- Email: [email protected]
The estrous cycle and induction of estrus in the australian feral sow (Sus scrofa)
Theriogenology 41:1181-1192, 1994 THE ESTROUS CYCLE AND INDUCTION OF ESTRUS IN THE AUSTRALIAN FERAL SOW (Sus scrofa) RJ. Kilgour and D. Choquenot NSW Agriculture, Agricultural Research Centre P.M.B. 19, Trangie, NSW, 2823, Australia
Received for Publication: April 20, 1993 Accepted: December 22, 1993 ABSTRACT In the first of 2 experiments, the estrous cycles of 11 Australian feral sows were studied. In 9 of the 11 sows the cycle was characterized by cyclic occurrences of low (0 to 2 ng/ml) and high (24.2 to 47.2 ng/ml) levels of progesterone. The low levels were associated, in all sows but one, with the standing response, lasting from 1 to 4 days, to a feral boar. Concomitant cyclic changes in vulval swelling and consistency of the vaginal mucus were also observed. Using intervals between the standing estrous response or the marked changes in the secretion of progesterone, the mean length of the cycle was calculated as 19.8 and 20.0 d, respectively. Two of the sows did not exhibit cyclic changes in any of the parameters measured, and on no occasion did either stand for service. In the second experiment, it was shown that estrus can be reliably induced in feral sows by either 1 of 2 methods: first, sows were induced to abort by prostaglandin injection. They were then administered gonadotrophin 48 to 72 h post abortion, and came into estrus 4 to 5 days later. Second, estrus was suppressed by feeding sows altrenogest, and was induced again about 9 days following altrenogest withdrawal. Key words: estrus, estrous cycle, progesterone, vaginal mucus, Australian feral sows
INTRODUCTION Feral pigs are widespread and abundant in Australia. Current estimates suggest that as many as 13 million feral pigs may inhabit the continent (10). Colonization by pigs closely coincided with European settlement of eastern and northern Australia (11), and feral pigs now occupy habitats ranging from semi-arid rangelands to sub-alpine highlands, to subtropical floodplains and forests (23). Throughout their range Australian feral pigs have reverted to wildtype morphological characteristics; they are typically more similar in size, shape and appearance to their progenitor type, the European wild boar, than they are to the domestic pigs from which they are directly evolved (2). If such reversion has occurred physiologically as well as morphologically, the estrous cycle of Australian feral sows would be expected to be more similar Acknowledgments We acknowledge the financial support of the Wildlife Exotic Disease Preparedness Fund of the Bureau of Rural Resources of the Department of Primary Energy and Industry; the assistance of Trevor ~,es for the construction of the restraining cage; the technical assistance of Brian Lukins and Margaret Szantar, and the donation of a kit for assay validation by Australian Laborotory Services, Rockdale, N.S.W., Australia.
Copyright © 1994 Butterworth-Heinemann
Theriogenology
1182 to that of European wild boar than that of domestic pigs.
The length of the estrous cycle in Australian feral sows is not known. Information about the estrous cycle is essential ff the cycle is to be manipulated in order to artificially bring sows into standing estrus. Estrus induction in feral sows is important for their use as bait to trap other feral pigs (3) or to enhance production for commercial harvest or recreational hunting (2). In this paper, we describe the estrous cycle of the Australian feral sow and the physiological changes associated with the cycle. We also look at the efficacy of 2 techniques for the induction of estrus in Australian feral sows. Results are discussed in relation to what is known of estrus in the European wild boar. MATERIALS AND METHODS The sows used in the experiments had either been caught in the wild and run in small paddocks for 3 yr or were the offspring of such sows. The sows were allocated at random to 1 of 3 groups so that the weight range in each group was as similar as possible. One of the groups was used to study the estrous cycle in naturally cycling sows, while the other 2 groups were used for the induction of estrus. The sows of the first group ( n = l l ) were housed in individual pens over the months of June to August (winter) and were fed either wheat grain or pelleted lucerne ad libitum. Water was freely available at all times. Each day for a total of 72 d, a jugular blood sample was taken from each sow using heparinized, evacuated blood collection tubes. At the time of blood collection, the appearance of the external genitalia and vaginal mucus were scored, and the sow was exposed to a teaser boar. The blood was centrifuged and the plasma stored at -20° C until assay. The vulva was scored as 1 = no vulval swelling, 3 = marked vulval swelling, sometimes with internal labia exposed; a score of 2 was given to sows showing an intermediate degree of swelling. The state of the vaginal mucus was assessed by taking a smear of the vaginal wall with a fiber glass rod. The mucus was scored as 1 = dry making insertion of the rod difficult, very little mucus taken; 2 = thick, creamy mucus which formed droplets on the rod; 3 = thin, copious mucus. Following examination of the vulva and mucus, the sows were exposed to a boar in groups of 3 and were observed until they showed either active avoidance of the boar's approaches or stood while he mounted to serve them. The teaser boar was an adult castrated male that had been treated with 1125 mg of testosterone as testosterone cypionate (Banrota, ICI, Sydney, Australia) 4 wk prior to the start of the observations and at 4-wk intervals thereafter. The assay for progesterone was a direct double-antibody radioimmunoassay for progesterone in human serum (Farmos Diagnostica, Turku, Finland). According to information supplied with the kit, the progesterone antiserum cross reacts 100% with progesterone; its cross-reaction with 17-hydroxyprogesterone, estrogens, pregnenolone and cortisol are less than 0.5%. The sensitivity of the assay was 0.3 ng/ml (def'med as the lower limit of the standard curve). Levels of progesterone in plasma samples taken from a sow on days when she stood for
Theriogenology
1183
the boar and at 3-d intervals gave values similar to those reported in the literature for sows in the follicular and luteal phases. The linear dilution of one of the luteal-phase samples gave progesterone values which decreased linearly. The second group of sows (n=14) was run with 3 fertile boars for a period of 10 wk over the months May to July (late autumn to early winter). At the end of this period, the sows were placed in individual pens where they were fed every day and allowed constant access to water. On the following day, each sow was adrnirtistered 10 mg of prostaglandin (Lutalysex, Upjohn, Sydney, Australia) to induce abortion. However, only 4 sows aborted by 48 h post injection, so the sows which had not aborted were given an additional 25 mg of prostaglandin. All sows which aborted were then treated with 1000 IU of a PMSG preparation (Pregnocolx, Heriot, Melbourne, Australia) no less than 48 and no more than 72 h post abortion. Every day for the next 10 d, a jugular blood sample was taken and the plasma was frozen until assayed for progesterone. Based on the results from the naturally-cycling group, a sow was deemed to be in estrus during the 2 d leading up to the rise in plasma progesterone concentration above undetectable levels. No notice could be taken of external genitalia or vulval mucosal scores since these animals had only recently aborted. Crown-rump lengths were measured on all intact fetuses and fetal age at abortion was estimated from the relationship between fetal age and crown-rump lengths as reported by Henry (9). Because we had to use 2 differing injections of prostaglandin to induce abortion, the protocol for this technique was deemed to be unreliable. Therefore, a supplementary group of sows (n=24) was placed with 4 fertile adult boars for 10 wk during the late spring and early summer (November to January). At the end of the breeding period, these sows were also placed in individual pens where they were fed every day and allowed constant access to water. On the following day, each sow was treated with a single 25 mg injection of prostaglandin to induce abortion. Fetal age at abortion was estimated from the crown-rump length.
The third group of sows (n=9) was housed in individual pens in early July where they were fed each day and given free access to water. Each day for 25 d, all the sows were given " 20 m g altrenogest (Regumatc x, Rousscl Uclaf, Sydney, Australia) which was dissolved in vegetable oil and mixed with their feed. Normally, treatment with altrenogest would have lasted for only 18 d (12), but we administered the treatment for a longer period because, on the day the sows were first fed altrenogest, 5 of them were suspected to be in estrus, as judged by the appearance of their vulvae. Each day, the appearance of the vulva and consistency of the vaginal mucus were scored. On the 26th day, altrenogest was withdrawn from the feed and these measurements continued for a further 10 d. Based on the data for the naturally-cycling sows, a sow was deemed to be in estrus when her progesterone levels increased above 10 ng/ml coincident with a score of 3 for either the external genitalia or vulval mucus. RESULTS
Naturally-cyclic Sows Levels of progesterone varied in cyclical fashion in 9 of the 11 sows, indicating the occurrence of regular estrous cycles. This was confirmed by the fact that on days when
Theriogenology
1184
3
i
2 1 0
*,3 0 C.)
~2 1
~0 120
~.I00 8O 6O 40
~ 20 •
0 0
10
20
30
40 Day
50
.
|
60
|
70
Figure 1. Changes in plasma progesterone level, vulval score and vaginal mucosal score over the duration of the study in a representative sow from the naturally cycling group. Triangles indicate days on which the sow stood for service.
Theriogenology
1185
progesterone levels were low the sows would stand for service by the boar (Figure 1). This pattern was characterized by the following changes: on days when the sows first allowed the boar to mount, progesterone levels were low, usually less than 2 ng/ml and often undetectable. On the last day on which the sows stood for service, progesterone levels had begun to rise again. These levels then reached a plateau over the next 1 to 4 d and remained relatively steady for 7 to 10 days after which time they fell rapidly again to low or undetectable levels. Progesterone then remained at these levels for periods which varied from 5 to 8 d until they increased once more, as described above. Taking the day on which the sows stood for service as Day 0 of the estrous cycle, progesterone levels were low to undetectable over the first 2 to 3 d of the cycle, after which time they rose rapidly. The rate of this increase generally slowed sometime between Days 6 and 9, so that levels reached a maximum, which ranged from 24.2 to 47.2 (mean 34.6) ng/ml, on Days 12 or 13. After this time there was a rapid decrease, with levels falling to nearly zero by Day 16 or 17, where they remained for 3 or 4 days until the sow again came into estrus. In 1 of the 2 sows that did not exhibit regular estrous cycles, progesterone levels fluctuated, with no obvious pattern and with levels varying from 2 to 30 ng/ml. In the other of these 2 sows, progesterone levels were low or undetectable throughout the study (Figure 2). Based on this pattern of progesterone secretion, the lengths of 21 estrous cycles from 9 sows showing regular patterns of progesterone secretion were determined as the intervals between the days on which progesterone levels showed their abrupt increase following the periods of low levels (Table 1). Mean ~ SEM) cycle length was 20.0 + 0.2 d with a coefficient of variation of 4.4%. For the first 9 d of the experiment, no sows were mounted by the teaser boar, which appeared to be intimidated by some of the sows. This boar was replaced on Day 10 by a boar which mounted sows from the first exposure. Sexual behavior of the boar and sows was similar to that described for domestic pigs (19);" the boar elicited series of short grunts when pursuing the sows, and sows in estrus stood rigidly when mounted, whereas those not in estrus actively discouraged the boar's attempts to mount. Over the duration of the study, the sows showed a cyclic pattern of sexual receptivity and nonreceptivity (Figure 1). Of the 11 sows, 2 failed to stand for the boar over the duration of the observations. These were the 2 sows that showed no obvious cyclic pattern in their secretion of progesterone. The 9 remaining sows stood for the boar on a total of 27 occasions, although on 1 occasion 1 sow failed to stand, even though her progesterone levels were low. The number of consecutive days on which the sows stood for the boar provide a measure of the duration of esu'us. One of the estrous periods was withdrawn from the data due to vaginal bleeding in 1 sow on the first day she stood to be mounted and we decided not to expose her to the boar on the subsequent days of that oestrus. Of the 26 remaining cycles, the sows stood for 1 d on 1 occasion, 2 d on 7 occasions, 3 d on 16 occasions and 4 d on 2 occasions.
Theriogenology
1186
IO0 8o
~
2O 0 I00
~
6O 4o 2o
0 0
10
20
30
40
50
60
70
Day Figure 2. Changes in plasma progesterone level over the duration of the study in the 2 sows of the naturally cycling group that did not stand for service.
Using the recurring pattern of sexual receptivity, the lengths of 19 estrous cycles from the 9 receptive sows were determined as being the number of days between the first day of one period of sexual receptivity and the first day of the next period (Table 1). Mean ~ SEM) cycle length was 19.8 + 0.3 d with a coefficient of variation of 6.0%. The 9 sows showing cyclic changes in progesterone levels also showed a cyclic pattern of change in the appearance of the vulva (Figure 1). On the days on which these sows stood for service, the vulva was usually swollen. The sows stood for service on a total of 75 occasions,
Theriogenology
1187
and on 59 (80%) of these, their vulvae were given a score of 3; on 4 occasions (7%) they were given a score of 2; and on 10 occasions (13%) they were given a score of 1. The sows also received scores of 3 on days when they did not stand for service, but these occasions were almost invariably associated with periods of sexual receptivity and rarely occurred more than 1 d before or I d after the period when they stood for service (Figure I). There was no evidence of synchrony of the occurrence of estrous between the sows.
Table I. Frequency of estrous cycles of various lengths in Australian feral sows as measured by cyclic changes in blood progesterone levels or in sexual behavior. Numbers in parentheses refer to the number of cycles on which the frequencies are based. Cycle length measured by Duration of cycle (days)
Progesterone levels (21)
Sexual behavior (19)
17
0.00
0.05
18
0.05
0.05
19
0.19
0.32
20
0.52
0.26
21
0.19
0.26
22
0.05
0.05
The 2 sows that did not stand for service over the entire period of the study each received vulval scores of 1 on all but 2 occasions. While less clearly defined than changes in progesterone or in the appearance of the vulva, vaginal mucus of the 9 cyclic sows also showed regular changes; on days when they stood for service their mucus was generally thin and copious (Score 3), while at other times during the cycle the scores varied from 1 to 2 but rarely was a score of 3 given (Figure 1). Of the 2 sows which showed no apparent estrous cycles, the sow with consistently low blood progesterone received a score of 1 on all but 4 occasions whereas the other sow received all 3 scores but with no obvious pattern. EstrusInduction Of the 14 sows in the early bred group, 1 sow aborted spontaneously before the administration of prostaglandin, 4 sows aborted 22 to 35 h after the fLrSt injection, and 5 sows aborted 22 to 44 h after the second injection. The 4 sows which did not abort were shown at autopsy to be nonprcgnam. Due to the intractable behavior of 1 sow, blood samples could not be collected from her, nor could observations on the vaginal mucus and vulval observations be performed.
Theriogenology
1188
Prior to prostaglandin treatment, plasma progesterone levels were high (50 to 80 ng/ml), but fell rapidly to undeteetable levels following prostaglandin injection (Figure 3). These levels remained undeteetable in all animals for a mean of 4.4 d (range 2 to 8 d) following the injection of gonadotrophin, indicating that the sows came into estrus 2 to 3 days after this injection (range 0 t o 6d). Of the 24 later-bred animals injected with a single 25-mg dose of prostaglandin, 18 aborted within 48 h of injection. At autopsy following euthanasia, 4 of the remaining sows were found to be nonpregnant, while the remaining 2 sows subsequently gave birth to live litters. Estimated age of the fetuses at abortion ranged from 65 to 114 d (Table 2).
80 70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
8
9
10
11
Days Figure 3. Changes in plasma progesterone over lime in 2 representative sows from the abortestrogen group. Day 0 is the day of administration of abortffacient, while the arrow indicates the day of administration of gonadotrophin.
In 8 of the 9 sows in the group in which oestrus was suppressed with altrenogest, progesterone levels were generally high (80 to 140 ng/ml) during the first 10 days of treatment, after which time they fell to almost undetectable levels (Figure 4). However, after a mean of 9.0 d following the cessation of feeding of altrenogest (range 8 to 11 d), progesterone levels began to rise once more. These changes in progesterone levels coincided with changes in the appearance of the external genitalia and in the consistency of the vaginal mucus. During the time of altrenogest feeding, the scores of sows were consistent with those observed for the non estrous sows, while 3 to 7 d following the cessation of feeding, the mucus and genitalia scores increased to those observed in estrous sows (Figure 4). Based on our observations in the naturally-cycling sows of this study, we concluded that these estrous-induced sows came into estrus about 7 d following
TheHogenology
1189
~e cessation of altrenogest treatment. One sow failed to respond to treatment with altrenogest ~ut displayed progesterone levels that were constantly elevated. This animal's vaginal mucus and ~xternal genitalia were almost constantly in a nonestrous state.
table 2. Day of gestation on which sows aborted. Data represent the number of sows aborting over a given gestational range, while figures in parentheses represent the proportion of these sows.
Range in days of gestation
Number of sows
65 to 69
1 (O.04)
70 to 74
0
75 to 79
0
80 to 84
1 (0.04)
85 to 89
2 (0.08)
9 0 t o 94
0
95 to 99
6 (0.23)
100 to 104
8 (0.31)
105 to 109
4 (0.15)
110 to 114
4 (0.15)
DISCUSSION The average length of the estrous cycle of Australian feral sows as measured in this study was 20 d. The cycle was characterized by standing estrus of 1 to 4 days, by cyclic changes in the levels of progesterone, and by cyclic changes in the appearance of the external genitalia and consistency of vaginal mucus. The mean length of the cycle of the sows in this study was shorter than that reported for the domestic sow, the average length of which varies from 20 to 22 d (1,6,14,16,18) or for the European wild boar in the United States, which averaged 23.8 and 21.8 d in the 2 studies by Henry (8). The variation in the length of the cycle was also lower than that reported for the domestic sows (16) or European wild sows (8). The pattern of progesterone secretion during the estrous cycle of the Australian feral sow was similar to that of the domestic sow. Minimal levels of progesterone were seen over the 3 or 4 d before estrus and were, more often than not, below the sensitivity of the assay used. In contrast, although progesterone has been shown to decline markedly over the same period in domestic sows, absolute levels were higher (range: <1 ng/ml to 8 ng/ml; 5,1,20,21,22) than those observed in this study.
Theriogenology
1190
Maximum levels of progesterone for feral sows in this study ranged from 24.2 to 47.2rig/m1 and oecunv,d on Day 13 or 14 of the cycle. Similar peak levels have been reported in domestic sows on Days 10 to 12 (13,22), Days 8 to 14 (21), Days 13 to 14 (5,17) and Days 14 and 15 (20).
~3
E1 .-~ 100 [
6°I /
/
4o
z0 , . . . . . . . . . . , . . . . . . . . . . . . . .
~" 0
!
~ 0
I0
20
30
40
Day of Treatment Figure 4. Changes in plasma progesterone level, vulval score and vaginal mucosal score over time in a representative sow from the altrenogest-treated group. Day 0 is the start of treatment, while the squares indicate the duration of altrenogest feeding.
Theriogenology
1191
The mean duration of estrus in mature domestic sows has been reported as 49.9 and 68.19 h (1), 52.9 and 58.8 h (18) and 59.3 h (16). Although the once-dally observation undertaken in the current study precluded such precise estimates of the duration of estrus, our range (1 to 4 d) encompassed these average values as well as the 1 to 3 d reported by Henry (8) for European wild sows. It would also appear that we observed one silent estrus during this study. Silent estruses have been reported in domestic sows (10) and, based on the data of Henry (8), probably occur in European wild boar. The cyclic changes in vulval swelling observed in this study have been reported for domesticated sows (16), where swelling 4 d before the first signs of estrus were noted. Our observations differ from this in that vulval swelling was rarely seen more than 1 d before or ld after the period of standing estrus. To our knowledge, cyclic changes in the consistency of the vaginal mucus observed in the current study have not been documented elsewhere, although cyclical changes in the crystalline appearance of the vaginal mucus have been described (15). In this study, there was no evidence of seasonal anestrus, which been reported elsewhere (4). This reported anestrus occurred during the months of summer and autumn in European wild boar, while whereas our studies mainly involved the months of winter and spring. However, in the supplementary group in which estrus was induced in late summer, there was no evidence of seasonally induced oestrus. In the current study, estrus was successfully induced in the Australian feral sow by both treatment with an aborlifacient followed by gonadotrophin and by suppression of estrus with a progestogen followed by withdrawal of this suppression. A single injection of 25 mg of prostaglandin led to abortion in 82% of pregnant sows, and treatment with a gonadotrophin preparation 2 to 3 d post abortion induced estrus within 4 to 5 d. This oectmeA over a wide range of stage of gestation (65 to 114 d). Similar results have been reported elsewhere for domestic sows (7), where oestms was induced in 87% of gilts 4 to 7 d after prostaglandin treatment. However, this occurred over the period 12 to 40 days of gestation. Treatment of sows with an oral progestogen suppressed estrus for the duration of" treatment. Within 8 to 11 d after withdrawal of the progestogen the sows returned to estrus. The interval from progestogen withdrawal to estms was longer than that previously reported (12), where estrus was induced in 96% of domestic gilts within 7 d of the cessation of treatment. Despite the reversion to a morphology similar to that of European wild boar, Australian feral sows have an estrous cycle length more closely resembling that of domestic sows. Feral sows in this study also showed similar cyclical changes in progesterone secretion and vulval swelling to those reported for domestic sows. In fact, the only observed difference in estrous cycles was that progesterone virtually disappeared at the time of standing estrus in feral sows, while detectable levels are reported to remain in the plasma of estrous domestic sows. Our capacity to contrast cyclical changes in Australian feral sows with those in European wild boar are limited by the availability of data. The only related studies on European wild boar come from work of naturalized animals in North America (1,9). However, these data did not include descriptions of cyclical changes in progesterone, vulval swelling or consistency of vaginal mucus. Finally, induction of estrus was successfully achieved using techniques involving abortion followed by PMSG treatment or estrus suppression.
Theriogenology
1192 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22.
23.
Burgex JF. Sex physiology of pigs. Onderspoort J Vet Res 1952; 25 (Supp] 2):2-217. Choquenot D, O'Brien P.H. Management of feral pigs in Australia. Proc XIXth hat Union Game Biol 1989; 503-509. Choquenot D, Kflgour R.I, Luldns B. Can oestrous sows be used to nap feral boars? Proc 9th Aust Vertebrate Pest Control Conference, Adelaide, 1991; 341-343. Delcroix I, Mauget R, Signoret J-P. Existence of sunchronization of reproduction at the level of the social group of the European wild boar (Sus Scrofa). J Reprod Fert 1990; 89:613-617. Edqvist LE, Lamm, AM. Progesterone levels in plasma during the oestrous cycle of the sow measured by a rapid competitive binding technique. J Reprod Fert 1971; 25:447-449. Guthrie I-H). Plasma estrogen, progesterone and luteinizing hormone prior to estrus and during early pregnancy in pigs. Endocrinol 1972; 91:675-679. Guthrie HD, Polge C. Treatment of pregnant gilts with a prostaglandin analogue, Cloprostenol, to control oestrus and fertility. J Reprod Fert 1978; 52: 271-273. Henry VG. Length of estrous cycle and gestation in European wild hogs. J Wildl Manage 1968; 32:406-408. Henry VG. Fetal development in European wild hogs. J Wildl Manage 1968; 32:966-970. Hone J. How many feral pigs in Australia? Aust Wildl Res 1990; 17:571-572. Lever C. Naturalized Mammals of the World. Longman, Essex, England, 1985. Martinat-Botte F, Bariteau F, Forgerit Y, Macar C, Moreau A, Terqui M, Signoret J-P. Control of oestrus in gilts. II. Synchronization of oestrus with a progestogen, altrenogest (Regumat¢): effect on fertility and litter size. Anim Reprod Sci 1990; 22:227-233. Parvizi N, Elsaesser F, Smidt D, EUendorf F. Plasma luteinizing hormone and progesterone in the adult female pig during the estrous cycle, late pregnancy and lactation, and after ovariectomy and pentobarbitone treatment. J Endocr 1976; 69:193-203. Robertson GL, Grummcr R_H, Casida LE, Chapman AB. Age at puberty and related phenomena in outbred Chester White and Poland China gilts. J Anim Sci 1951; 10:647-656. Sate M, Niwa T. Changes in the crystal configulation of vaginal mucus of gilts during estrous cycle and pregnancy. Jap J Anita Reprod 1980; 26:126-129. Schmidt K, Bretschneider W. Ubcr den ausseren Ablauf des Sexualsyklus bci der San. Tierzucht 1954; 8:119-125. Shearer IJ, thn-vis K, Jcnkin G, Haynes NB. Peripheral plasma progesterone and cestradiol-17 levels before and after puberty in gilts. J Reprod Fert 1972; 30:347-360. Signoret J-P. Durt~ du cycle cestrien et de l'oestrus chez la truie, action du benzoate d'ocstradiol chez la femelle ovariectomis~e. Ann Biol anim Biophys Biochim 1967; 7:407-421. Signoret J-P. Reproductive behaviour of pigs. J Reprod Fert 1970; Suppl 11:105-117. Stabenfeldt GH, Aldns EL, Ewing LL, Morrissette MC. Peripheral progesterone levels in pigs during the oestrous cycle. J Reprod Fert 1969; 20:443-449. Tillson SA, Erb RE. Progesterone concenwation in peripheral blood plasma of the domestic sow prior to and during early pregnancy. J Anim Sci 1967; 26:1366-1368. Tillson SA, Erb RE, Niswender GD. Comparison of luteinizing hormone and progesterone in blood and metabolites of progesterone in urine of domestic sows during the estrous cycle and early pregnancy. J Anita Sci 1967; 30:795-805. Tisdell CA. Wild pigs: environmental pest or economic resource? Pergamon Press, Sydney, Australia, 1982.
Received for Publication: April 20, 1993 Accepted: December 22, 1993 ABSTRACT In the first of 2 experiments, the estrous cycles of 11 Australian feral sows were studied. In 9 of the 11 sows the cycle was characterized by cyclic occurrences of low (0 to 2 ng/ml) and high (24.2 to 47.2 ng/ml) levels of progesterone. The low levels were associated, in all sows but one, with the standing response, lasting from 1 to 4 days, to a feral boar. Concomitant cyclic changes in vulval swelling and consistency of the vaginal mucus were also observed. Using intervals between the standing estrous response or the marked changes in the secretion of progesterone, the mean length of the cycle was calculated as 19.8 and 20.0 d, respectively. Two of the sows did not exhibit cyclic changes in any of the parameters measured, and on no occasion did either stand for service. In the second experiment, it was shown that estrus can be reliably induced in feral sows by either 1 of 2 methods: first, sows were induced to abort by prostaglandin injection. They were then administered gonadotrophin 48 to 72 h post abortion, and came into estrus 4 to 5 days later. Second, estrus was suppressed by feeding sows altrenogest, and was induced again about 9 days following altrenogest withdrawal. Key words: estrus, estrous cycle, progesterone, vaginal mucus, Australian feral sows
INTRODUCTION Feral pigs are widespread and abundant in Australia. Current estimates suggest that as many as 13 million feral pigs may inhabit the continent (10). Colonization by pigs closely coincided with European settlement of eastern and northern Australia (11), and feral pigs now occupy habitats ranging from semi-arid rangelands to sub-alpine highlands, to subtropical floodplains and forests (23). Throughout their range Australian feral pigs have reverted to wildtype morphological characteristics; they are typically more similar in size, shape and appearance to their progenitor type, the European wild boar, than they are to the domestic pigs from which they are directly evolved (2). If such reversion has occurred physiologically as well as morphologically, the estrous cycle of Australian feral sows would be expected to be more similar Acknowledgments We acknowledge the financial support of the Wildlife Exotic Disease Preparedness Fund of the Bureau of Rural Resources of the Department of Primary Energy and Industry; the assistance of Trevor ~,es for the construction of the restraining cage; the technical assistance of Brian Lukins and Margaret Szantar, and the donation of a kit for assay validation by Australian Laborotory Services, Rockdale, N.S.W., Australia.
Copyright © 1994 Butterworth-Heinemann
Theriogenology
1182 to that of European wild boar than that of domestic pigs.
The length of the estrous cycle in Australian feral sows is not known. Information about the estrous cycle is essential ff the cycle is to be manipulated in order to artificially bring sows into standing estrus. Estrus induction in feral sows is important for their use as bait to trap other feral pigs (3) or to enhance production for commercial harvest or recreational hunting (2). In this paper, we describe the estrous cycle of the Australian feral sow and the physiological changes associated with the cycle. We also look at the efficacy of 2 techniques for the induction of estrus in Australian feral sows. Results are discussed in relation to what is known of estrus in the European wild boar. MATERIALS AND METHODS The sows used in the experiments had either been caught in the wild and run in small paddocks for 3 yr or were the offspring of such sows. The sows were allocated at random to 1 of 3 groups so that the weight range in each group was as similar as possible. One of the groups was used to study the estrous cycle in naturally cycling sows, while the other 2 groups were used for the induction of estrus. The sows of the first group ( n = l l ) were housed in individual pens over the months of June to August (winter) and were fed either wheat grain or pelleted lucerne ad libitum. Water was freely available at all times. Each day for a total of 72 d, a jugular blood sample was taken from each sow using heparinized, evacuated blood collection tubes. At the time of blood collection, the appearance of the external genitalia and vaginal mucus were scored, and the sow was exposed to a teaser boar. The blood was centrifuged and the plasma stored at -20° C until assay. The vulva was scored as 1 = no vulval swelling, 3 = marked vulval swelling, sometimes with internal labia exposed; a score of 2 was given to sows showing an intermediate degree of swelling. The state of the vaginal mucus was assessed by taking a smear of the vaginal wall with a fiber glass rod. The mucus was scored as 1 = dry making insertion of the rod difficult, very little mucus taken; 2 = thick, creamy mucus which formed droplets on the rod; 3 = thin, copious mucus. Following examination of the vulva and mucus, the sows were exposed to a boar in groups of 3 and were observed until they showed either active avoidance of the boar's approaches or stood while he mounted to serve them. The teaser boar was an adult castrated male that had been treated with 1125 mg of testosterone as testosterone cypionate (Banrota, ICI, Sydney, Australia) 4 wk prior to the start of the observations and at 4-wk intervals thereafter. The assay for progesterone was a direct double-antibody radioimmunoassay for progesterone in human serum (Farmos Diagnostica, Turku, Finland). According to information supplied with the kit, the progesterone antiserum cross reacts 100% with progesterone; its cross-reaction with 17-hydroxyprogesterone, estrogens, pregnenolone and cortisol are less than 0.5%. The sensitivity of the assay was 0.3 ng/ml (def'med as the lower limit of the standard curve). Levels of progesterone in plasma samples taken from a sow on days when she stood for
Theriogenology
1183
the boar and at 3-d intervals gave values similar to those reported in the literature for sows in the follicular and luteal phases. The linear dilution of one of the luteal-phase samples gave progesterone values which decreased linearly. The second group of sows (n=14) was run with 3 fertile boars for a period of 10 wk over the months May to July (late autumn to early winter). At the end of this period, the sows were placed in individual pens where they were fed every day and allowed constant access to water. On the following day, each sow was adrnirtistered 10 mg of prostaglandin (Lutalysex, Upjohn, Sydney, Australia) to induce abortion. However, only 4 sows aborted by 48 h post injection, so the sows which had not aborted were given an additional 25 mg of prostaglandin. All sows which aborted were then treated with 1000 IU of a PMSG preparation (Pregnocolx, Heriot, Melbourne, Australia) no less than 48 and no more than 72 h post abortion. Every day for the next 10 d, a jugular blood sample was taken and the plasma was frozen until assayed for progesterone. Based on the results from the naturally-cycling group, a sow was deemed to be in estrus during the 2 d leading up to the rise in plasma progesterone concentration above undetectable levels. No notice could be taken of external genitalia or vulval mucosal scores since these animals had only recently aborted. Crown-rump lengths were measured on all intact fetuses and fetal age at abortion was estimated from the relationship between fetal age and crown-rump lengths as reported by Henry (9). Because we had to use 2 differing injections of prostaglandin to induce abortion, the protocol for this technique was deemed to be unreliable. Therefore, a supplementary group of sows (n=24) was placed with 4 fertile adult boars for 10 wk during the late spring and early summer (November to January). At the end of the breeding period, these sows were also placed in individual pens where they were fed every day and allowed constant access to water. On the following day, each sow was treated with a single 25 mg injection of prostaglandin to induce abortion. Fetal age at abortion was estimated from the crown-rump length.
The third group of sows (n=9) was housed in individual pens in early July where they were fed each day and given free access to water. Each day for 25 d, all the sows were given " 20 m g altrenogest (Regumatc x, Rousscl Uclaf, Sydney, Australia) which was dissolved in vegetable oil and mixed with their feed. Normally, treatment with altrenogest would have lasted for only 18 d (12), but we administered the treatment for a longer period because, on the day the sows were first fed altrenogest, 5 of them were suspected to be in estrus, as judged by the appearance of their vulvae. Each day, the appearance of the vulva and consistency of the vaginal mucus were scored. On the 26th day, altrenogest was withdrawn from the feed and these measurements continued for a further 10 d. Based on the data for the naturally-cycling sows, a sow was deemed to be in estrus when her progesterone levels increased above 10 ng/ml coincident with a score of 3 for either the external genitalia or vulval mucus. RESULTS
Naturally-cyclic Sows Levels of progesterone varied in cyclical fashion in 9 of the 11 sows, indicating the occurrence of regular estrous cycles. This was confirmed by the fact that on days when
Theriogenology
1184
3
i
2 1 0
*,3 0 C.)
~2 1
~0 120
~.I00 8O 6O 40
~ 20 •
0 0
10
20
30
40 Day
50
.
|
60
|
70
Figure 1. Changes in plasma progesterone level, vulval score and vaginal mucosal score over the duration of the study in a representative sow from the naturally cycling group. Triangles indicate days on which the sow stood for service.
Theriogenology
1185
progesterone levels were low the sows would stand for service by the boar (Figure 1). This pattern was characterized by the following changes: on days when the sows first allowed the boar to mount, progesterone levels were low, usually less than 2 ng/ml and often undetectable. On the last day on which the sows stood for service, progesterone levels had begun to rise again. These levels then reached a plateau over the next 1 to 4 d and remained relatively steady for 7 to 10 days after which time they fell rapidly again to low or undetectable levels. Progesterone then remained at these levels for periods which varied from 5 to 8 d until they increased once more, as described above. Taking the day on which the sows stood for service as Day 0 of the estrous cycle, progesterone levels were low to undetectable over the first 2 to 3 d of the cycle, after which time they rose rapidly. The rate of this increase generally slowed sometime between Days 6 and 9, so that levels reached a maximum, which ranged from 24.2 to 47.2 (mean 34.6) ng/ml, on Days 12 or 13. After this time there was a rapid decrease, with levels falling to nearly zero by Day 16 or 17, where they remained for 3 or 4 days until the sow again came into estrus. In 1 of the 2 sows that did not exhibit regular estrous cycles, progesterone levels fluctuated, with no obvious pattern and with levels varying from 2 to 30 ng/ml. In the other of these 2 sows, progesterone levels were low or undetectable throughout the study (Figure 2). Based on this pattern of progesterone secretion, the lengths of 21 estrous cycles from 9 sows showing regular patterns of progesterone secretion were determined as the intervals between the days on which progesterone levels showed their abrupt increase following the periods of low levels (Table 1). Mean ~ SEM) cycle length was 20.0 + 0.2 d with a coefficient of variation of 4.4%. For the first 9 d of the experiment, no sows were mounted by the teaser boar, which appeared to be intimidated by some of the sows. This boar was replaced on Day 10 by a boar which mounted sows from the first exposure. Sexual behavior of the boar and sows was similar to that described for domestic pigs (19);" the boar elicited series of short grunts when pursuing the sows, and sows in estrus stood rigidly when mounted, whereas those not in estrus actively discouraged the boar's attempts to mount. Over the duration of the study, the sows showed a cyclic pattern of sexual receptivity and nonreceptivity (Figure 1). Of the 11 sows, 2 failed to stand for the boar over the duration of the observations. These were the 2 sows that showed no obvious cyclic pattern in their secretion of progesterone. The 9 remaining sows stood for the boar on a total of 27 occasions, although on 1 occasion 1 sow failed to stand, even though her progesterone levels were low. The number of consecutive days on which the sows stood for the boar provide a measure of the duration of esu'us. One of the estrous periods was withdrawn from the data due to vaginal bleeding in 1 sow on the first day she stood to be mounted and we decided not to expose her to the boar on the subsequent days of that oestrus. Of the 26 remaining cycles, the sows stood for 1 d on 1 occasion, 2 d on 7 occasions, 3 d on 16 occasions and 4 d on 2 occasions.
Theriogenology
1186
IO0 8o
~
2O 0 I00
~
6O 4o 2o
0 0
10
20
30
40
50
60
70
Day Figure 2. Changes in plasma progesterone level over the duration of the study in the 2 sows of the naturally cycling group that did not stand for service.
Using the recurring pattern of sexual receptivity, the lengths of 19 estrous cycles from the 9 receptive sows were determined as being the number of days between the first day of one period of sexual receptivity and the first day of the next period (Table 1). Mean ~ SEM) cycle length was 19.8 + 0.3 d with a coefficient of variation of 6.0%. The 9 sows showing cyclic changes in progesterone levels also showed a cyclic pattern of change in the appearance of the vulva (Figure 1). On the days on which these sows stood for service, the vulva was usually swollen. The sows stood for service on a total of 75 occasions,
Theriogenology
1187
and on 59 (80%) of these, their vulvae were given a score of 3; on 4 occasions (7%) they were given a score of 2; and on 10 occasions (13%) they were given a score of 1. The sows also received scores of 3 on days when they did not stand for service, but these occasions were almost invariably associated with periods of sexual receptivity and rarely occurred more than 1 d before or I d after the period when they stood for service (Figure I). There was no evidence of synchrony of the occurrence of estrous between the sows.
Table I. Frequency of estrous cycles of various lengths in Australian feral sows as measured by cyclic changes in blood progesterone levels or in sexual behavior. Numbers in parentheses refer to the number of cycles on which the frequencies are based. Cycle length measured by Duration of cycle (days)
Progesterone levels (21)
Sexual behavior (19)
17
0.00
0.05
18
0.05
0.05
19
0.19
0.32
20
0.52
0.26
21
0.19
0.26
22
0.05
0.05
The 2 sows that did not stand for service over the entire period of the study each received vulval scores of 1 on all but 2 occasions. While less clearly defined than changes in progesterone or in the appearance of the vulva, vaginal mucus of the 9 cyclic sows also showed regular changes; on days when they stood for service their mucus was generally thin and copious (Score 3), while at other times during the cycle the scores varied from 1 to 2 but rarely was a score of 3 given (Figure 1). Of the 2 sows which showed no apparent estrous cycles, the sow with consistently low blood progesterone received a score of 1 on all but 4 occasions whereas the other sow received all 3 scores but with no obvious pattern. EstrusInduction Of the 14 sows in the early bred group, 1 sow aborted spontaneously before the administration of prostaglandin, 4 sows aborted 22 to 35 h after the fLrSt injection, and 5 sows aborted 22 to 44 h after the second injection. The 4 sows which did not abort were shown at autopsy to be nonprcgnam. Due to the intractable behavior of 1 sow, blood samples could not be collected from her, nor could observations on the vaginal mucus and vulval observations be performed.
Theriogenology
1188
Prior to prostaglandin treatment, plasma progesterone levels were high (50 to 80 ng/ml), but fell rapidly to undeteetable levels following prostaglandin injection (Figure 3). These levels remained undeteetable in all animals for a mean of 4.4 d (range 2 to 8 d) following the injection of gonadotrophin, indicating that the sows came into estrus 2 to 3 days after this injection (range 0 t o 6d). Of the 24 later-bred animals injected with a single 25-mg dose of prostaglandin, 18 aborted within 48 h of injection. At autopsy following euthanasia, 4 of the remaining sows were found to be nonpregnant, while the remaining 2 sows subsequently gave birth to live litters. Estimated age of the fetuses at abortion ranged from 65 to 114 d (Table 2).
80 70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
8
9
10
11
Days Figure 3. Changes in plasma progesterone over lime in 2 representative sows from the abortestrogen group. Day 0 is the day of administration of abortffacient, while the arrow indicates the day of administration of gonadotrophin.
In 8 of the 9 sows in the group in which oestrus was suppressed with altrenogest, progesterone levels were generally high (80 to 140 ng/ml) during the first 10 days of treatment, after which time they fell to almost undetectable levels (Figure 4). However, after a mean of 9.0 d following the cessation of feeding of altrenogest (range 8 to 11 d), progesterone levels began to rise once more. These changes in progesterone levels coincided with changes in the appearance of the external genitalia and in the consistency of the vaginal mucus. During the time of altrenogest feeding, the scores of sows were consistent with those observed for the non estrous sows, while 3 to 7 d following the cessation of feeding, the mucus and genitalia scores increased to those observed in estrous sows (Figure 4). Based on our observations in the naturally-cycling sows of this study, we concluded that these estrous-induced sows came into estrus about 7 d following
TheHogenology
1189
~e cessation of altrenogest treatment. One sow failed to respond to treatment with altrenogest ~ut displayed progesterone levels that were constantly elevated. This animal's vaginal mucus and ~xternal genitalia were almost constantly in a nonestrous state.
table 2. Day of gestation on which sows aborted. Data represent the number of sows aborting over a given gestational range, while figures in parentheses represent the proportion of these sows.
Range in days of gestation
Number of sows
65 to 69
1 (O.04)
70 to 74
0
75 to 79
0
80 to 84
1 (0.04)
85 to 89
2 (0.08)
9 0 t o 94
0
95 to 99
6 (0.23)
100 to 104
8 (0.31)
105 to 109
4 (0.15)
110 to 114
4 (0.15)
DISCUSSION The average length of the estrous cycle of Australian feral sows as measured in this study was 20 d. The cycle was characterized by standing estrus of 1 to 4 days, by cyclic changes in the levels of progesterone, and by cyclic changes in the appearance of the external genitalia and consistency of vaginal mucus. The mean length of the cycle of the sows in this study was shorter than that reported for the domestic sow, the average length of which varies from 20 to 22 d (1,6,14,16,18) or for the European wild boar in the United States, which averaged 23.8 and 21.8 d in the 2 studies by Henry (8). The variation in the length of the cycle was also lower than that reported for the domestic sows (16) or European wild sows (8). The pattern of progesterone secretion during the estrous cycle of the Australian feral sow was similar to that of the domestic sow. Minimal levels of progesterone were seen over the 3 or 4 d before estrus and were, more often than not, below the sensitivity of the assay used. In contrast, although progesterone has been shown to decline markedly over the same period in domestic sows, absolute levels were higher (range: <1 ng/ml to 8 ng/ml; 5,1,20,21,22) than those observed in this study.
Theriogenology
1190
Maximum levels of progesterone for feral sows in this study ranged from 24.2 to 47.2rig/m1 and oecunv,d on Day 13 or 14 of the cycle. Similar peak levels have been reported in domestic sows on Days 10 to 12 (13,22), Days 8 to 14 (21), Days 13 to 14 (5,17) and Days 14 and 15 (20).
~3
E1 .-~ 100 [
6°I /
/
4o
z0 , . . . . . . . . . . , . . . . . . . . . . . . . .
~" 0
!
~ 0
I0
20
30
40
Day of Treatment Figure 4. Changes in plasma progesterone level, vulval score and vaginal mucosal score over time in a representative sow from the altrenogest-treated group. Day 0 is the start of treatment, while the squares indicate the duration of altrenogest feeding.
Theriogenology
1191
The mean duration of estrus in mature domestic sows has been reported as 49.9 and 68.19 h (1), 52.9 and 58.8 h (18) and 59.3 h (16). Although the once-dally observation undertaken in the current study precluded such precise estimates of the duration of estrus, our range (1 to 4 d) encompassed these average values as well as the 1 to 3 d reported by Henry (8) for European wild sows. It would also appear that we observed one silent estrus during this study. Silent estruses have been reported in domestic sows (10) and, based on the data of Henry (8), probably occur in European wild boar. The cyclic changes in vulval swelling observed in this study have been reported for domesticated sows (16), where swelling 4 d before the first signs of estrus were noted. Our observations differ from this in that vulval swelling was rarely seen more than 1 d before or ld after the period of standing estrus. To our knowledge, cyclic changes in the consistency of the vaginal mucus observed in the current study have not been documented elsewhere, although cyclical changes in the crystalline appearance of the vaginal mucus have been described (15). In this study, there was no evidence of seasonal anestrus, which been reported elsewhere (4). This reported anestrus occurred during the months of summer and autumn in European wild boar, while whereas our studies mainly involved the months of winter and spring. However, in the supplementary group in which estrus was induced in late summer, there was no evidence of seasonally induced oestrus. In the current study, estrus was successfully induced in the Australian feral sow by both treatment with an aborlifacient followed by gonadotrophin and by suppression of estrus with a progestogen followed by withdrawal of this suppression. A single injection of 25 mg of prostaglandin led to abortion in 82% of pregnant sows, and treatment with a gonadotrophin preparation 2 to 3 d post abortion induced estrus within 4 to 5 d. This oectmeA over a wide range of stage of gestation (65 to 114 d). Similar results have been reported elsewhere for domestic sows (7), where oestms was induced in 87% of gilts 4 to 7 d after prostaglandin treatment. However, this occurred over the period 12 to 40 days of gestation. Treatment of sows with an oral progestogen suppressed estrus for the duration of" treatment. Within 8 to 11 d after withdrawal of the progestogen the sows returned to estrus. The interval from progestogen withdrawal to estms was longer than that previously reported (12), where estrus was induced in 96% of domestic gilts within 7 d of the cessation of treatment. Despite the reversion to a morphology similar to that of European wild boar, Australian feral sows have an estrous cycle length more closely resembling that of domestic sows. Feral sows in this study also showed similar cyclical changes in progesterone secretion and vulval swelling to those reported for domestic sows. In fact, the only observed difference in estrous cycles was that progesterone virtually disappeared at the time of standing estrus in feral sows, while detectable levels are reported to remain in the plasma of estrous domestic sows. Our capacity to contrast cyclical changes in Australian feral sows with those in European wild boar are limited by the availability of data. The only related studies on European wild boar come from work of naturalized animals in North America (1,9). However, these data did not include descriptions of cyclical changes in progesterone, vulval swelling or consistency of vaginal mucus. Finally, induction of estrus was successfully achieved using techniques involving abortion followed by PMSG treatment or estrus suppression.
Theriogenology
1192 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22.
23.
Burgex JF. Sex physiology of pigs. Onderspoort J Vet Res 1952; 25 (Supp] 2):2-217. Choquenot D, O'Brien P.H. Management of feral pigs in Australia. Proc XIXth hat Union Game Biol 1989; 503-509. Choquenot D, Kflgour R.I, Luldns B. Can oestrous sows be used to nap feral boars? Proc 9th Aust Vertebrate Pest Control Conference, Adelaide, 1991; 341-343. Delcroix I, Mauget R, Signoret J-P. Existence of sunchronization of reproduction at the level of the social group of the European wild boar (Sus Scrofa). J Reprod Fert 1990; 89:613-617. Edqvist LE, Lamm, AM. Progesterone levels in plasma during the oestrous cycle of the sow measured by a rapid competitive binding technique. J Reprod Fert 1971; 25:447-449. Guthrie I-H). Plasma estrogen, progesterone and luteinizing hormone prior to estrus and during early pregnancy in pigs. Endocrinol 1972; 91:675-679. Guthrie HD, Polge C. Treatment of pregnant gilts with a prostaglandin analogue, Cloprostenol, to control oestrus and fertility. J Reprod Fert 1978; 52: 271-273. Henry VG. Length of estrous cycle and gestation in European wild hogs. J Wildl Manage 1968; 32:406-408. Henry VG. Fetal development in European wild hogs. J Wildl Manage 1968; 32:966-970. Hone J. How many feral pigs in Australia? Aust Wildl Res 1990; 17:571-572. Lever C. Naturalized Mammals of the World. Longman, Essex, England, 1985. Martinat-Botte F, Bariteau F, Forgerit Y, Macar C, Moreau A, Terqui M, Signoret J-P. Control of oestrus in gilts. II. Synchronization of oestrus with a progestogen, altrenogest (Regumat¢): effect on fertility and litter size. Anim Reprod Sci 1990; 22:227-233. Parvizi N, Elsaesser F, Smidt D, EUendorf F. Plasma luteinizing hormone and progesterone in the adult female pig during the estrous cycle, late pregnancy and lactation, and after ovariectomy and pentobarbitone treatment. J Endocr 1976; 69:193-203. Robertson GL, Grummcr R_H, Casida LE, Chapman AB. Age at puberty and related phenomena in outbred Chester White and Poland China gilts. J Anim Sci 1951; 10:647-656. Sate M, Niwa T. Changes in the crystal configulation of vaginal mucus of gilts during estrous cycle and pregnancy. Jap J Anita Reprod 1980; 26:126-129. Schmidt K, Bretschneider W. Ubcr den ausseren Ablauf des Sexualsyklus bci der San. Tierzucht 1954; 8:119-125. Shearer IJ, thn-vis K, Jcnkin G, Haynes NB. Peripheral plasma progesterone and cestradiol-17 levels before and after puberty in gilts. J Reprod Fert 1972; 30:347-360. Signoret J-P. Durt~ du cycle cestrien et de l'oestrus chez la truie, action du benzoate d'ocstradiol chez la femelle ovariectomis~e. Ann Biol anim Biophys Biochim 1967; 7:407-421. Signoret J-P. Reproductive behaviour of pigs. J Reprod Fert 1970; Suppl 11:105-117. Stabenfeldt GH, Aldns EL, Ewing LL, Morrissette MC. Peripheral progesterone levels in pigs during the oestrous cycle. J Reprod Fert 1969; 20:443-449. Tillson SA, Erb RE. Progesterone concenwation in peripheral blood plasma of the domestic sow prior to and during early pregnancy. J Anim Sci 1967; 26:1366-1368. Tillson SA, Erb RE, Niswender GD. Comparison of luteinizing hormone and progesterone in blood and metabolites of progesterone in urine of domestic sows during the estrous cycle and early pregnancy. J Anita Sci 1967; 30:795-805. Tisdell CA. Wild pigs: environmental pest or economic resource? Pergamon Press, Sydney, Australia, 1982.