The effect of pregnancy on the interval between one oestrus and the next in the tammar , eugenii J. C. Merchant Division of Wildlife Research, CSIRO, P.O. Box 84, Lyneham, A.C.T. 2602,

Summary. In females mated to vasectomized males the interval between oestrous periods was 30\m=.\6\m=+-\1\m=.\17(s.d.) days but when mated to intact males and pregnancy intervened, it was 29\m=.\4\m=+-\1\m=.\26days (P < 0\m=.\025).After removal of pouch young, females carrying diapausing blastocysts gave birth 26\m=.\2days later and came into oestrus 26\m=.\4\m=+-\0\m=.\57(s.d.) days later. When post-partum fertilization was prevented, removal of pouch young was followed by oestrus 30\m=.\4\m=+-\0\m=.\99days later (P < 0\m=.\0005).These results indicate an influence of the conceptus upon the oestrous cycle of Macropus eugenii.

Introduction

It is well known that the young exerts a powerful influence on the mother after birth, but in a review of marsupial reproduction Sharman (1970) points out that it has long been thought that it has no effect on the mother before birth and that the placenta has no endocrine function. However, the placenta of the exerts a morphogenetic effect on the endometrium (Renfree, 1972), possibly as a result of the indirect evidence that the yolk sac placenta of the and the tammar in vitro has the capacity to convert pregnenolone to progesterone (Bradshaw, McDonald, Hahnel & Heller, 1975; Renfree & Heap, 1977). Since gestation in these species is accommodated within the duration of one oestrous cycle it seemed probable that the endocrine function of the placenta is limited to effects on the immediate environment of the uterus but may not affect ovarian events, particularly the life-span of the corpus luteum or the next oestrus and ovulation. Indeed, Tyndale-Biscoe, Hearn & Renfree (1974) proposed that the control of pregnancy and the oestrous cycle in macropodid may be independent of each other except for the initial part of the cycle when both are controlled by the young corpus luteum. In the agile wallaby, Macropus agilis, which has a reproductive pattern similar to that of the tammar, M. eugenii (Group 2 of Sharman, Calaby & Poole, 1966) it was observed that the interval between two consecutive oestrous periods was significantly shorter when a pregnancy intervened than it was in unmated females (Merchant, 1976). Since the data were not obtained from sequential observations on the same females, individual differences or the effect of copulation could have obscured the effect of pregnancy itself. The present experiments were undertaken to test the hypothesis that some aspect of pregnancy can hasten the onset of oestrus in the tammar wallaby.

Materials and Methods All the used in the experiments came from a breeding colony of tammar , held in CSIRO enclosures at Canberra, A.C.T., and derived from animals caught on Kangaroo Island, South Australia. They were kept outdoors in two large pens grassed with Phalaris tuberosa and were given a supplementary feed mix of ground lucerne, hay and oats.

Downloaded from Bioscientifica.com at 09/28/2021 10:41:14AM via free access AU females were at least 2 years old and had bred before the start of the experiments. The males were adults of proven fertility, 2 of which were vasectomized 1 month before the experiments. The experiments were carried out in series over 2 breeding seasons, early January to late July (Sharman & Berger, 1969), in 1976 (Series 1) and 1977 (Series 2).

Series 1 Eleven females that were known to have been with males during a post-partum oestrus and could be expected to be carrying a diapausing blastocyst had their young removed from the pouch (RPY) to initiate reproductive activity culminating in birth and post-partum oestrus (Text- fig, la). This provided the first set of intervals—RPY to post-partum oestrus—in which there was an intervening pregnancy. At post-partum oestrus, however, the females were allowed access only to vasectomized males, thus preventing fertilization and aiding diagnosis of the day of oestrus. When the pouch young were removed 8-15 days later, therefore, there was no diapausing blastocyst present to be activated and thus no intervening pregnancy before the next oestrus occurred.

RPY |o RPY · (a) U il I I

Jan. Feb. Mar. Apr. May Jun. 1976

lo RPY O · I·

J_I_I_I Jan. Feb. Mar. Apr. May Jun. 1.977 Text-fig. 1. Diagram of the experimental procedure in females of (a) Series 1, (b) Series 2, i.e. those which gave birth at the end of the non-breeding season, and (c) Series 2, i.e. those which did not give birth at the end of the non-breeding season. RPY = removal of pouch young; = birth; O = oestrus with vasectomized male; · = oestrus with intact male; solid line = period of daily examination; hatched bar = pregnant cycle; hollow bar = non-pregnant cycle. Series 2 Twelve females, without pouch young, were examined daily from the end of the non-breeding season until birth and/or mating occurred: 6 gave birth to young derived from blastocysts carried over the non-breeding period (Text-fig. lb) and all 12 mated with vasectomized males. The pouch young of those females which gave birth were removed 8-15 days later to initiate the delayed cycle of reproduction. At the next oestrus they were again mated with vasectomized males and underwent non-pregnant cycles. At the end of this cycle they were mated to fertile males and underwent pregnant cycles. The 6 females which mated with a vasectomized male without giving birth (Text-fig. lc) then entered non-pregnant cycles, at the end of which they were mated to intact males and provided pregnant cycles. In order to minimize disturbance to the females they were kept together in groups of no more than 6 in the same pen throughout one series of experiments; pairs of intact or vasectomized males were introduced to or removed from the pens as required. Furthermore, examination of

Downloaded from Bioscientifica.com at 09/28/2021 10:41:14AM via free access females was restricted to once per day and not begun until Day 24 after RPY, from which time birth and oestrus could be expected from previous knowledge of the reproductive cycle of this species (Berger, 1970). These examinations were continued on each of 4 days after mating. The day of birth ± 12 h was determined by examining the pouch for newborn young and the day of oestrus from examination of vaginal smears, using the methods of Poole & Catling (1974). A plug of coagulated semen is frequently found in the urogenital sinus and is the best evidence of copulation but spermatozoa and prostatic bodies in the vaginal smear provided additional confirmation. In Series 1 the onset of the cycle was fixed precisely by the time of RPY and the intervals to birth and oestrus were accurate to ±12 h. In Series 2, however, the initial oestrus was known only to an accuracy of ±12 h and so the intervals to birth and next oestrus were accurate to ±24 h.

Results The results of the Series 1 experiments are presented in Table 1. The mean intervals from RPY to birth and RPY to post-partum oestrus were the same because in most animals birth and post- partum oestrus occurred within the same 24-h period. From other studies the actual interval from birth to onset of oestrus is known to be 10-16 h (Tyndale-Biscoe & Rodger, 1978). The interval from RPY to oestrus in the same females after being mated to vasectomized males was 4 days longer, the difference being highly significant (P < 0-0005).

Table 1. Intervals (days) from removal of pouch young (RPY) to birth, to next oestrus and to the next oestrus after no pregnancy in female tammar wallabies

Mated to intact male Mated to vasectomized male

Animal RPY to birth RPY to next oestrus RPY to next oestrus

3945 26-5 26-5 30-5 3947 26-5 26-5 31-5 3955 27-5 27-5 31-5 3956 26-5 26-5 28-5 3975 25-5 26-5 30-5 4032 25-5 25-5 29-5 3960 26-5 26-5 31-5 3721 25-5 26-5 29-5 3892 26-5 26-5 30-5 4158 25-5 25-5 30-5 3825* 28-5 28-5 30-5,38-5,43-5 Overall 26-2 ± 0-67 f26-4±0-57 f30-4 ± 0-99 mean ±s.d. (N = 10) * Female excluded from analysis (see text). t Means used in one-tailed / test; observed t value 11 11 ; < 0-0005. · One female (No. 3825) was excluded from the analysis because she exhibited oestrous changes in the vaginal smear by Day 31 but did not mate then. However, she mated on Day 39 and Day 44 after RPY. As no other female was observed to mate other than on the day of oestrus, as determined by vaginal smear, the behaviour of this female cast doubt on when oestrus occurred. Nevertheless, whichever figure was used it would have been in excess of the interval for her pregnant cycle, 28-5 days, and if included would have exaggerated the difference between the two conditions.

Downloaded from Bioscientifica.com at 09/28/2021 10:41:14AM via free access The results of Series 2 are presented in Table 2 and again show the close proximity of post- partum mating to birth. In this series the difference between the two intervals was less marked, being about 1 day, but it was statistically significant (P < 0-025). This smaller difference, as compared to Series 1, is partly due to the less accurate method of determining the interval. Again two females (Nos 3721 and 4233) were excluded from the analysis. They failed to give birth after mating with a fertile male, which could have been due to an unsuccessful pregnancy or loss of young at parturition (Table 2). Both females had non-pregnant intervals of 33 days after mating with a vasectomized male, but after mating with a fertile male, one female next mated 28 days later and the other showed oestrous-like changes in the vaginal smear at Day 29 but did not mate. Both values, 28 and 29 days, for these 'non-pregnant' intervals are among the shortest in the pregnant group and shorter than all except two of the others in the non-pregnant group (Table 2). Table 2. Intervals (days) from oestrus to birth, to next oestrus and to the next oestrus after no pregnancy in female tammar wallabies

Mated to intact male Mated to vasectomized male

Oestrus Oestrus to to birth next oestrus Oestrus to next oestrus

3947 28 28 29 3955 29 29 31 3960 29 29 29 4216 31 32 33 4223 30 30 30 4236 30 30 31 4238 28 28 31 4311 28 28 31 86986 30 30 31 86996 30 30 30 3721* 28 33 4233* — 29 33

— Overall 29-3 ± 1-06 t29-4 ± 1-26 f30-6±l-17 mean ± s.d. (N = 10) * Females excluded from analysis (see text). t Means used in one-tailed I test; observed t value 2-22; < 0-025.

Discussion The results for the tammar wallaby reported here confirm the conclusion previously reached for the agile wallaby (Merchant, 1976) that some aspect of a successful pregnancy affects the length of the interval between removal of a pouch young and oestrus and between one oestrus and the next. In view of these results it is likely that other macropodid species with a similar reproductive pattern may exhibit the same effect of pregnancy. The one species for which there are sufficiently detailed data for comparison is the red kangaroo. However, Sharman & Calaby (1964) state that "although the interval from fertile mating until post-partum oestrus slightly exceeded the length of the oestrous cycle it was not significantly different from it. There is thus no evidence that fertiliz¬ ation, followed by pregnancy, birth and attachment of a young to a teat in the pouch, in any way delayed the onset of the next oestrus". This suggestion, contrary to the present results, was qualified by the comment that most of the data were derived from young females which tended to have shorter oestrous cycles than older females. The interval between oestrus and post-partum

Downloaded from Bioscientifica.com at 09/28/2021 10:41:14AM via free access oestrous on the other hand was established using a higher proportion of older females. Sharman & Calaby (1964) further suggest that there may be instrinsic differences in cycle length between different females and that there was only partial correspondence between the females used for establishing each set of data. These factors, then, may contribute to a reduction in any differences which may exist and it remains possible that a closer examination of the red kangaroo, using the same females to provide each set of data and vasectomized males at the appropriate oestrous periods would show the same effect as that presented here for the tammar wallaby. The short intervals observed in the two females in Series 2 which mated with a fertile male but apparently did not give birth is highly suggestive of a pregnancy effect, despite the apparent failure of the pregnancy to go to term. This emphasizes the need to know definitely whether a female is pregnant or not and the inadvisability of relying solely on the presence of a pouch young to determine it retrospectively. The particular aspect of pregnancy that brings about the observed effect is at present unknown, but it is presumably related to the hormonal status of the female. Oestrus usually occurs during the same 24-h period as birth and some aspect of the changing hormonal status at the end of pregnancy could therefore act as a trigger to initiate oestrous behaviour. Hearn (1972) observed that the preovulatory peak of plasma gonadotrophin coincided with oestrus, so that it is likely that the phenomenon reported here relates not only to the observed behaviour but to the whole sequence of birth, oestrus and ovulation. The present results favour the view that the timing of this sequence is influenced by the preceding pregnancy and to this extent are evidence for a form of maternal recognition of pregnancy hitherto only suspected in the agile wallaby.

I am greatly indebted to Dr C. H. Tyndale-Biscoe for helpful and constructive criticism. Mr N. G. Simms and R. Moore provided technical assistance with animal handling and maintenance and Mrs. W. Guy typed the manuscript. Mr F. Knight drew the figure.

References

Berger, PJ. (1970) The reproductive biology of the the marsupial Macropus eugenii. Theriogenology 8, tammar wallaby, Macropus eugenii Desmarest 164. (Marsupialia). Ph.D. thesis, Tulane University, New Sharman, G.B. (1970) Reproductive physiology of Orleans. marsupials. Science,N.Y. 167, 1221-1228. Bradshaw, S.D., McDonald, J.R., Hahnel, R. & Heller, Sharman, G.B. & Berger, PJ. (1969) Embryonic H. (1975) Synthesis of progesterone by the placenta diapause in marsupials. Adv. Reprod. Physiol. 4, of a marsupial. J. Endocr. 65,451-452. 211-240. Hearn, J.P. (1972) The pituitary gland and reproduction Sharman, G.B. & Calaby, J.H. (1964) Reproductive in the marsupial Macropus eugenii. Ph.D. thesis, behaviour in the red kangaroo, Megaleia rufa, in Australian National University. captivity. CSIRO Wildl. Res. 9, 58-85. Merchant, J.C (1976) Breeding biology of the agile Sharman, G.B., Calaby, J.H. & Poole, W.E. (1966) wallaby, Macropus agilis (Gould) (Marsupialia: Patterns of reproduction in female diprotodont ), in captivity. Aust. Wild!. Res. , 93- marsupials. Symp. zool. Soc. Lond. 15, 205-232. 103. Tyndale-Biscoe, CH. & Rodger, J.C (1978) Differential Poole, W.E. & Catling, P.C. (1974) Reproduction in the transport of spermatozoa into the two sides of the two species of grey kangaroos Macropus giganteus genital tract of a monovular marsupial, the tammar Shaw and M. fuliginosus (Desmarest). I. Sexual wallaby (Macropus eugenii). J. Reprod. Fert. 52, 37- maturity and oestrus. Aust. J. Zool. 22, 277-302. 43. Renfree, M.B. (1972) Influence of the embryo on the Tyndale-Biscoe, C.H., Hearn, J.P. & Renfree, M.B. marsupial uterus. Nature, Lond. 240, 475-477. (1974) Control of reproduction in macropodid Renfree, M.B. & Heap, R.B. (1977) Steroid metabolism marsupials. /. Endocr. 63, 589-614. in the placenta, corpus luteum and endometrium in Received 24 August 1978

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