Bazer and H. D. Wallace. 1973b. Porcine uterine response to progesterone therapy. J. Anim. Sci. 36:211. (Abstr.). Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. Reddy, V. B., J. F. Lasley and D. T. Mayer. 1958. Hormonal modification of the intrauterine environment in swine and its effect on embryonic viability. Mo. Agr. Exp. Sta. Res. Bull. 667. Sammelwitz, P. H., P. J. Dziuk and A. V. Nalbandov. 1956. Effects of progesterone on embryonal mortality of rats and swine. J. Anita. Sci. 15:1211. (Abstr.). Spies, H. G., D. R. Zimmerman, H. L. Self and L. E. Casida. 1959. The effect of exogenous progesterone on formation and maintenance of the corpora lutea and on early embryo survival in pregnant swine. J. Anim. Sci. 18:163. Steel, R. G. D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill. New York. Warwick, B. L. 1928. Prenatal growth of swine. J. Morph. 46:59. Webel, S. K., T. J. Reimers, P. Martin and P. J. Dziuk. 1972. Plasma progesterone and embryo survival in gilts. J. Anim. Sci. 32:256. (Abstr.).