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Exogenous steroid effects on litter size and early embryonic survival in swine
THERIOGENOLOCY
EXOGENOUS STEROID EFFECTS ON LITTER SIZE AND EARLY EMBRYONIC SURVIVAL IN SWINE W. F. de Sa, P. Pleumsamran, C. B. Morcom and W. Richard Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48824 Received for publication:
7124180
ABSTRACT These studies confirm the efficacy of 12.5 pg estrone and 25 mg progesterone, administered on Days 16 and 17 of pregnancy, on increased litter size in swine. Substitution of estradiol (with ten-fold greater biological activity) for estrone in this regimen eliminated the positive effect on litter size and estradiol given alone at dosages of 3.1 and 6.3 ug were without effect. However, when estradiol was given in combination with progesterone (25 mg) at a dosage equivalent to 10% the effective estrone dosage (1.25 ug), a 22.6% increase in total pigs born per litter over controls was observed. Estrone alone at dosages of 6.25 and 12.5 ug did not alter litter size. At a dosage of 18.75 ug, an 8.7% decrease in total pigs born was noted. The amniotic fluid from sows treated for ten days with 12.5 pg estrone and 25 mg progesterone near the time of implantation, had significantly higher protein concentration, pH and osmolality. INTRODUCTION Conflicting results have been reported on the effects of exogenous progesterone and estrogen on embryonic mortality in swine. In 1956, Sammelwitz et al. (1) treated gilts with progesterone at 50, 100, 200 and 400 mg daily for 26 days after mating with little beneficial effect. Later, others reported that either raising plasma progesterone concentrations two-fold or reducing them by half did not affect embryonic survival (2, 3, 4). In contrast, a positive response (5) to progesterone and estrone on embryonic mortality was reported (6). In this study, two ratios of progesterone to estrone were used, 1000:1 and 2000:l. In the latter group, with estrone and progesterone given at dosages of 12.5 pg and 25 mg daily, respectively, (for ten days beginning either on Day 4 or 14). there was a highly significant increase in litter size by 55 days of gestation. Exogenous progesterone and estradiol, given from Days 11 to 25 or 17 to 25, had a trend towards increased litter size; however, the difference was not significant. Three treatments were used, all at a progesterone:estradiol ratio of 500:1 (5). The authors acknowledge the technical assistance of Drs. P. T. McGovern, D. E. Wildt, W. T. Magee, and Mrs. Julie Howe-Baughman during the conduct of these studies. Supporting funds were received from EMBRAPA (Empresa Brasileira de pesquisa Agro-pecuaria) and the Agency for International Development.
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THERIOGENOLOGY
Wildt et al. (7) repeated earlier studies (5) with modification and reported that a dose of 25 mg progesterone and 12.5 ug estrone, given for 2, 5 or 10 consecutive days near implantation, resulted in a significant increase in litter size at term. However, estrone or progesterone alone, as well as higher dosages of estrone and progesterone in combination, had no effect. The present study was designed to further investigate the temporal relationships between the time of steroid administration and litter size. MATERIALS AND METHODS Experiment 1: Initially a field trial was carried out at the Michigan State University Swine Facility. A total of 88 multiparous sows, which were held in confinement and hand-mated, were used. Every other sow that came into estrus served as a control (2 ml cottonseed oil (CSO); whereas, treated sows received 25 mg progesterone and 12.5 ng estrone in 2 ml CSO (5). All injections were IM into either the ham or anterior scapular region and all injections were given on Day 16 and 17 of gestation based on earlier studies (7). Experiment 2: This series of trials was designed to examine the substitution of estradiol for estrone (with an approximate biological activity ten-fold higher) alone, and in combinat'?n with progesterone. All animals were sows of the Duroc,Yorkshire and Hampshire breeds and Yorkshire-Hampshire crossbreds. Animals were randomly assigned to treatment within breed types. All injections were in 2 ml arachis oil (AO) on Days 16 and 17 of gestation. The change in the control vehicle was to standardize this factor with other ongoing research. No differences in the rate of absorption of steroids between CSO and A0 are known. Control animals received the vehicle alone. The total number of pigs born and the number born alive, as well as litter weight at birth and piglet birth weight, were recorded. In this series of experiments the procedure of least squares was used to obtain estimates of treatment means (adjusted for breed of sow, breed of boar and parity) and the error mean square. Student's t test was used to compare the mean of each treated group to the control group. In Trial C, estrone alone was tested at dosages of 6.25, 12.50 and lg.75 pg with appropriate controls. In Trial D, which was conducted totally with Yorkshire sows, the effect of a single-day injection schedule was examined. All injections were with 2 ml A0 administered IM in the neck region on Day 16 of gestation. Sows received estrone alone (12.5 up>, progesterone alone (25 mg) or a combination of the two steroids at the dosages indicated. The significance was measured by Student's t test. Experiment 3: Previous studies (8) have shown that sows treated with 25 mg progesterone and 12.5 pg estrone daily between Days 14 and 23 of gestation showed an increase of 15% for the chorionic surface area and an increase in allantoic fluid volume when examined at 30 to 35 days of gestation. In contrast, there were no significant differences in these parameters in sows examined at 46 to 50 days of gestation. In the
246
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THERIOCENOLOGY present experiment, allantoic and amniotic fluid samples from the sows which were killed at 30 to 35 days of gestation were examined for protein bands on electrophoresed acrylamide gels. The electrophogesis technique has been previously described (9). For separation, 7+.5% acrylamide lower gel was used with tris-glycine buffer at pH 8.6. Proteins were separated by applying a constant current of 2.0 mA per tube for two hours and then staining with Amid0 black, followed by destaining. Rf values for migrating protein bands were calculated and compared for treated and control animals. RESULTS Experiment 1: Table 1 shows the mean number of pigs (total born and born alive) for the treated and controf sows. Table 1:
Effect of 12.5 pg estrone and 25 mg progesterone (given on Days 16 and 17 of gestation) on sow litter size.
Treatment
No. of sows
MeanL/sow born alive
Mean/sow total born
Control Total treated Ram injected Neck injected
45 43 18 25
10.0 10.9 10.5 11.2
10.9 11.7 11.1 12.1
+ 0.4 T 0.4 '7 0.5 - 0.6
+ + & +
0.3 0.4 0.5 0.6
1 mean + S.E. The overall distribution of litter sizes in field trials is more significant than total increases in pigs born. Many factors affect litter size, and several small litters can mask a significant increment in litter size. Tables 2 and 3 show the distribution of litters arranged by size categories. The mean number of total pigs born was significantly (PcO.05) different from controls (neck injections). Table 2.
Effect of 12.5 pg estrone and 25 mg progesterone (given on Days 16 and 17 of gestation) on distribution of litter size based on total pigs born.
Litter size
<7
7-a
9-11
12-13
.13
Control Treated
1 (2.2%) 1 (2.3%)
2 (4.4%) 4 (9.3%)
24 (53.3%) 12 (27.9%)
11 (24.2%) 16 (37.3%)
7 (15.6%) 10 (23.2%)
Table 3.
Effect of 12.5 ug estrone and 25 mg progesterone (given on Days 16 and 17 of gestation) on distribution of litter size based on pigs born alive.
Litter size
<7
7-a
9-11
12-13
>13
Control Treated
1 (2.2%) 2 (4.7%)
5 (11.1%) 4 (9.3%)
30 (66.7%) 19 (44.2%)
5 (11.1%) 13 (30.2%)
4 (8.9%) 5 (11.6%)
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THERIOCENOKICY
It was found preferable to make all injections in the neck region (anterior to the scapula), not only based on litter size results, but also because of occasionally observed leakage of injection material following injections into the ham. Experiment 2: (Trial A, estradiol (12.5 ug) alone, progesterone (25 mg) alone and in combination). The percentage of sows becoming pregnant at first breeding to the control and three treatments given above was 85.0, 83.3, 93.8 and 93.8% respectively. The results of the experiment are shown in Table 4. Table 4.
Treatment
Effect of estradiol (12.5 ng) and progesterone (25 mg) alone and in combination (Days 16 and 17) on litter size and birth weight.l
No. of sows
Control Estradiol alone Progesterone alone Combination
17 13 15 15
Total pigs born 12.7 14.2 13.4 12.5
+ T T +
0.7 0.72 0.7 0.7
Live pigs born
Mean/sow birth wt/ litter (kg)
Mean/sow birth wt/ pig (kg)
11.7 13.3 12.0 11.3
17.6 17.8 17.2 17.3
1.6 1.4 1.7 1.8
-I0.7 T 0.82 T 0.7 5 0.7
+ T T 5
1.0 1.1 1.1 1.1
+ 0.4 +- 0.4 7- 0.4 z 0.4
, >alues are least squares regression means f. S.E. Significantly (P
Effect of estradiol alone at dosages of 3.125 and 6.250 ug (Days 16 and 17) on litter size and birth weight.1
Treatment Control Estradiol (3.125pg) Estradiol (6.25Opg)
No. of sows 17 6 9
Total pigs born
Live pigs born
12.7 + 0.7 11.7 + 0.7 13.1 T 1.1 12.2 'T 1.1 12.1 F 0.9 11.0 5 0.9
Mean/sow Mean/sow birth wtf birth wt/ litter(kg) pig (kg) 17.6 + 1.0 1.6 + 0.4 17.3 + 1.7 1.6 T 0.6 17.9 5 1.4 2.2 - 0.5
1
Values are least square regressions 2 S.E.
No significant differences were observed in litter size or birth weights due to treatments at these dosages.
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THERIOCENOLOGY In the subsidiary trial to Trial B (estradiol at a dosage of 1.25 pg with and without progesterone at 25 mg) injection of estradiol alone did not alter litter size or birth weight. The combination of estradiol and progesterone resulted in a 22.6% increase in total pigs per litter and a 17.9% increase in live pigs per litter (Table 6). Table 6.
Effect of estradiol (1.25 ng) alone and in combination with progesterone (25 mg), on Days 16 and 17, on litter size and birth weighta
No. of sows
Treatment Control Estradiol alone (1.25 ug) Combination
11 11 8
Total pigs born
Live pigs born
Mean/sow birth wt/ litter (kg)
8.4 + 0.8 7.8 4 0.7 10.3 + 0.8
8.3 + 0.9 7.4 + 0.7 9.8 + 0.7
14.3 + 1.2 12.3 2 1.4 13.8 + 1.7
1 Values expressed as raw means + S.E. Trial C (estrone alone at dosages of 6.25, 12.50 and 18.75 ug). The percentage of sows becoming pregnant after first breeding in the control and three treatment groups listed above were 85.0, 83.3, 76.9 and 83.3%, respectively. The results of the experiment are shown in Table 7. Table 7.
Effects of estrone alone at dosages of 7.25, 12.50 and 18.75vg (Days 16 and 17) on litter size and birth weight.1
Treatment Control Estrone (6.25 up) Estrone (12.50 ug) Estrone (18.75 ug)
No. of sows 17 10 10 10
Total pigs born
Live pigs born
Mean/sow Mean/sow birth wt/ birth wt/ litter (kg) pig (kg)
12.7 + 13.0 + 13.12 11.6 +
11.7 12.0 12.3 10.5
17.6 17.0 17.4 16.2
0.7 0.9 1.0 0.9
2 2 + +
0.7 0.9 0.9 0.7
4 + 4 t
1.0 1.4 1.3 1.3
1.6 1.4 1.8 1.7
+ 0.4 ? 0.5 2,0.5 + 0.5 _
'Values are least square regressions 2 S.E. Administration of estrone at the two lower dosages did not alter litter size or birth weights. On the other hand, estrone at 18.75 ug resulted in a reduction of both total pigs born and pigs born alive. The percent reduction for this treatment on those two parameters was 8.7 and 10.3% respectively. Trial D (estrone (12.5 ng) and progesterone (25 mg) alone and in combination given only on Day 16). Treatment with estrone alone resulted in increases for the total pigs born and live pigs born of 20.4 and 7.4% respectively (Table 8). Corresponding values for the combination treatment were 9.7 and 12.0% respectively. These were not statistically significant. Birth weights were not available on these pigs.
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THERIOGENOLOGY
Table 8.
Effect of estrone (12.5 ug) and progesterone (25 mg) alone and in combination, given only on Day 16, on litter size.1 No.
Treatment
of sows
Control 12 Estrone alone 8 Progesterone alone 6 Combination 7
Total pigs born
Live pigs born
11.3 13.6 11.3 12.4
10.8 11.6 12.1 12.1
+ T + z
0.9 1.1 1.1 1.1
+ + + +
0.9 0.8 1.2 1.0
lValues expressed as raw means + S.E. Experiment 3: The protein concentration, pH and osmolarity measured in fetal fluids recovered between Days 30 and 35 of gestation on sows which received a ten day course of injections with 12.5 pg estrone and 25 mg progesterone daily are shown in Table 9. A significant (PcO.01) increase was found in the pH of the treated sows allantoic fluid when compared to that of the control animals. Allantoic fluid protein concentration was significantly higher in control animals (PcO.05). In the amniotic fluid, the protein concentration was three-fold higher in treated sows than in controls (P
250
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THERIOGENOLOGY
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THElUOCENOLOCY
and 25% of the above dose: (Table 5) did not result in a significant increase in litter size. When a dosage of estradiol, 10% that of estrone, is given in combination with progesterone (1.25 pg estradiol and 25 rag progesterone) (Table 6), there is a substantial increase in both total pigs per litter (22.6%) and live pigs per litter (17.9%). This dosage corresponds, in terms of biological activity, to the effective ratio of estrone and progesterone. Estradiol alone, at this dosage had no effect which indicates the importance of the synergistic relationship. The results of Trial C (experiment 2) confirm the lack of effect from using estrone alone (Table 7) with the exception that the highest dosage (18.75 ug) obviously exerts an adverse effect on implantation. Wildt et al. (7) also noted that estrone alone (12.5 pg administered for ten days of gestation) had no effect on litter size. The role of estrogen in controlling litter size remains unclear. However, a synergistic action with progesterone may exert the effect. There are several lines of evidence which lend credence to this hypothesis. Pig blastocysts are capable of synthesizing estrogen from progesterone and this might be involved in the process of implantation (9). Furthermore, it is known that exogenous estrogen in rats causes the release of histamine, prostaglandin and serotonin, which induce a local increase in capillary permeability. The increase in capillary permeability is a necessary prerequisite for the induction of implantation in most species (10). That the effect is on the implantation process itself, rather than on increased placental function (thereby reducing embryonic death loss), has been strengthened by the work of McGovern et al. (8). When estrone (12.5 pg) is given with progesterone (25 mg) as a single injection on Day 16, there is a 9.7% increase in total litter size, which is a nonsignificant difference (Table 8) but when estrone, at the same dosage is given alone the increase is 20.4%. 'Sinceplacental microvilli formation is just beginning at this stage of gestation, this could reflect a changing requirement for the availability of estrogen relative to the plasma concentration of progesterone between Day 16 and Day 17 (estrone given at this level for both Day 16 and 17 does not alter litter size (Table 7), but the confirmation of this concept will require further study. The results showing alteration in the pH, protein concentration and osmolality of the fetal fluids suggest increased secretory activity with estrone-progesterone treatment over a ten day period. The reduction of the protefn concentration, as well as the lack of a significant effect on osmolality by steroid treatment, in the allantoic fluid, appears to be associated with its increase in volume in the conceptuses from pigs by Day 30 of gestation (8, 11). In the present studies, two protein bands in allantoic or amniotic fluid were less often observed in the treated than in control group. The presence of functional corpora lutea is necessary for the secretion of two uterine proteins (Fraction IV and V) during the estrous cycle of swine (12). Studies further showed that progesterone alone or progesterone and estrogen in combination were key hormones controlling the secretion of the specific protein components from the secretory cells of the uterine endometrium (13, 14). A possible role of these protein changes may relate to embryonic mortality during early pregnancy. Future evaluation of this hypothesis must await identification and quantitation of the specific proteins of the allantoic and amniotic fluids during early pregnancy.
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THERIOCENOLOCY REFERENCES 1.
Sammelwitz, P. H., Dziuk, P. J. and Nalbandov, A. V. Effect of progesterone on embryonal mortality of rats and swine. .I.Anim. Sci. 15:1211 (1956).
2.
Haines, C. E., Warnick, A. C., Wallace, H. D. and Edwards, G. M. The effect of level of feeding and progesterone injection on reproductive performance in gilts. J. Anim. Sci. s:1099 (1957).
3.
Spies, H. G., Zimmerman, D. R., Self, H. L. and Casida, L. E. The effect of exogenous progesterone on formation and maintenance of the corpora lutea and on early survival in pregnant swine. .I.Anim. Sci. x:163 (1959).
4.
The lack of relaWebel, S. K., Reimers, I. J. and Dziuk, P. 3. tionship between plasma progesterone levels and the number of embryos and their survival in the pig. Biol. Reprod. -13:177 (1975).
5.
Reddy, V. B., Mayer, D. T. and Lasley, J. F. Hormonal modification of the intra-uterine environment in swine and its effects on embryonic viability. Res. Bull. MO. Agri. Exp. Sta. 667:5 (1958).
6.
Day, B. N., Romack, F. E. and Lasley, J. F. Influence of progesterone estrogen implants on early embryonic mortality in swine. J. Anim. Sci. -22:637 (1963).
7.
Wildt, D. W., Culver, A. A., Morcom, C. B. and Dukelow, W. R. Effect of administration of progesterone and estrogen on litter size in pigs. .I.Reprod. Fert. -48:209 (1976).
8.
McGovern, P. T., Morcom, C. B., de Sa, W. F. and Dukelow, W. R. Chorionic surface area in conceptuses from sows treated with progesterone and oestrogen during early pregnancy. J. Reprod. Fert. (submitted) (1980).
9.
Perry, J. S., Heap, R. B. and Amoroso, E. C. Steroid hormone production by pig blastocysts. Nature -245:45 (1973.
10.
Dickman, Z., Gupta, J. S. and Dey, S. K. Does "blastocyst estrogen" initiate implantation? Science m:687 (1977).
11.
Knight, J. W., Bazer, F. W., Thatcher, W. W., Franke, D. E. and Wallace, H. D. Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts: interrelations among hormonal status, placental development, fetal fluids and fetal growth. J. Anim. Sci: -44:620 (1977).
12.
Murray, F. A., Bazer, F. W., Wallace, H. D. and Warnick, A. C. Quantitative and qualitative variation in the secretion of protein by the porcine uterus during the estrous cycle. Biol, Reprod. 7: 314 (1972).
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13.
Chen, T. T., Baser, F. W., CetorelLi, J. J., Pollard, W. R. and Roberts, R. M. Purification and properties of a progesterone induced basic glycoprotein from the uterine fluids of pigs. J. Biol. Chem. 248:8560 (1973).
14.
Knight, J. W., Bazer, F. W. and Wallace, H. D. Hormonal regulation of porcine uterine protein secretion. J. Anim. Sci. -36:546 (1973).
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EXOGENOUS STEROID EFFECTS ON LITTER SIZE AND EARLY EMBRYONIC SURVIVAL IN SWINE W. F. de Sa, P. Pleumsamran, C. B. Morcom and W. Richard Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48824 Received for publication:
7124180
ABSTRACT These studies confirm the efficacy of 12.5 pg estrone and 25 mg progesterone, administered on Days 16 and 17 of pregnancy, on increased litter size in swine. Substitution of estradiol (with ten-fold greater biological activity) for estrone in this regimen eliminated the positive effect on litter size and estradiol given alone at dosages of 3.1 and 6.3 ug were without effect. However, when estradiol was given in combination with progesterone (25 mg) at a dosage equivalent to 10% the effective estrone dosage (1.25 ug), a 22.6% increase in total pigs born per litter over controls was observed. Estrone alone at dosages of 6.25 and 12.5 ug did not alter litter size. At a dosage of 18.75 ug, an 8.7% decrease in total pigs born was noted. The amniotic fluid from sows treated for ten days with 12.5 pg estrone and 25 mg progesterone near the time of implantation, had significantly higher protein concentration, pH and osmolality. INTRODUCTION Conflicting results have been reported on the effects of exogenous progesterone and estrogen on embryonic mortality in swine. In 1956, Sammelwitz et al. (1) treated gilts with progesterone at 50, 100, 200 and 400 mg daily for 26 days after mating with little beneficial effect. Later, others reported that either raising plasma progesterone concentrations two-fold or reducing them by half did not affect embryonic survival (2, 3, 4). In contrast, a positive response (5) to progesterone and estrone on embryonic mortality was reported (6). In this study, two ratios of progesterone to estrone were used, 1000:1 and 2000:l. In the latter group, with estrone and progesterone given at dosages of 12.5 pg and 25 mg daily, respectively, (for ten days beginning either on Day 4 or 14). there was a highly significant increase in litter size by 55 days of gestation. Exogenous progesterone and estradiol, given from Days 11 to 25 or 17 to 25, had a trend towards increased litter size; however, the difference was not significant. Three treatments were used, all at a progesterone:estradiol ratio of 500:1 (5). The authors acknowledge the technical assistance of Drs. P. T. McGovern, D. E. Wildt, W. T. Magee, and Mrs. Julie Howe-Baughman during the conduct of these studies. Supporting funds were received from EMBRAPA (Empresa Brasileira de pesquisa Agro-pecuaria) and the Agency for International Development.
MARCH
1981 VOL. 15 NO. 3
245
THERIOGENOLOGY
Wildt et al. (7) repeated earlier studies (5) with modification and reported that a dose of 25 mg progesterone and 12.5 ug estrone, given for 2, 5 or 10 consecutive days near implantation, resulted in a significant increase in litter size at term. However, estrone or progesterone alone, as well as higher dosages of estrone and progesterone in combination, had no effect. The present study was designed to further investigate the temporal relationships between the time of steroid administration and litter size. MATERIALS AND METHODS Experiment 1: Initially a field trial was carried out at the Michigan State University Swine Facility. A total of 88 multiparous sows, which were held in confinement and hand-mated, were used. Every other sow that came into estrus served as a control (2 ml cottonseed oil (CSO); whereas, treated sows received 25 mg progesterone and 12.5 ng estrone in 2 ml CSO (5). All injections were IM into either the ham or anterior scapular region and all injections were given on Day 16 and 17 of gestation based on earlier studies (7). Experiment 2: This series of trials was designed to examine the substitution of estradiol for estrone (with an approximate biological activity ten-fold higher) alone, and in combinat'?n with progesterone. All animals were sows of the Duroc,Yorkshire and Hampshire breeds and Yorkshire-Hampshire crossbreds. Animals were randomly assigned to treatment within breed types. All injections were in 2 ml arachis oil (AO) on Days 16 and 17 of gestation. The change in the control vehicle was to standardize this factor with other ongoing research. No differences in the rate of absorption of steroids between CSO and A0 are known. Control animals received the vehicle alone. The total number of pigs born and the number born alive, as well as litter weight at birth and piglet birth weight, were recorded. In this series of experiments the procedure of least squares was used to obtain estimates of treatment means (adjusted for breed of sow, breed of boar and parity) and the error mean square. Student's t test was used to compare the mean of each treated group to the control group. In Trial C, estrone alone was tested at dosages of 6.25, 12.50 and lg.75 pg with appropriate controls. In Trial D, which was conducted totally with Yorkshire sows, the effect of a single-day injection schedule was examined. All injections were with 2 ml A0 administered IM in the neck region on Day 16 of gestation. Sows received estrone alone (12.5 up>, progesterone alone (25 mg) or a combination of the two steroids at the dosages indicated. The significance was measured by Student's t test. Experiment 3: Previous studies (8) have shown that sows treated with 25 mg progesterone and 12.5 pg estrone daily between Days 14 and 23 of gestation showed an increase of 15% for the chorionic surface area and an increase in allantoic fluid volume when examined at 30 to 35 days of gestation. In contrast, there were no significant differences in these parameters in sows examined at 46 to 50 days of gestation. In the
246
MARCH
1981 VOL. 15 NO. 3
THERIOCENOLOGY present experiment, allantoic and amniotic fluid samples from the sows which were killed at 30 to 35 days of gestation were examined for protein bands on electrophoresed acrylamide gels. The electrophogesis technique has been previously described (9). For separation, 7+.5% acrylamide lower gel was used with tris-glycine buffer at pH 8.6. Proteins were separated by applying a constant current of 2.0 mA per tube for two hours and then staining with Amid0 black, followed by destaining. Rf values for migrating protein bands were calculated and compared for treated and control animals. RESULTS Experiment 1: Table 1 shows the mean number of pigs (total born and born alive) for the treated and controf sows. Table 1:
Effect of 12.5 pg estrone and 25 mg progesterone (given on Days 16 and 17 of gestation) on sow litter size.
Treatment
No. of sows
MeanL/sow born alive
Mean/sow total born
Control Total treated Ram injected Neck injected
45 43 18 25
10.0 10.9 10.5 11.2
10.9 11.7 11.1 12.1
+ 0.4 T 0.4 '7 0.5 - 0.6
+ + & +
0.3 0.4 0.5 0.6
1 mean + S.E. The overall distribution of litter sizes in field trials is more significant than total increases in pigs born. Many factors affect litter size, and several small litters can mask a significant increment in litter size. Tables 2 and 3 show the distribution of litters arranged by size categories. The mean number of total pigs born was significantly (PcO.05) different from controls (neck injections). Table 2.
Effect of 12.5 pg estrone and 25 mg progesterone (given on Days 16 and 17 of gestation) on distribution of litter size based on total pigs born.
Litter size
<7
7-a
9-11
12-13
.13
Control Treated
1 (2.2%) 1 (2.3%)
2 (4.4%) 4 (9.3%)
24 (53.3%) 12 (27.9%)
11 (24.2%) 16 (37.3%)
7 (15.6%) 10 (23.2%)
Table 3.
Effect of 12.5 ug estrone and 25 mg progesterone (given on Days 16 and 17 of gestation) on distribution of litter size based on pigs born alive.
Litter size
<7
7-a
9-11
12-13
>13
Control Treated
1 (2.2%) 2 (4.7%)
5 (11.1%) 4 (9.3%)
30 (66.7%) 19 (44.2%)
5 (11.1%) 13 (30.2%)
4 (8.9%) 5 (11.6%)
MARCH 1981 VOL. 15 NO. 3
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THERIOCENOKICY
It was found preferable to make all injections in the neck region (anterior to the scapula), not only based on litter size results, but also because of occasionally observed leakage of injection material following injections into the ham. Experiment 2: (Trial A, estradiol (12.5 ug) alone, progesterone (25 mg) alone and in combination). The percentage of sows becoming pregnant at first breeding to the control and three treatments given above was 85.0, 83.3, 93.8 and 93.8% respectively. The results of the experiment are shown in Table 4. Table 4.
Treatment
Effect of estradiol (12.5 ng) and progesterone (25 mg) alone and in combination (Days 16 and 17) on litter size and birth weight.l
No. of sows
Control Estradiol alone Progesterone alone Combination
17 13 15 15
Total pigs born 12.7 14.2 13.4 12.5
+ T T +
0.7 0.72 0.7 0.7
Live pigs born
Mean/sow birth wt/ litter (kg)
Mean/sow birth wt/ pig (kg)
11.7 13.3 12.0 11.3
17.6 17.8 17.2 17.3
1.6 1.4 1.7 1.8
-I0.7 T 0.82 T 0.7 5 0.7
+ T T 5
1.0 1.1 1.1 1.1
+ 0.4 +- 0.4 7- 0.4 z 0.4
, >alues are least squares regression means f. S.E. Significantly (P
Effect of estradiol alone at dosages of 3.125 and 6.250 ug (Days 16 and 17) on litter size and birth weight.1
Treatment Control Estradiol (3.125pg) Estradiol (6.25Opg)
No. of sows 17 6 9
Total pigs born
Live pigs born
12.7 + 0.7 11.7 + 0.7 13.1 T 1.1 12.2 'T 1.1 12.1 F 0.9 11.0 5 0.9
Mean/sow Mean/sow birth wtf birth wt/ litter(kg) pig (kg) 17.6 + 1.0 1.6 + 0.4 17.3 + 1.7 1.6 T 0.6 17.9 5 1.4 2.2 - 0.5
1
Values are least square regressions 2 S.E.
No significant differences were observed in litter size or birth weights due to treatments at these dosages.
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THERIOCENOLOGY In the subsidiary trial to Trial B (estradiol at a dosage of 1.25 pg with and without progesterone at 25 mg) injection of estradiol alone did not alter litter size or birth weight. The combination of estradiol and progesterone resulted in a 22.6% increase in total pigs per litter and a 17.9% increase in live pigs per litter (Table 6). Table 6.
Effect of estradiol (1.25 ng) alone and in combination with progesterone (25 mg), on Days 16 and 17, on litter size and birth weighta
No. of sows
Treatment Control Estradiol alone (1.25 ug) Combination
11 11 8
Total pigs born
Live pigs born
Mean/sow birth wt/ litter (kg)
8.4 + 0.8 7.8 4 0.7 10.3 + 0.8
8.3 + 0.9 7.4 + 0.7 9.8 + 0.7
14.3 + 1.2 12.3 2 1.4 13.8 + 1.7
1 Values expressed as raw means + S.E. Trial C (estrone alone at dosages of 6.25, 12.50 and 18.75 ug). The percentage of sows becoming pregnant after first breeding in the control and three treatment groups listed above were 85.0, 83.3, 76.9 and 83.3%, respectively. The results of the experiment are shown in Table 7. Table 7.
Effects of estrone alone at dosages of 7.25, 12.50 and 18.75vg (Days 16 and 17) on litter size and birth weight.1
Treatment Control Estrone (6.25 up) Estrone (12.50 ug) Estrone (18.75 ug)
No. of sows 17 10 10 10
Total pigs born
Live pigs born
Mean/sow Mean/sow birth wt/ birth wt/ litter (kg) pig (kg)
12.7 + 13.0 + 13.12 11.6 +
11.7 12.0 12.3 10.5
17.6 17.0 17.4 16.2
0.7 0.9 1.0 0.9
2 2 + +
0.7 0.9 0.9 0.7
4 + 4 t
1.0 1.4 1.3 1.3
1.6 1.4 1.8 1.7
+ 0.4 ? 0.5 2,0.5 + 0.5 _
'Values are least square regressions 2 S.E. Administration of estrone at the two lower dosages did not alter litter size or birth weights. On the other hand, estrone at 18.75 ug resulted in a reduction of both total pigs born and pigs born alive. The percent reduction for this treatment on those two parameters was 8.7 and 10.3% respectively. Trial D (estrone (12.5 ng) and progesterone (25 mg) alone and in combination given only on Day 16). Treatment with estrone alone resulted in increases for the total pigs born and live pigs born of 20.4 and 7.4% respectively (Table 8). Corresponding values for the combination treatment were 9.7 and 12.0% respectively. These were not statistically significant. Birth weights were not available on these pigs.
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THERIOGENOLOGY
Table 8.
Effect of estrone (12.5 ug) and progesterone (25 mg) alone and in combination, given only on Day 16, on litter size.1 No.
Treatment
of sows
Control 12 Estrone alone 8 Progesterone alone 6 Combination 7
Total pigs born
Live pigs born
11.3 13.6 11.3 12.4
10.8 11.6 12.1 12.1
+ T + z
0.9 1.1 1.1 1.1
+ + + +
0.9 0.8 1.2 1.0
lValues expressed as raw means + S.E. Experiment 3: The protein concentration, pH and osmolarity measured in fetal fluids recovered between Days 30 and 35 of gestation on sows which received a ten day course of injections with 12.5 pg estrone and 25 mg progesterone daily are shown in Table 9. A significant (PcO.01) increase was found in the pH of the treated sows allantoic fluid when compared to that of the control animals. Allantoic fluid protein concentration was significantly higher in control animals (PcO.05). In the amniotic fluid, the protein concentration was three-fold higher in treated sows than in controls (P
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THERIOGENOLOGY
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THElUOCENOLOCY
and 25% of the above dose: (Table 5) did not result in a significant increase in litter size. When a dosage of estradiol, 10% that of estrone, is given in combination with progesterone (1.25 pg estradiol and 25 rag progesterone) (Table 6), there is a substantial increase in both total pigs per litter (22.6%) and live pigs per litter (17.9%). This dosage corresponds, in terms of biological activity, to the effective ratio of estrone and progesterone. Estradiol alone, at this dosage had no effect which indicates the importance of the synergistic relationship. The results of Trial C (experiment 2) confirm the lack of effect from using estrone alone (Table 7) with the exception that the highest dosage (18.75 ug) obviously exerts an adverse effect on implantation. Wildt et al. (7) also noted that estrone alone (12.5 pg administered for ten days of gestation) had no effect on litter size. The role of estrogen in controlling litter size remains unclear. However, a synergistic action with progesterone may exert the effect. There are several lines of evidence which lend credence to this hypothesis. Pig blastocysts are capable of synthesizing estrogen from progesterone and this might be involved in the process of implantation (9). Furthermore, it is known that exogenous estrogen in rats causes the release of histamine, prostaglandin and serotonin, which induce a local increase in capillary permeability. The increase in capillary permeability is a necessary prerequisite for the induction of implantation in most species (10). That the effect is on the implantation process itself, rather than on increased placental function (thereby reducing embryonic death loss), has been strengthened by the work of McGovern et al. (8). When estrone (12.5 pg) is given with progesterone (25 mg) as a single injection on Day 16, there is a 9.7% increase in total litter size, which is a nonsignificant difference (Table 8) but when estrone, at the same dosage is given alone the increase is 20.4%. 'Sinceplacental microvilli formation is just beginning at this stage of gestation, this could reflect a changing requirement for the availability of estrogen relative to the plasma concentration of progesterone between Day 16 and Day 17 (estrone given at this level for both Day 16 and 17 does not alter litter size (Table 7), but the confirmation of this concept will require further study. The results showing alteration in the pH, protein concentration and osmolality of the fetal fluids suggest increased secretory activity with estrone-progesterone treatment over a ten day period. The reduction of the protefn concentration, as well as the lack of a significant effect on osmolality by steroid treatment, in the allantoic fluid, appears to be associated with its increase in volume in the conceptuses from pigs by Day 30 of gestation (8, 11). In the present studies, two protein bands in allantoic or amniotic fluid were less often observed in the treated than in control group. The presence of functional corpora lutea is necessary for the secretion of two uterine proteins (Fraction IV and V) during the estrous cycle of swine (12). Studies further showed that progesterone alone or progesterone and estrogen in combination were key hormones controlling the secretion of the specific protein components from the secretory cells of the uterine endometrium (13, 14). A possible role of these protein changes may relate to embryonic mortality during early pregnancy. Future evaluation of this hypothesis must await identification and quantitation of the specific proteins of the allantoic and amniotic fluids during early pregnancy.
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THERIOCENOLOCY REFERENCES 1.
Sammelwitz, P. H., Dziuk, P. J. and Nalbandov, A. V. Effect of progesterone on embryonal mortality of rats and swine. .I.Anim. Sci. 15:1211 (1956).
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Haines, C. E., Warnick, A. C., Wallace, H. D. and Edwards, G. M. The effect of level of feeding and progesterone injection on reproductive performance in gilts. J. Anim. Sci. s:1099 (1957).
3.
Spies, H. G., Zimmerman, D. R., Self, H. L. and Casida, L. E. The effect of exogenous progesterone on formation and maintenance of the corpora lutea and on early survival in pregnant swine. .I.Anim. Sci. x:163 (1959).
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The lack of relaWebel, S. K., Reimers, I. J. and Dziuk, P. 3. tionship between plasma progesterone levels and the number of embryos and their survival in the pig. Biol. Reprod. -13:177 (1975).
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Reddy, V. B., Mayer, D. T. and Lasley, J. F. Hormonal modification of the intra-uterine environment in swine and its effects on embryonic viability. Res. Bull. MO. Agri. Exp. Sta. 667:5 (1958).
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Day, B. N., Romack, F. E. and Lasley, J. F. Influence of progesterone estrogen implants on early embryonic mortality in swine. J. Anim. Sci. -22:637 (1963).
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Wildt, D. W., Culver, A. A., Morcom, C. B. and Dukelow, W. R. Effect of administration of progesterone and estrogen on litter size in pigs. .I.Reprod. Fert. -48:209 (1976).
8.
McGovern, P. T., Morcom, C. B., de Sa, W. F. and Dukelow, W. R. Chorionic surface area in conceptuses from sows treated with progesterone and oestrogen during early pregnancy. J. Reprod. Fert. (submitted) (1980).
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Perry, J. S., Heap, R. B. and Amoroso, E. C. Steroid hormone production by pig blastocysts. Nature -245:45 (1973.
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Dickman, Z., Gupta, J. S. and Dey, S. K. Does "blastocyst estrogen" initiate implantation? Science m:687 (1977).
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Knight, J. W., Bazer, F. W., Thatcher, W. W., Franke, D. E. and Wallace, H. D. Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts: interrelations among hormonal status, placental development, fetal fluids and fetal growth. J. Anim. Sci: -44:620 (1977).
12.
Murray, F. A., Bazer, F. W., Wallace, H. D. and Warnick, A. C. Quantitative and qualitative variation in the secretion of protein by the porcine uterus during the estrous cycle. Biol, Reprod. 7: 314 (1972).
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Chen, T. T., Baser, F. W., CetorelLi, J. J., Pollard, W. R. and Roberts, R. M. Purification and properties of a progesterone induced basic glycoprotein from the uterine fluids of pigs. J. Biol. Chem. 248:8560 (1973).
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Knight, J. W., Bazer, F. W. and Wallace, H. D. Hormonal regulation of porcine uterine protein secretion. J. Anim. Sci. -36:546 (1973).
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