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Effect of fetal number on the concentrations of circulating maternal serum progesterone and estradiol of does during late pregnancy
Small Ruminant Research ELSEVIER
Small Ruminant Research 23 (1996) 117-124
Effect of fetal number on the concentrations of circulating maternal serum progesterone and estradiol of does during late pregnancy W. Manalu *, M.Y. Sumaryadi I, N. Kusumorini Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural Universiry, Jalan Taman Kencana 3, Bogor 16151, Indonesia
Accepted 20 June 1996
Abstract Twenty Etawah-cross does with similar body weights (20-22 kg) and ages (2-3 years) were used to study the effects of fetal number (single or twin) on serum concentrations of progesterone and estradiol during the last two months of pregnancy. Average serum progesterone concentrations during the last two months of gestation period in the single- and twin-bearing markedly increased (P < 0.01) by 1302 and 259 I%, respectively, as compared to those non-pregnant does (5.791 and 11.113 vs. 0.413 ng/ml). Serum progesterone concentrations increased by 92.4% (P < 0.01) as fetal number increased from single to twin. Concentrations of serum estradiol during the last two months of gestation period in the single- and twin-bearing increased (P < 0.01) by 684 and 2006%, respectively as compared to those in the non-pregnant does (68.84 and 184.93 vs. 8.78 pg/ml). Serum estradiol concentrations increased by 169% (P < 0.01) as fetal number increased from single to twin. Average serum progesterone and estradiol concentrations of aborted does had decreased to a very low level, but were still higher than those of non-pregnant does, at least seven weeks prior to abortion date (I .691 ng/ml and 14.46 pg/ml for aborted vs. 0.413 ng/ml and 8.78 pg/ml for non-pregnant does). It was concluded that hormonal stimulation for mammary gland growth and development may have increased with the increased fetal number during pregnancy in preparation for more milk synthesis for the newborn kids. Keywords: Progesterone; Estradiol; Pregnancy; Goat
1. Introduction
mammary tissue (Cowie, 1971, Anderson
A major amount of mammary development is associated with hormonal stimuli of pregnancy, and the absence of these stimuli reduces the amount of
et al., 198 1). Exogenous administration of mammogenic hormones, including prolactin, growth hormone, estrogen, progesterone and relaxin, (Harness and Anderson, 1977a,b,Wright and Anderson, 1982, Wahab and Anderson, 1989) stimulated mammary gland growth and development.
Corresponding author. Phone: (0251) 328 487. Fax: (0251) 323 161. ’ Present address: Laboratory of Physiology and Reproduction, Faculty of Animal Science, Jenderal Soedirman University, P.O. Box 10 Poerwokerto 53123, Central Java, Indonesia.
Mammary gland growth and development in small ruminant animals (sheep and goat) dramatically increased during the last two months of the gestation period (Rattray et al., 1974, Anderson, 1975b,Ander-
l
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son et al., 1981). Anderson et al. (1981) reported that mammary cell DNA in goats was higher than in sheep (Anderson, 1975b) at comparable days of pregnancy and lactation. This difference was suggested (Anderson et al., 1981) to be due to the higher twinning rate occurring in goats compared to sheep. Anderson et al. (1981) suggested that the presence of more placental tissue in goats was related to more placental lactogen secretion to stimulate mammary gland growth, accounting for greater mammary development in goats than in sheep. The role of placental lactogen was demonstrated in mammary gland development in the rat (Anderson, 1975a) and in the goat (Hayden et al., 1979). Hayden et al. (1979) reported a positive correlation between litter size (i.e., feto-placental mass> and serum concentrations of placental lactogen and mammary gland growth and development in goats. Butler et al. (1981) observed an increased maternal serum placental lactogen level in sheep bearing different litter size. Rattray et al. (1974) also reported a higher mammary gland growth in ewes bearing different fetal numbers. The difference found in mammary gland growth and development in different litter size, however, was not solely due to the greater placental tissue. The increased number of corpora lutea was suggested to contribute more progesterone and estrogen secretion that eventually increased hormonal stimulation of mammary gland growth and development in sheep or goats bearing more than one fetus. Increased litter size means increased number of ovulating follicle and fertilized ova. It was documented that litter size positively correlated with ovulation rate (Piper and Bindon, 1984, Bradford, 1985). Increased ovulation rate would increase the number of corpora lutea secreting progesterone and estrogen during the gestation period. Increased placental mass with the increased litter size would increase placental glands secreting progesterone and estradiol during the second half of the gestation period (after placentation stage). It was reported that plasma concentrations of progesterone during pregnancy increased with the increased litter size in sheep (Butler et al., 198 1). Serum e&one sulfate was found positively correlated with the number of fetuses during the gestation period in does (Refsal et al., 1991). However, the correlation between progesterone concentra-
tions and fetal number have not been documented in goat species. The increased hormonal stimulation for mammary gland growth and development during the gestation period could explain the observed increased mammary gland growth in sheep (Rattray et al., 1974) and in goats (Hayden et al., 1979) with increased litter size. The increased mammary gland growth and development in ewes and does bearing different fetal numbers further confirm the observations of higher milk production in goats (Hayden et al., 1979) and sheep (Butler et al., 1981) having more than one kid or lamb. It was hypothesized that the secretion of endogenous progesterone and estradiol increased with the increased number of fetuses carried. The objective of this experiment was to study the profile of serum progesterone and estradiol concentrations during the fastest growing period of mammary glands (the second half of the gestation period) in does bearing different fetal numbers.
2. Materials and methods 2.1. Enuironmental ment
conditions
and animal manage-
This experiment was conducted in the village site during the period of October 1992 to May 1993. The whole experiment was conducted during the wet season of the year. Average daily ambient temperature and relative humidity ranged from 25 to 32°C and from 70 to 80%, respectively. The Etawah-cross goat (Etawah X Kacang, with undetermined degree of crossing) is an economically important breed of goat in the Indonesian small ruminant industry. This breed, medium in size and milk production (between Etawah and Kacang), is multipurpose in production (either for milk or meat production) and nonseasonal in reproduction. However, this breed of goat is primarily raised for meat production, as it is true for the small ruminant industry in Indonesia. Experimental does were kept in a pen with a raised floor as typical of small ruminant housing in the region. Fresh field grass was fed ad libitum with supplementation of table salt block. The animals were provided drinking water ad libitum. This feed-
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ing regiment was adopted to simulate the prevailing management practice of the small-holder small-ruminant farmer in the region. 2.2. Experimental design and protocol Twenty Etawah-cross does with similar weight (20-22 kg) and age (2-3 years) were maintained in an experimental pen with a 2 month adaptation to the experimental conditions prior to the mating period. During the mating period, the experimental does were mixed with five bucks for a month, the experimental does were maintained in a group of three per pen. Blood samples were drawn weekly after the breeding period. At the end of the experimental period, only eight does gave birth to normal kids (five singles and three twins). Three does aborted near the expected parturition date and were included in the statistical analyses of the data. The others were non-pregnant during the experimental period, and four of those were included in data analyses as a control for pregnancy. Experimental does were grouped into four groups: 0 (non-pregnant does as a control for pregnancy); Al3 (aborted does); 1 (does carrying a single fetus) and 2 (does carrying twin fetuses), with n = 4, 3, 5, and 3, respectively.
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USA) with a slight modification for ovine progesterone concentration ranges (Manalu et al., 1995). The radioactivity of progesterone-bound tubes was counted with an automatic gamma counter. The concentrations of standard progesterone used to construct the standard curve ranged from 0.1 to 20 ng/ml. A sample volume of 100 ~1 serum was used in the assay for samples with progesterone concentrations ranging from 0.1 to 20 ng/ml. For samples with progesterone concentrations lower than 0.1 ng/ml, sample volume was increased to 200 p,l to bring the progesterone concentrations to the range of standard used. All sample progesterone concentrations were within the range of concentrations of standard progesterone used to construct the standard curve. Inter- and intra-assay variation coefficients were 7.5, and 2.0%, respectively. The concentrations of progesterone were parallel in the sample volumes of 50, 100, and 200 (11.
A 10 ml volume of blood was drawn with a plain vacutainer or a sterile syringe from the jugular vein in the time period from around 0900 to 1000 h on Thursdays weekly. The bleeding time was conducted consistently prior to feeding, and the day of week was chosen merely for convenience. The blood samples were allowed to clot in a cool ice box and transported to the laboratory for further separation of serum by centrifugation. The serum samples were then kept frozen for further hormone analyses. Weeks prior to parturition were counted back from the last blood sampling prior to the parturition date.
2.4.2. Estradiol Concentration of serum estradiol in duplicate was measured by the solid-phase technique radioimmunoassay (Diagnostic Products) with a slight modification for ovine estradiol concentration ranges (Manalu et al., 1995). The radioactivity of estradiolbound tubes was counted with an automatic gamma counter. The concentrations of standard estradiol used to construct the standard curve ranged from 20 to 500 pg/ml. A sample volume of 100 l.~l serum was used in the assay for samples with estradiol concentrations ranging from 20 to 500 pg/ml. For samples with estradiol concentrations lower than 20 pg/ml, sample volume was increased to 200 c1.1to bring the estradiol concentrations to the range of standard used. All sample estradiol concentrations were within the range of concentrations of standard estradiol used to construct the standard curve. Inter- and intra-assay variation coefficients were 10.2, and 5.0%, respectively. The concentrations of estradiol were parallel in the sample volumes of 50, 100, and 200 ~1.
2.4. Hormone analyses
2.5. Statistical analyses
2.4.1. Progesterone
Data collected during the seven-week measurement were analyzed for main effect (week prior to parturition or abortion, and status of pregnancy: non-pregnant, aborted, carrying single and twin) and
2.3. Blood sampling and processing
Concentration of serum progesterone in duplicate was measured by the solid-phase technique radioimmunoassay (Diagnostic Products, Los Angeles, CA,
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their interactions with a randomized design with factorial arrangement with unequal n. The difference between means of main effects were tested using least significant differences (Snedecor and Cohran, 1982).
of the progesterone concentration were far lower than average serum progesterone concentrations of pregnant does but higher than those of non-pregnant does. Serum progesterone concentrations were dramatically higher in the pregnant (5.79 1 and 11.113 ng/ml for single- and twin-bearing does, respectively) (P < 0.01) as compared to the non-pregnant does (0.413 ng/ml). The results of the experiment showed that the average serum progesterone concentration during the last two months of the gestation period in the twin-bearing does increased (P < 0.01) by 91.9% as compared to those in the single-bearing does (11 .l 1 vs. 5.79 ng/ml). It was important to note that serum progesterone concentrations of aborted does had decreased, approaching the levels of non-pregnant does seven weeks prior to the abortion date (1.69 1 for aborted vs. 0.413 ng/ml for non-pregnant does).
2.6. Results 2.6.1. Progesterone Average weekly serum progesterone concentrations of experimental does are presented in Table 1. There was no difference in the concentration of progesterone among weeks of blood sampling in pregnant and aborted groups, i.e., serum progesterone seemed to be at a concentration plateau in the seven weeks period of measurement prior to parturition or abortion. There was no interaction between fetal number and weeks prior to parturition or abortion. Average serum progesterone concentration in the non-pregnant does in the seven week measurement period prior to parturition was high. This high mean was due to the consistently high serum progesterone concentration in two does (numbers 2 and 4) as was verified by several replications. Individual weekly variations in serum progesterone concentration were found in aborted does. However, the peaks
Table 1 Concentrations
of serum progesterone
Fetal number ’
in non-pregnant,
Progesterone (ng/ml) Weeks prior to parturition 7 6
2.6.2. Estradiol Average weekly serum estradiol concentrations of experimental does are presented in Table 2. There was no difference in the concentrations of estradiol between weeks prior to parturition within the same fetal number group from seven weeks to two weeks
aborted, single- and twin-bearing
or abortion 5
does seven weeks prior to parturition
or abortion d
f 4
3
2
1
Mean g
0.067 0.004
0.059 0.003
0.065 0.004
0.413 a 0.325
0
Mean SE
2.525 1.317
0.059 0.001
0.049 0.004
0.067 0.007
AB
Mean SE
2.621 2.545
0.270 0.205
1.648 0.789
0.45 0.404
I
1.456
2.407 2.348
2.580 2.503
1.691 a 0.450
Mean SE
5.917
1.473
5.215 1.324
5.638 0.717
6.397 0.569
6.392 0.823
5.069 0.324
5.869 1.226
5.791 b 0.197
Mean SE
13.855 1.559
9.139 0.397
9.068 0.434
11.848 0.700
10.859 0.576
10.861 0.494
12.164 2.628
11.113 c 0.643
1
2
1.858
* Means and SE of 4, 5, 3, and 3 does for fetal number 0, 1, and 2, and aborted does, respectively. e Fetal number 0, 1, and 2 refers to non-pregnant, single- and twin-bearing does, respectively. AB, refers to aborted does. f Weeks prior to parturition only applied to single- and twin-bearing does. For aborted does this term means weeks prior to abortion. non-pregnant does, this term means nothing but is a control for hormonal profile at the same period of blood sampling. g Different superscripts in this column refer to differences between fetal number (P < 0.01).
For
W. Manalu et al./Small Table 2 Concentrations Fetal number
of serum estradiol e
in non-pregnant,
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aborted, single- and twin-beating
Estradiol (pg/ml) Weeks prior to parturition or abortion f 7 6 5 4
does seven weeks prior to parturition
3
1
2
or abortion d
Mean g
0
Mean SE
9.40 0.96
9.05 0.72
8.85 0.78
8.96 0.86
10.45 2.41
8.16 0.52
6.59 0.15
8.78 a 0.45
AB
Mean SE
9.14 0.56
11.26 1.21
12.99
1.41
18.15 5.59
14.83 2.03
16.72 3.57
18.15 1.51
14.46 a 1.1 I
1
Mean SE
30.87 5.07
40.07 8.82
53.15 7.32
53.01 5.67
76.36 14.55
89.66 19.50
138.76 47.14
68.84 b 13.93
2
Mean SE
117.56 19.06
87.33 8.57
126.0 1 27.86
151.38 18.92
164.43 37.23
170.09 35.98
477.72 219.09
184.93 50.01
’
d Means and SE of 4, 5, 3, and 3 does for fetal number 0, 1, and 2, and aborted does, respectively. e Fetal number 0, 1. and 2 refers to non-pregnant, single- and twin-bearing does, respectively. AB, refers to aborted does. f Weeks prior to parturition only applied to single- and twin-bearing does. For aborted does this term means weeks prior to abortion. For non-pregnant does, this term means nothing but is a control for hormonal profile at the same period of blood sampling. g Different superscripts in this column refer to differences between fetal number (P < 0.01).
prior to parturition. Serum estradiol concentrations seemed to slowly rise and reached peak concentrations (P < 0.01) in the week of parturition. The increased serum estradiol concentration one week prior to parturition was subject to a very high individual variation, giving very high SE values (greater than the mean values for a certain week of measurements). Serum estradiol concentrations were dramatically higher in the pregnant (68.839 and 184.932 pg/ml, for single- and twin-bearing does, respectively) (P < 0.01) as compared to those in the nonpregnant does (8.782 pg/ml). Concentrations of serum estradiol during the last two months of gestation in the twin-bearing does increased (P < 0.01) by 169% compared to those in the single-bearing does (184.932 vs. 68.839 pg/ml). Serum estradiol concentrations of aborted does had decreased, approaching those of the basal non-pregnant level seven weeks prior to abortion date (14.46 pg/ml for aborted vs. 8.78 pg/ml for non-pregnant does).
3. Discussion The results of the experiment showed that average maternal serum progesterone concentrations during the last two months of the gestation period for twins
was almost twice as much as found for single-bearing does. Concentrations of maternal serum estradiol at the same period was almost trite for twin- compared with single-bearing does. Even though progesterone and estradiol concentrations were not measured from the beginning of the gestation period, the results of this experiment implied a dramatic increase in hormonal stimulation for mammary gland growth and development when fetal numbers increased from one to two. The source of the increased serum concentrations of progesterone and estradiol in response to different fetal numbers was unclear, i.e., whether it was due to the increased number of corpora lutea or to the increased mass of placental tissue or a combination of both. Non-pregnant does had basal flat average progesterone and estradiol concentration during the same period of measurement, with slight spikes during the estrous cycles. The existence of corpora lutea and placenta during pregnancy dramatically increased serum progesterone and estradiol concentrations. Ranges of progesterone and estradiol concentrations found by others in goats at comparable ages of pregnancy (Umo et al., 1976) agreed with our results. In comparison to the profile of progesterone concentrations in sheep with the same litter size (Butler et al., 19811, goat progesterone concentrations observed in this study were slightly higher.
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However, Butler et al. (1981) found that there were no significant effects of fetal number on maternal serum estradiol concentrations in sheep, with average estradiol concentrations substantially lower than those found in goat species in this experiment. Maternal serum progesterone and estradiol concentrations in sheep (Manalu et al., 1995) were substantially lower than those reported in goats in this experiment. The differences in the profiles of progesterone and estradiol during pregnancy between goats and sheep were probably related to the difference in the role of placenta as a source of progesterone and estradiol in the different species (Heap and Flint, 1984). Since progesterone and estradiol are hormones that play critical roles in mammary gland growth and development during pregnancy (Anderson, 1986, Tucker, 1986, Knight and Peaker, 1982, Tucker, 1987), the increased fetal number or litter size would increase hormonal stimulation for mammary gland growth and development in preparation of more milk synthesis for the newborn kids. The increased hormonal stimulation of mammary gland growth and development with the increased litter size were accompanied by the increased mammary gland growth and development (Rattray et al., 1974, Hayden et al., 1979, Butler et al., 1981). Therefore the increased endogenous mammogenic hormones, especially progesterone, estrogen and placental lactogen, could increase mammary gland growth and development and ultimately milk production. These phenomena could be used as alternative means of improving milk production in small ruminant animals either for human consumption or for nourishing the new born kids. In Indonesia, sheep and goats play an important role in animal husbandry. However the mortality of the kids and lambs are so high that kids and lambs survival rates are basically very low (Obst et al., 1980). The low survival rate is strongly related to the low birth weight C3nama et al., 1988, Sutama, 1992) and milk production (Tiesnamurti, 1992). Lower birth weight is probably due to the lower uterine gland growth and development to secrete nutrients to nourish the fetus (McDonald, 1980), in addition to other factors such as nutrient deficiency and parasitic diseases. Serum progesterone and estradiol concentrations of aborted does approached those found in the non-
pregnant does seven weeks prior to abortion date. It was surprising that the does were still capable of maintaining the fetus for seven weeks with such low levels of maternal serum progesterone and estradiol. It is still unclear whether the increased progesterone and estradiol serum concentrations in does bearing two fetuses is due to the placental mass or the number of corpora lutea formed during the estrous cycle or combination of both. Even though sheep and goats are closely related phylogenetically and have morphologically similar forms of placentation, these two species differ in terms of the role of the placenta as a source of progesterone during pregnancy. In the ewe the placenta takes over progesterone production but in goats the ovaries and pituitary are necessary during the whole pregnancy and a doe will abort promptly following the removal of either (Heap and Flint, 1984). Since the placenta does not play a critical role in secreting progesterone during late pregnancy in does, it was suggested that the increased serum progesterone and estrogen concentrations in does bearing more than one fetus were not due to the increased placental mass, but related to the increased number of maintained corpora lutea during pregnancy. In sheep it was reported that litter size was highly correlated with the ovulation rate (Bradford et al., 1986). It is suggested that the research be extended to differentiate between the effects of fetal number and the number of surviving corpora lutea during pregnancy on the increased maternal serum progesterone and estradiol concentrations in sheep and goats. Progesterone concentrations were doubled in the twin-bearing as compared to single-bearing does, and estradiol concentrations in the twin-bearing as compared to those in the single-bearing does were more than doubled. The results of this experiment indicated that progesterone and estradiol could have been secreted by different glands during late pregnancy in the goat. If the primary source of both hormones are the same, ie., corpora lutea, why did estradiol concentrations increase more than progesterone concentrations in pregnancies with the same fetal number? This fact is probably related to the different function of estradiol during late pregnancy and parturition. In general, estradiol concentrations increased with the advance of pregnancy approaching parturi-
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tion date (Umo et al., 1976, Rice et al., 1984). The physiological significance of the increased estradiol concentrations prepartum is probably associated with the preparation of mammary glands to initiate milk secretion by stimulating prolactin secretion as was indicated by a previous report (Umo et al., 1976). Progesterone and estradiol concentrations during the second half of pregnancy could be used as strong parameters to predict whether the does are carrying single or twin fetuses. This information could be used in devising appropriate feeding strategies for pregnant does to meet the nutrient requirements of the mother and the fast-growing fetus(es) during late pregnancy. In addition, maternal serum progesterone and estradiol concentrations could be used as indicators of the possibility of abortion at least seven weeks prior to the abortion date.
4. Conclusion The results of this experiment indicate that maternal serum progesterone and estradiol concentrations increase with an increase in fetal number from one to two. Maternal serum progesterone and estradiol could be used as strong indices of selection for higher litter size, and to distinguish does carrying single or multiple fetuses during late pregnancy in goats. Progesterone and estradiol could have been secreted by different sources during late pregnancy in the goat. The increased maternal serum concentrations of progesterone and estradiol suggested increased hormonal stimulation for mammary gland growth and development during the second half of pregnancy with increased fetal number from single to twin. Correlation between the increased maternal serum progesterone or estradiol concentrations with the mammary gland growth and development as well as milk production are being studied to devise a novel means of increasing mammary gland growth and development.
Acknowledgements
This experiment was funded by a grant provided by The Agency for Agricultural Research and Development, Agricultural Research Management Project
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under the contract No. PL.420.206.69 11/P4N/ 1992. The authors extend a deep appreciation to T. Supriawan, Sumirat, Sardju and Asmarida for technical assistance during the experiment.
References Anderson, R.R., 1975a. Mammary gland growth in the hypophysectomized pregnant rat. Proc. Sot. Exp. Biol. Med., 148: 283-287. Anderson, R.R., 1975b. Mammary gland growth in sheep. J. Anim. Sci., 41: 118-123. Anderson, R.R., 1986. Mammary gland. In: B. Larson (Editor), Lactation, Iowa State University Press, Ames, pp. 3-38. Anderson, R.R., Harness, J.R., Sinead, A.F. and Salah, M.S., 198 1. Mammary growth pattern in goats during pregnancy and lactation. J. Dairy Sci., 64: 427-432. Bradford, GE., 1985. Selection for litter size. In: R.B. Land and D.W. Robinson (Editors). Genetics of Reproduction in Sheep, Butterworth, London, pp. 3- 17. Bradford, G.E., Quirke, J.F., Sitorus, P., Inounu, I., Tiesnamurti, B., Bell, F.L., Fletcher, I.C. and Torrell, D.T., 1986. Reproduction in Javanese sheep: evidence for a gene with large effect on ovulation rate and litter size. J. Anim. Sci., 63:418431. Butler, W.R., Fullenkamp, S.M., Capiello, L.A. and Handwerger, S., 1981. The relationship between breed and litter size in sheep and maternal serum concentrations of placental lactogen, estradiol and progesterone. J. Anim. Sci., 53: 1077-1081. Cowie, A.T., 1971. Influence of hormones on mammary growth and milk secretion. In: I.R. Falconer (Editor), Lactation, Pennsylvania University Press, University Park, pp. 127. Harness, J.R. and Anderson, R.R., 1977a. Effect of relaxin in combination with prolactin and ovarian steroids on mammary growth in hypophysectomized rats. Proc. Sot. Exp. Biol. Med., 156: 354-360. Harness, J.R. and Anderson, R.R., 1977b. Effect of relaxin and somatotropin in combination with ovarian steroids on mammary glands in rats. Biol. Reprod., 17: 599-604. Hayden, T.J., Thomas, C.R. and Forsyth, LA., 1979. Effect of number of young born (litter size) on milk yield of goats: role of placental lactogen. I. Dairy Sci., 62: 53-57. Heap, R.B. and Flint, A.P.F., 1984. Pregnancy. In: Austin and Short (Editors), Reproduction in Mammals: 3. Hormonal Control of Reproduction, Cambridge University Press, Cambridge, pp. 153-194. Knight, C.H. and Peaker, M., 1982. Development of the mammary gland. J. Reprod. Fertil., 65: 521-536. Manalu, W., Sumaryadi, M.Y. and Kusumorini, N., 1995. The effects of fetal number on maternal semm progesterone and estradiol of ewes during pregnancy. Bull. Anim. Sci. Spec. Edn., pp. 237-241. McDonald, L.E., 1980. Veterinary Endocrinology and Reproduction, 3rd edn. Lea and Febriger, Philadelphia, 560 pp.
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Obst, J.M, Boyes, T. and Chaniago, T., 1980. Reproductive performance of Indonesian sheep and goats. hoc. Aust. Sot. Anim. Prod., 13: 321-324. Piper, L.R. and Bindon, B.M., 1984. Ovulation rate as a selection criterion for improving litter size in Merino sheep. In: D.L. Lindsay and D.T. Pearce (Editors), Reproduction in Sheep, Cambridge University Press, Cambridge, pp. 237-239. Rattray, P.V., Garret, W.N., East, N.E. and Hinman, N., 1974. Growth, development and composition of the ovine conceptus and mammary gland during pregnancy. J. Anim. Sci., 38: 613-626. Refsal, K.R., Marteniuk, J.V., Williams, C.S.F. and Nachreiner, R.F., 1991. Concentrations of estrone sulfate in peripheral semm of pregnant goats: relationships with gestation length, fetal number and the occurrence of fetal death in utero. Theriogenology, 36: 449-461. Rice, G.E., Leach Harper, C.M., Hooper, S. and Thorbum, G.D., 1984. Endocrinology of pregnancy and parturition. In: D.R. Lindsay and D.T. Pearce (Editors), Reproduction in Sheep, Cambridge University Press, Cambridge, pp. 165-173. Snedecor, G.W. and Cohran, WC., 1982. Statistical Methods, 7th edn. Iowa State University Press, Ames, pp. 234-235. Sutama, I.K., 1992. Reproductive development and performance of small ruminants in Indonesia. In: P. Ludgar and S. Scolz (Editors), New Technology for Small Ruminant Production in Indonesia, Winrock International Institute for Agricultural Development, Morrilton, Arkansas, pp. 7- 14.
Summa, I.K., Edey, T.N. and Fletcher, I.C., 1988. Studies on reproduction of Javanese Thin-tail ewes. Aust. J. Agric. Res., 39: 703-7 11. Tiesnamurti, B., 1992. Reducing the preweaning mortality rate of Javanese Thin-tail sheep. In: P. Ludgar and S. Scolz (Editors), New Technology for Small Ruminant Production in Indonesia, Winrock International Institute for Agricultural Development, Morrilton, Arkansas, pp. 71-80. Tucker, H.A., 1986. Endocrine and neural control of the mammary gland. In: B. Larson (Editor), Lactation, Iowa State University Press, Ames, pp. 39-79. Tucker, H.A., 1987. Quantitative estimates of mammary growth during various physiological states: a review. J. Dairy Sci., 70: 1958-1966. Umo, I., Fitzpatrick, R.J. and Ward, W.R., 1976. Parturition in the goat: plasma concentrations of prostaglandin F and steroid hormones and uterine activity during late pregnancy and parturition. J. Endocrinol., 68: 383-389. Wahab, I.M. and Anderson, R.R., 1989. Physiologic role of relaxin on mammary gland growth in rats. Proc. Sot. Exp. Biol. Med., 192: 285-289. Wright L.C. and Anderson, R.R., 1982. Effect of relaxin on mammary growth in the hypophysectomized rat. In: R.R. Anderson (Editor), Relaxin, Plenum Press, New York, pp. 341-353.
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Effect of fetal number on the concentrations of circulating maternal serum progesterone and estradiol of does during late pregnancy W. Manalu *, M.Y. Sumaryadi I, N. Kusumorini Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural Universiry, Jalan Taman Kencana 3, Bogor 16151, Indonesia
Accepted 20 June 1996
Abstract Twenty Etawah-cross does with similar body weights (20-22 kg) and ages (2-3 years) were used to study the effects of fetal number (single or twin) on serum concentrations of progesterone and estradiol during the last two months of pregnancy. Average serum progesterone concentrations during the last two months of gestation period in the single- and twin-bearing markedly increased (P < 0.01) by 1302 and 259 I%, respectively, as compared to those non-pregnant does (5.791 and 11.113 vs. 0.413 ng/ml). Serum progesterone concentrations increased by 92.4% (P < 0.01) as fetal number increased from single to twin. Concentrations of serum estradiol during the last two months of gestation period in the single- and twin-bearing increased (P < 0.01) by 684 and 2006%, respectively as compared to those in the non-pregnant does (68.84 and 184.93 vs. 8.78 pg/ml). Serum estradiol concentrations increased by 169% (P < 0.01) as fetal number increased from single to twin. Average serum progesterone and estradiol concentrations of aborted does had decreased to a very low level, but were still higher than those of non-pregnant does, at least seven weeks prior to abortion date (I .691 ng/ml and 14.46 pg/ml for aborted vs. 0.413 ng/ml and 8.78 pg/ml for non-pregnant does). It was concluded that hormonal stimulation for mammary gland growth and development may have increased with the increased fetal number during pregnancy in preparation for more milk synthesis for the newborn kids. Keywords: Progesterone; Estradiol; Pregnancy; Goat
1. Introduction
mammary tissue (Cowie, 1971, Anderson
A major amount of mammary development is associated with hormonal stimuli of pregnancy, and the absence of these stimuli reduces the amount of
et al., 198 1). Exogenous administration of mammogenic hormones, including prolactin, growth hormone, estrogen, progesterone and relaxin, (Harness and Anderson, 1977a,b,Wright and Anderson, 1982, Wahab and Anderson, 1989) stimulated mammary gland growth and development.
Corresponding author. Phone: (0251) 328 487. Fax: (0251) 323 161. ’ Present address: Laboratory of Physiology and Reproduction, Faculty of Animal Science, Jenderal Soedirman University, P.O. Box 10 Poerwokerto 53123, Central Java, Indonesia.
Mammary gland growth and development in small ruminant animals (sheep and goat) dramatically increased during the last two months of the gestation period (Rattray et al., 1974, Anderson, 1975b,Ander-
l
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son et al., 1981). Anderson et al. (1981) reported that mammary cell DNA in goats was higher than in sheep (Anderson, 1975b) at comparable days of pregnancy and lactation. This difference was suggested (Anderson et al., 1981) to be due to the higher twinning rate occurring in goats compared to sheep. Anderson et al. (1981) suggested that the presence of more placental tissue in goats was related to more placental lactogen secretion to stimulate mammary gland growth, accounting for greater mammary development in goats than in sheep. The role of placental lactogen was demonstrated in mammary gland development in the rat (Anderson, 1975a) and in the goat (Hayden et al., 1979). Hayden et al. (1979) reported a positive correlation between litter size (i.e., feto-placental mass> and serum concentrations of placental lactogen and mammary gland growth and development in goats. Butler et al. (1981) observed an increased maternal serum placental lactogen level in sheep bearing different litter size. Rattray et al. (1974) also reported a higher mammary gland growth in ewes bearing different fetal numbers. The difference found in mammary gland growth and development in different litter size, however, was not solely due to the greater placental tissue. The increased number of corpora lutea was suggested to contribute more progesterone and estrogen secretion that eventually increased hormonal stimulation of mammary gland growth and development in sheep or goats bearing more than one fetus. Increased litter size means increased number of ovulating follicle and fertilized ova. It was documented that litter size positively correlated with ovulation rate (Piper and Bindon, 1984, Bradford, 1985). Increased ovulation rate would increase the number of corpora lutea secreting progesterone and estrogen during the gestation period. Increased placental mass with the increased litter size would increase placental glands secreting progesterone and estradiol during the second half of the gestation period (after placentation stage). It was reported that plasma concentrations of progesterone during pregnancy increased with the increased litter size in sheep (Butler et al., 198 1). Serum e&one sulfate was found positively correlated with the number of fetuses during the gestation period in does (Refsal et al., 1991). However, the correlation between progesterone concentra-
tions and fetal number have not been documented in goat species. The increased hormonal stimulation for mammary gland growth and development during the gestation period could explain the observed increased mammary gland growth in sheep (Rattray et al., 1974) and in goats (Hayden et al., 1979) with increased litter size. The increased mammary gland growth and development in ewes and does bearing different fetal numbers further confirm the observations of higher milk production in goats (Hayden et al., 1979) and sheep (Butler et al., 1981) having more than one kid or lamb. It was hypothesized that the secretion of endogenous progesterone and estradiol increased with the increased number of fetuses carried. The objective of this experiment was to study the profile of serum progesterone and estradiol concentrations during the fastest growing period of mammary glands (the second half of the gestation period) in does bearing different fetal numbers.
2. Materials and methods 2.1. Enuironmental ment
conditions
and animal manage-
This experiment was conducted in the village site during the period of October 1992 to May 1993. The whole experiment was conducted during the wet season of the year. Average daily ambient temperature and relative humidity ranged from 25 to 32°C and from 70 to 80%, respectively. The Etawah-cross goat (Etawah X Kacang, with undetermined degree of crossing) is an economically important breed of goat in the Indonesian small ruminant industry. This breed, medium in size and milk production (between Etawah and Kacang), is multipurpose in production (either for milk or meat production) and nonseasonal in reproduction. However, this breed of goat is primarily raised for meat production, as it is true for the small ruminant industry in Indonesia. Experimental does were kept in a pen with a raised floor as typical of small ruminant housing in the region. Fresh field grass was fed ad libitum with supplementation of table salt block. The animals were provided drinking water ad libitum. This feed-
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ing regiment was adopted to simulate the prevailing management practice of the small-holder small-ruminant farmer in the region. 2.2. Experimental design and protocol Twenty Etawah-cross does with similar weight (20-22 kg) and age (2-3 years) were maintained in an experimental pen with a 2 month adaptation to the experimental conditions prior to the mating period. During the mating period, the experimental does were mixed with five bucks for a month, the experimental does were maintained in a group of three per pen. Blood samples were drawn weekly after the breeding period. At the end of the experimental period, only eight does gave birth to normal kids (five singles and three twins). Three does aborted near the expected parturition date and were included in the statistical analyses of the data. The others were non-pregnant during the experimental period, and four of those were included in data analyses as a control for pregnancy. Experimental does were grouped into four groups: 0 (non-pregnant does as a control for pregnancy); Al3 (aborted does); 1 (does carrying a single fetus) and 2 (does carrying twin fetuses), with n = 4, 3, 5, and 3, respectively.
119
USA) with a slight modification for ovine progesterone concentration ranges (Manalu et al., 1995). The radioactivity of progesterone-bound tubes was counted with an automatic gamma counter. The concentrations of standard progesterone used to construct the standard curve ranged from 0.1 to 20 ng/ml. A sample volume of 100 ~1 serum was used in the assay for samples with progesterone concentrations ranging from 0.1 to 20 ng/ml. For samples with progesterone concentrations lower than 0.1 ng/ml, sample volume was increased to 200 p,l to bring the progesterone concentrations to the range of standard used. All sample progesterone concentrations were within the range of concentrations of standard progesterone used to construct the standard curve. Inter- and intra-assay variation coefficients were 7.5, and 2.0%, respectively. The concentrations of progesterone were parallel in the sample volumes of 50, 100, and 200 (11.
A 10 ml volume of blood was drawn with a plain vacutainer or a sterile syringe from the jugular vein in the time period from around 0900 to 1000 h on Thursdays weekly. The bleeding time was conducted consistently prior to feeding, and the day of week was chosen merely for convenience. The blood samples were allowed to clot in a cool ice box and transported to the laboratory for further separation of serum by centrifugation. The serum samples were then kept frozen for further hormone analyses. Weeks prior to parturition were counted back from the last blood sampling prior to the parturition date.
2.4.2. Estradiol Concentration of serum estradiol in duplicate was measured by the solid-phase technique radioimmunoassay (Diagnostic Products) with a slight modification for ovine estradiol concentration ranges (Manalu et al., 1995). The radioactivity of estradiolbound tubes was counted with an automatic gamma counter. The concentrations of standard estradiol used to construct the standard curve ranged from 20 to 500 pg/ml. A sample volume of 100 l.~l serum was used in the assay for samples with estradiol concentrations ranging from 20 to 500 pg/ml. For samples with estradiol concentrations lower than 20 pg/ml, sample volume was increased to 200 c1.1to bring the estradiol concentrations to the range of standard used. All sample estradiol concentrations were within the range of concentrations of standard estradiol used to construct the standard curve. Inter- and intra-assay variation coefficients were 10.2, and 5.0%, respectively. The concentrations of estradiol were parallel in the sample volumes of 50, 100, and 200 ~1.
2.4. Hormone analyses
2.5. Statistical analyses
2.4.1. Progesterone
Data collected during the seven-week measurement were analyzed for main effect (week prior to parturition or abortion, and status of pregnancy: non-pregnant, aborted, carrying single and twin) and
2.3. Blood sampling and processing
Concentration of serum progesterone in duplicate was measured by the solid-phase technique radioimmunoassay (Diagnostic Products, Los Angeles, CA,
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their interactions with a randomized design with factorial arrangement with unequal n. The difference between means of main effects were tested using least significant differences (Snedecor and Cohran, 1982).
of the progesterone concentration were far lower than average serum progesterone concentrations of pregnant does but higher than those of non-pregnant does. Serum progesterone concentrations were dramatically higher in the pregnant (5.79 1 and 11.113 ng/ml for single- and twin-bearing does, respectively) (P < 0.01) as compared to the non-pregnant does (0.413 ng/ml). The results of the experiment showed that the average serum progesterone concentration during the last two months of the gestation period in the twin-bearing does increased (P < 0.01) by 91.9% as compared to those in the single-bearing does (11 .l 1 vs. 5.79 ng/ml). It was important to note that serum progesterone concentrations of aborted does had decreased, approaching the levels of non-pregnant does seven weeks prior to the abortion date (1.69 1 for aborted vs. 0.413 ng/ml for non-pregnant does).
2.6. Results 2.6.1. Progesterone Average weekly serum progesterone concentrations of experimental does are presented in Table 1. There was no difference in the concentration of progesterone among weeks of blood sampling in pregnant and aborted groups, i.e., serum progesterone seemed to be at a concentration plateau in the seven weeks period of measurement prior to parturition or abortion. There was no interaction between fetal number and weeks prior to parturition or abortion. Average serum progesterone concentration in the non-pregnant does in the seven week measurement period prior to parturition was high. This high mean was due to the consistently high serum progesterone concentration in two does (numbers 2 and 4) as was verified by several replications. Individual weekly variations in serum progesterone concentration were found in aborted does. However, the peaks
Table 1 Concentrations
of serum progesterone
Fetal number ’
in non-pregnant,
Progesterone (ng/ml) Weeks prior to parturition 7 6
2.6.2. Estradiol Average weekly serum estradiol concentrations of experimental does are presented in Table 2. There was no difference in the concentrations of estradiol between weeks prior to parturition within the same fetal number group from seven weeks to two weeks
aborted, single- and twin-bearing
or abortion 5
does seven weeks prior to parturition
or abortion d
f 4
3
2
1
Mean g
0.067 0.004
0.059 0.003
0.065 0.004
0.413 a 0.325
0
Mean SE
2.525 1.317
0.059 0.001
0.049 0.004
0.067 0.007
AB
Mean SE
2.621 2.545
0.270 0.205
1.648 0.789
0.45 0.404
I
1.456
2.407 2.348
2.580 2.503
1.691 a 0.450
Mean SE
5.917
1.473
5.215 1.324
5.638 0.717
6.397 0.569
6.392 0.823
5.069 0.324
5.869 1.226
5.791 b 0.197
Mean SE
13.855 1.559
9.139 0.397
9.068 0.434
11.848 0.700
10.859 0.576
10.861 0.494
12.164 2.628
11.113 c 0.643
1
2
1.858
* Means and SE of 4, 5, 3, and 3 does for fetal number 0, 1, and 2, and aborted does, respectively. e Fetal number 0, 1, and 2 refers to non-pregnant, single- and twin-bearing does, respectively. AB, refers to aborted does. f Weeks prior to parturition only applied to single- and twin-bearing does. For aborted does this term means weeks prior to abortion. non-pregnant does, this term means nothing but is a control for hormonal profile at the same period of blood sampling. g Different superscripts in this column refer to differences between fetal number (P < 0.01).
For
W. Manalu et al./Small Table 2 Concentrations Fetal number
of serum estradiol e
in non-pregnant,
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aborted, single- and twin-beating
Estradiol (pg/ml) Weeks prior to parturition or abortion f 7 6 5 4
does seven weeks prior to parturition
3
1
2
or abortion d
Mean g
0
Mean SE
9.40 0.96
9.05 0.72
8.85 0.78
8.96 0.86
10.45 2.41
8.16 0.52
6.59 0.15
8.78 a 0.45
AB
Mean SE
9.14 0.56
11.26 1.21
12.99
1.41
18.15 5.59
14.83 2.03
16.72 3.57
18.15 1.51
14.46 a 1.1 I
1
Mean SE
30.87 5.07
40.07 8.82
53.15 7.32
53.01 5.67
76.36 14.55
89.66 19.50
138.76 47.14
68.84 b 13.93
2
Mean SE
117.56 19.06
87.33 8.57
126.0 1 27.86
151.38 18.92
164.43 37.23
170.09 35.98
477.72 219.09
184.93 50.01
’
d Means and SE of 4, 5, 3, and 3 does for fetal number 0, 1, and 2, and aborted does, respectively. e Fetal number 0, 1. and 2 refers to non-pregnant, single- and twin-bearing does, respectively. AB, refers to aborted does. f Weeks prior to parturition only applied to single- and twin-bearing does. For aborted does this term means weeks prior to abortion. For non-pregnant does, this term means nothing but is a control for hormonal profile at the same period of blood sampling. g Different superscripts in this column refer to differences between fetal number (P < 0.01).
prior to parturition. Serum estradiol concentrations seemed to slowly rise and reached peak concentrations (P < 0.01) in the week of parturition. The increased serum estradiol concentration one week prior to parturition was subject to a very high individual variation, giving very high SE values (greater than the mean values for a certain week of measurements). Serum estradiol concentrations were dramatically higher in the pregnant (68.839 and 184.932 pg/ml, for single- and twin-bearing does, respectively) (P < 0.01) as compared to those in the nonpregnant does (8.782 pg/ml). Concentrations of serum estradiol during the last two months of gestation in the twin-bearing does increased (P < 0.01) by 169% compared to those in the single-bearing does (184.932 vs. 68.839 pg/ml). Serum estradiol concentrations of aborted does had decreased, approaching those of the basal non-pregnant level seven weeks prior to abortion date (14.46 pg/ml for aborted vs. 8.78 pg/ml for non-pregnant does).
3. Discussion The results of the experiment showed that average maternal serum progesterone concentrations during the last two months of the gestation period for twins
was almost twice as much as found for single-bearing does. Concentrations of maternal serum estradiol at the same period was almost trite for twin- compared with single-bearing does. Even though progesterone and estradiol concentrations were not measured from the beginning of the gestation period, the results of this experiment implied a dramatic increase in hormonal stimulation for mammary gland growth and development when fetal numbers increased from one to two. The source of the increased serum concentrations of progesterone and estradiol in response to different fetal numbers was unclear, i.e., whether it was due to the increased number of corpora lutea or to the increased mass of placental tissue or a combination of both. Non-pregnant does had basal flat average progesterone and estradiol concentration during the same period of measurement, with slight spikes during the estrous cycles. The existence of corpora lutea and placenta during pregnancy dramatically increased serum progesterone and estradiol concentrations. Ranges of progesterone and estradiol concentrations found by others in goats at comparable ages of pregnancy (Umo et al., 1976) agreed with our results. In comparison to the profile of progesterone concentrations in sheep with the same litter size (Butler et al., 19811, goat progesterone concentrations observed in this study were slightly higher.
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However, Butler et al. (1981) found that there were no significant effects of fetal number on maternal serum estradiol concentrations in sheep, with average estradiol concentrations substantially lower than those found in goat species in this experiment. Maternal serum progesterone and estradiol concentrations in sheep (Manalu et al., 1995) were substantially lower than those reported in goats in this experiment. The differences in the profiles of progesterone and estradiol during pregnancy between goats and sheep were probably related to the difference in the role of placenta as a source of progesterone and estradiol in the different species (Heap and Flint, 1984). Since progesterone and estradiol are hormones that play critical roles in mammary gland growth and development during pregnancy (Anderson, 1986, Tucker, 1986, Knight and Peaker, 1982, Tucker, 1987), the increased fetal number or litter size would increase hormonal stimulation for mammary gland growth and development in preparation of more milk synthesis for the newborn kids. The increased hormonal stimulation of mammary gland growth and development with the increased litter size were accompanied by the increased mammary gland growth and development (Rattray et al., 1974, Hayden et al., 1979, Butler et al., 1981). Therefore the increased endogenous mammogenic hormones, especially progesterone, estrogen and placental lactogen, could increase mammary gland growth and development and ultimately milk production. These phenomena could be used as alternative means of improving milk production in small ruminant animals either for human consumption or for nourishing the new born kids. In Indonesia, sheep and goats play an important role in animal husbandry. However the mortality of the kids and lambs are so high that kids and lambs survival rates are basically very low (Obst et al., 1980). The low survival rate is strongly related to the low birth weight C3nama et al., 1988, Sutama, 1992) and milk production (Tiesnamurti, 1992). Lower birth weight is probably due to the lower uterine gland growth and development to secrete nutrients to nourish the fetus (McDonald, 1980), in addition to other factors such as nutrient deficiency and parasitic diseases. Serum progesterone and estradiol concentrations of aborted does approached those found in the non-
pregnant does seven weeks prior to abortion date. It was surprising that the does were still capable of maintaining the fetus for seven weeks with such low levels of maternal serum progesterone and estradiol. It is still unclear whether the increased progesterone and estradiol serum concentrations in does bearing two fetuses is due to the placental mass or the number of corpora lutea formed during the estrous cycle or combination of both. Even though sheep and goats are closely related phylogenetically and have morphologically similar forms of placentation, these two species differ in terms of the role of the placenta as a source of progesterone during pregnancy. In the ewe the placenta takes over progesterone production but in goats the ovaries and pituitary are necessary during the whole pregnancy and a doe will abort promptly following the removal of either (Heap and Flint, 1984). Since the placenta does not play a critical role in secreting progesterone during late pregnancy in does, it was suggested that the increased serum progesterone and estrogen concentrations in does bearing more than one fetus were not due to the increased placental mass, but related to the increased number of maintained corpora lutea during pregnancy. In sheep it was reported that litter size was highly correlated with the ovulation rate (Bradford et al., 1986). It is suggested that the research be extended to differentiate between the effects of fetal number and the number of surviving corpora lutea during pregnancy on the increased maternal serum progesterone and estradiol concentrations in sheep and goats. Progesterone concentrations were doubled in the twin-bearing as compared to single-bearing does, and estradiol concentrations in the twin-bearing as compared to those in the single-bearing does were more than doubled. The results of this experiment indicated that progesterone and estradiol could have been secreted by different glands during late pregnancy in the goat. If the primary source of both hormones are the same, ie., corpora lutea, why did estradiol concentrations increase more than progesterone concentrations in pregnancies with the same fetal number? This fact is probably related to the different function of estradiol during late pregnancy and parturition. In general, estradiol concentrations increased with the advance of pregnancy approaching parturi-
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tion date (Umo et al., 1976, Rice et al., 1984). The physiological significance of the increased estradiol concentrations prepartum is probably associated with the preparation of mammary glands to initiate milk secretion by stimulating prolactin secretion as was indicated by a previous report (Umo et al., 1976). Progesterone and estradiol concentrations during the second half of pregnancy could be used as strong parameters to predict whether the does are carrying single or twin fetuses. This information could be used in devising appropriate feeding strategies for pregnant does to meet the nutrient requirements of the mother and the fast-growing fetus(es) during late pregnancy. In addition, maternal serum progesterone and estradiol concentrations could be used as indicators of the possibility of abortion at least seven weeks prior to the abortion date.
4. Conclusion The results of this experiment indicate that maternal serum progesterone and estradiol concentrations increase with an increase in fetal number from one to two. Maternal serum progesterone and estradiol could be used as strong indices of selection for higher litter size, and to distinguish does carrying single or multiple fetuses during late pregnancy in goats. Progesterone and estradiol could have been secreted by different sources during late pregnancy in the goat. The increased maternal serum concentrations of progesterone and estradiol suggested increased hormonal stimulation for mammary gland growth and development during the second half of pregnancy with increased fetal number from single to twin. Correlation between the increased maternal serum progesterone or estradiol concentrations with the mammary gland growth and development as well as milk production are being studied to devise a novel means of increasing mammary gland growth and development.
Acknowledgements
This experiment was funded by a grant provided by The Agency for Agricultural Research and Development, Agricultural Research Management Project
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under the contract No. PL.420.206.69 11/P4N/ 1992. The authors extend a deep appreciation to T. Supriawan, Sumirat, Sardju and Asmarida for technical assistance during the experiment.
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Summa, I.K., Edey, T.N. and Fletcher, I.C., 1988. Studies on reproduction of Javanese Thin-tail ewes. Aust. J. Agric. Res., 39: 703-7 11. Tiesnamurti, B., 1992. Reducing the preweaning mortality rate of Javanese Thin-tail sheep. In: P. Ludgar and S. Scolz (Editors), New Technology for Small Ruminant Production in Indonesia, Winrock International Institute for Agricultural Development, Morrilton, Arkansas, pp. 71-80. Tucker, H.A., 1986. Endocrine and neural control of the mammary gland. In: B. Larson (Editor), Lactation, Iowa State University Press, Ames, pp. 39-79. Tucker, H.A., 1987. Quantitative estimates of mammary growth during various physiological states: a review. J. Dairy Sci., 70: 1958-1966. Umo, I., Fitzpatrick, R.J. and Ward, W.R., 1976. Parturition in the goat: plasma concentrations of prostaglandin F and steroid hormones and uterine activity during late pregnancy and parturition. J. Endocrinol., 68: 383-389. Wahab, I.M. and Anderson, R.R., 1989. Physiologic role of relaxin on mammary gland growth in rats. Proc. Sot. Exp. Biol. Med., 192: 285-289. Wright L.C. and Anderson, R.R., 1982. Effect of relaxin on mammary growth in the hypophysectomized rat. In: R.R. Anderson (Editor), Relaxin, Plenum Press, New York, pp. 341-353.