- Email: [email protected]
Post partum ovarian activity and uterine involution in the suckled swamp buffalo (Bubalus bubalis)
Animal Reproduction Science, 5 (1982/1983) 181--190
181
Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
POST PARTUM OVARIAN ACTIVITY AND UTERINE INVOLUTION IN THE SUCKLED SWAMP BUFFALO (BUBALUS BUBALIS)
M.R. JAINUDEEN, T~A. BONGSO and H.S. TAN
Department of Veterinary Clinical Studies, Universiti Pertanian Malaysia, Serdang, Selangor (Malaysia) (Accepted 11 October 1982)
ABSTRACT Jainudeen, M.R., Bongso, T.A. and Tan, H.S., 1983. Post partum ovarian activity and uterine involution in the suckled swamp buffalo (Bubalus bubalis). Anita. Reprod. Sci., 5: 181--190. Ovarian activity and uterine involution were monitored b y rectal palpation, oestrus detection and plasma progesterone analysis from 3 to 4 days to approx|m~tely 150 days post p a r t u m in 38 suckled swamp buffaloes (Bubalus bubalis). The intervals from parturition t o regression of the corpus luteum (CL) o f pregnancy and involution o f the uterus were 10 and 28 ± 6 (S.D.) days respectively. First detected oestrus and first elevation of plasma progesterone (> 0.7 ng/ml) occurred at 88 -+ 26 and 96 -+ 22 days in 8 and 12 buffaloes respectively. During the f'n'st 150 days post partum, 26 o f 38 suckling buffaloes (88°£) were acyclic (anoestrus) and o f 12 animals (32%) exhibiting ovarian cycles, 4 were n o t detected in oestrus. The tentative diagnosis, based on rectal palpation, that CL were present between days 30 and 90 after parturition (without concurrent luteal levels o f progesterone in plasma) suggests that confirmation should be by laparoscopy. It is concluded that a delay in the resumption of ovarian cyclicity post partum represents an imp o r t a n t factor contributing to the prolonged calving to conception interval in the suckled swamp buffalo.
INTRODUCTION
Problems of oestrus detection and long calving intervals are among the major constraints that impede genetic prowess and productivity of the swamp buffalo (Bubalus bubalis). The calving to conception interval of 190--200 days in the suckled swamp buffalo (Jainudeen, 1977) may be related to a delay in the resumption of post partum ovarian activity, abnormalities of oestrus or the oestrous cycle, and low conception rates. Uterine involution and ovarian activity have been investigated in cattle by rectal palpation (Morrow et al., 1969; Wagner and Hansel, 1969) and plasma or milk progesterone profiles have been determined in post partum dairy (Bulman and Lamming, 1978; Boyd and Munro, 1979) and beef (Radford et al., 1978; Rawlings et al., 1980) cows. Hitherto, no similar studies have been conducted in the swamp buffalo. 0378-4320183/0000--0000/$03.00
© 1983 Elsevier Scientific Publishing Company
182 Since swamp buffaloes suckle their calves for 6- to 9-month periods, a knowledge of the events that lead to the resumption of post partum ovarian cyclicity in beef cattle may be useful in studying the endocrinology of the post partum swamp buffalo. This information could assist us in reducing the contribution made by post partum anoestrus to the long calving interval in this species. As a first step in investigating this problem, a study was undertaken to determine the time of uterine involution and the resumption of post partum ovarian cyclicity in the suckled swamp buffalo. MATERIALS AND METHODS The experimental animals were 38 suckled swamp buffaloes (Bubalus bubalis), 4--9 years old, which calved normally between October 1979 and October 1980 and were managed in paddocks with access to salt blocks containing phosphorus and trace minerals. Each buffalo was weighed, the uterus and ovaries palpated per rectum and jugular blood collected in heparinized evacuated tubes at weekly intervals, commencing 3--4 days after parturition and continuing until 90--150 days post partum. The position of the uterus in the body cavity and the diameter at the external bifurcation of each uterine horn were recorded. Uterine involution was complete when both uterine horns reached normal nongravid size and position. The ovaries were palpated to estimate size and to record palpable structures (follicles or CL). The day of ovulation was estimated to be 4 days before the time a CL was first palpable. Buffaloes were penned and tested daily for oestrus between 20.00 and 22.00 h using a vasectomized buffalo fitted with a chinball marking device. The male remained with the cows until the next morning. A female was judged as in oestrus (day 0) if mating was observed or she was marked by the male. Rectal palpation of the ovaries and collection of heparinized blood samples were performed daily from four suckled buffaloes during six oestrous cycles.
Progesterone assays Blood samples were centrifuged and the plasma stored at --20°C until assayed. The progesterone levels in the plasma were determined by the radioimmunoassay technique as adapted for the buffalo (Perera et al., 1978; Kamonpatana et al., 1979). Progesterone was extracted from 500 #l plasma with petroleum ether and assayed directly using antiserum against 1 la-hydroxyprogesterone hemisuccinate-BSA-conjugate. All determinations were performed in duplicate and corrected for procedural losses and volume of plasma used. The assay standard curve ranged from 25 to 1000 pg per assay tube with an assay sensitivity of 0.20 ng/ml plasma, i.e. the lowest concentration of unlabelled progesterone that significantly displaced labelled progesterone (P < 0.05). The average extraction efficiency of labelled progesterone was
183
80 ± 8.3% (S.D., n = 67). The intra-assay coefficients of variation were 9% (n = 30) at 1.07 + 0.10 ng/ml and 12% (n =30) at 1.78 + 0.22 ng/ml. The inter-assay coefficients of variation were 10% (n = 5) at 1.09 + 0.11 ng/ml and 19% (n = 16} at 1.82 + 0.34 ng/ml. RESULTS
The mean -+ S.D. (n = 38) liveweight (kg) of the buffaloes immediately after calving was 419 + 65. Changes in body weight during the post partum period expressed as a percentage of the body weight at calving (Fig. 1) showed that animals lost weight between days 60 and 130 post partum (P < 0.05) and regained it thereafter. Uterine involution. The uterine horns were palpable cranial and ventral to the pelvic brim from day 7 to 10 post partum. Thereafter, as the uterus gradually decreased in size, luminal distension decreased, caruncles were not palpable, the horns became clearly demarcated and the uterus attained its normal shape and position. Uterine lochia was recognized in 42% of the animals as a white mucoid discharge until day 18 post partum; it became clear and ceased by day 28 post partum. The cervix which was 4--5 cm in diameter and soft during the first week post partum, gradually reduced in size to 2--3 cm diameter, became firm and its outline could be defined as involution progressed. Involution was completed by day 16 to 39 (mean + S.D. = day 28 + 6) post partum (Table I and Fig. 1). Ovarian activity The ovaries of the swamp buffalo were ovoid and firm. On rectal palpaTABLE I Uterine involution and ovarian activity in post partum swamp buffaloes Item
No. o f observations
Mean (-+ S.D.) (days)
Post partum interval to: Uterine involution Detected oestrus Elevation o f plasma progesterone (> 0.7 ng/ml) for more than I week
38 8
28 ± 6 88 -+ 26
12
96
Ovarian activity 1 (day 3 to 150 post partum) Cycling with detected oestrus Cycling with non-detected oestrus Anoestrus (non-functioning ovaries)
8 (21%) 4 (II%) 26 (68%)
~Based on plasma progesterone profiles.
±
22
184 A
f -10 .C
-15
r-
LP •
P
¢-
a
O O
14
28
42
56
70
84
98
1 ?2
126
140
154
Days post partum Fig. 1. Temporal changes in body weight and occurrence (mean -+ S.D.) o f ovarian activity and uterine involution in 38 suckled swamp buffaloes. Shaded area (mean + S.D.) represents increase (+) or decrease (--) in body weight as a percent o f weight at day 3 or 4 post partum. UI = uterine involution; E = 8 animals with detected oestrus; LP = 12 animals with luteal levels of progesterone (> 0.7 ng/ml).
tion, they measured 2--3 cm in length, 1--1.5 cm from surface to surface and 1--2 cm from the attached to the free border, and were located within the pelvic cavity, caudal and lateral to the uterine horns.
Oestrous cycle. The mean plasma progesterone levels in six complete oestrous cycles, that ranged from 19 to 22 days in length, are presented in Fig. 2. During oestrus (day 0), a follicle (10 mm in diameter) was palpable as a turgid area on the ovarian surface and the progesterone level in plasma was 0.24 + 0.12 ng/ml (S.D.). Following ovulation, the progesterone level increased gradually to 0.42 + 0.21 ng/ml (days 4 to 6) when a CL was palpable as a soft protrusion. With further growth, the CL was clearly demarcated from the rest of the ovary as a firm projection measuring 5--10 mm in diameter (days 8 to 16) and the plasma levels of progesterone increased rapidly to reach a peak concentration o f 1.51 + 0.35 ng/ml (day 14). On or about day 17, the CL decreased in size, became hard and nodular and was accompanied by a sharp decline in plasma progesterone level to reach 0.25 + 0.18 ng/ml at the next oestrus.
Post partum period. The CL of pregnancy (corpus albicans) regressed very rapidly following parturition and by day 10 post partum was palpable as a hard small protuberance (< 3 mm diameter) on the ovarian surface. During the first m o n t h post partum, the ovaries were less than 1 cm in length, smooth and were devoid of either follicles or CL. The plasma concentrations of progesterone were below 0.5 ng/ml. Between days 29 and 56 (day 40 + 7) post partum, 24 of 38 buffaloes possessed mature ovarian follicles, marked uterine tone and a discharge of cervical mucus w i t h o u t estrus. However, ovarian structures palpated as CL were n o t associated with luteal phase progesterone levels.
185 1.8
1.6
E
i-4
4) c-
1.2
o I,=.
e U} 1.0 0
,,t-.
@ ~, 0.8
= 06
,:
l
0"4
0.2
'
-4
-2
'
I
I
I
I
0
2
4
5
8 10 12 14 16 18 20 22 24
|
I
I
I
I
I
i
I
I
26
Days of o e s t r o u s cycle Fig. 2. Mean (+- S.D.) peripheral blood plasma levels of progesterone during six oestrous cycles (19--22 days) from four suckled sw~m'lp buffaloes. E = oes~'us.
Regular ovarian cyclicity, characterized by palpable CL and plasma progesterone levels of 0.7 ng/ml or more on at least two successive samplings (7-day-interval), was initiated at a mean time of 96 +- 22 days (Table I) in 12 of 38 buffaloes. Of the 12 cycling buffaloes, eight were detected in oestrus with an average post partum interval to first oestrus of 88 + 26 days (Table I). The remaining 26 buffaloes (68%) were acyclic (anoestrus), had smooth ovaries with no palpable ovarian structures and plasma concentrations of progesterone were less than 0.5 ng/ml. Wide variations existed in the patterns of pl_~sma progesterone levels in the post partum swamp buffalo (Fig. 3). Over a 90 0.7 ng/ml). On day 85 post partum, the progesterone level declined to basal levels and increased to luteal phase levels thereafter. The progesterone profile suggested that the return to service on day 85 was not detected by the vasectomized bull.
186
2L 'L
Buffalo 8 0 2 8
1
i
30
40
50
60
70
80
90
100
110
Buffalo 8027
1
•.
. . . . . . . . . . .
~.
.
.
.
"- ' - ' L - --- ' -- ~
.
.*'/"
"~.-,"-,
i
30
E O) cO
:f
40
50
60
70
Buffalo 3007
80
90
100
110
•
~1
•
./'~
.....
•,_..
...!!:./
20
40
60
80
100
120
140
40
80
80
100
120
140
Buffllo 8037
0
E {e O.
I
20
Buffalo 3034
,~
..,....~,~,.. e _ _ 20
iL:
~,,.'-.\
40
,u,,.,o 3oo,
100
:\
110
60
,
120
: .,.,
80
100
~ j > j , . *. / . , 130
.;'.,..f\. " "'\
i / Il . , .41 . / % / ""
120
..... 140
""\ / . .""/\ . / \ .: 140
150
180
Days post partum
Fig. 3. Plasma progesterone profiles of suckled swamp buffaloes to illustrate post partum anoestrus (buffaloes 8028 and 8027), resumption of the oestrous cycle and luteal activity (buffaloes 3007, 3034 and 3002). Note palpable CL with low levels of progesterone (< 0.5 ng/ml) before first oestrus (buffalo 3007 and 3034). • = palpable CL; E = detected oestrus; I = artificial insemination.
A CL was palpated on days 43, 52 and 65 in buffalo 8037 but this could n o t be confirmed by plasma concentrations of progesterone which were below 0.25 ng/ml. At the first oestrus detected on day 73 the animal was inseminated but progesterone levels persisted at basal levels until day 83. This indicated failure of ovulation. A peak of progesterone was recorded on day 85 but the animal returned to service on day 90 after a 17
187 observed only after first oestrus. The animal conceived to insemination at this oestrus. Buffalo 3002 showed a small peak of progesterone (0.5 ng/ml) before oestrus and 21 days after insemination the level of progesterone was 1.4 ng/ml which persisted thereafter. The animal was confirmed pregnant by rectal palpation. DISCUSSION The present study employed both rectal palpation of the ovaries and patterns of plasma concentrations of progesterone to diagnose cyclicity and acyclicity during the post partum period in the suckled buffalo. Our data confirm that plasma concentrations of progesterone during the oestrous cycle are considerably lower in buffalo (Perera et al., 1978; Kamonpatana et al., 1979) than in cattle (Boyd and Munro, 1979; Webb et al., 1980). Plasma concentrations of progesterone during the post partum period remained at baseline values in anoestrous buffaloes whereas they fluctuated -- periods of low progesterone (~ 0.25 ng/ml) alternating with periods of high progesterone (~> 0.7 ng/ml) -- in cycling animals. The present study shows that plasma progesterone analysis is a convenient technique for monitoring the resumption of ovarian activity in the suckled buffalo. Rectal findings accorded well with plasma concentrations of progesterone in the diagnosis of anoestrus. There was also a close agreement between the palpation of a CL and plasma concentrations of progesterone in those buffaloes which were detected in oestrus or had luteal phase concentrations of progesterone beyond 60 days post partum. However, between days 30 and 60 post partum, rectal palpation revealed follicular activity and apparently normal CL development in 24 of 38 suckled buffaloes, but subsequent luteal function, as indicated by plasma concentrations of progesterone, did n o t reach normal luteal phase values of cycling buffaloes. This situation is comparable to that observed after LH-RH treatment for anoestrus in ewes (Haresign et al., 1975) and suckled beef cows (Webb et al., 1977). At least two possibilities exist for the apparent discrepancy between progesterone concentrations and rectal findings during the early post partum period in the buffalo. One possibility is that an error in diagnosing a CL per rectum could have occurred. This could n o t be verified as the accuracy of rectal palpation in the diagnosis of ovarian structures in the buffalo has, as yet, n o t been determined. But in cattle, rectal palpation of the ovaries was 89% accurate in diagnosing a CL (Dawson, 1975) or 77--85% accurate in diagnosing functional luteal tissue based on plasma or milk progesterone concentrations (Boyd and Munro, 1979; Watson and Munro, 1980). A further possibility is that these early CL failed to secrete progesterone. In cattle, the first two successive ovulation post partum are followed by the development of abnormally short-lived CL, undersized and with subnormal production of progesterone (Erb et al., 1971; Webb et ah, 1980). Our results indicate that a similar but longer sequence may occur frequently in the buffalo but that setting a
188 discrete level of plasma progesterone (> 0.7 ng/ml) for the presence of a CL introduced a potential error. Studies are, therefore, needed to observe ovarian contents of the buffalo by laparotomy or laparoscopy (Jainudeen et al., 1982) during the first 3 months post partum. This may provide information on the accuracy of rectal palpation in the assessment of luteal function in the buffalo. The resumption of ovarian cyclicity in this study was based on functional activity (progesterone concentrations) rather than on the morphological presence (rectal palpation) of a CL. The interval from parturition to first ovulation was 96 +- 22 days in 32% of buffaloes and, in most cases, ovulation was preceded by oestrus. Over 68% of suckled buffaloes were in anoestrus by 150 days post partum. This period of anoestrus is not due to a maintenance of the CL of pregnancy or a delay in uterine involution. The CL of pregnancy regressed rapidly and progesterone levels remained at basal levels during the first 30 days post partum. Uterine involution was also completed by 28 days post partum as compared to 39--41 days in dairy buffaloes (Roy and Luktuke, 1962; El-Sheikh and Mohamed, 1977). Post partum anoestrus is an important cause of infertility in the suckled buffalo but whether it is due to inactive ovaries or those that are cycling but not detected in oestrus has not been established. Progesterone profiles in this study provide convincing evidence that post partum anoestrus is due to a failure in the resumption of ovarian cyclicity in the suckled buffalo. Once cyclicity is re-established, buffaloes are able to conceive readily to natural service at the first or second oestrus (unpublished data), although only 2 of 8 buffaloes conceived to insemination with frozen semen in this study. Several factors contribute to anoestrus in suckled beef cows. Suckling regulates the resumption of post partum ovarian activity and the intervals from parturition to first oestrus, ovulation and conception are shorter in non-suckled than suckled cows (Radford et al., 1978; Wettemann et al., 1978; Carter et al., 1980). Increasing the frequency of milking or suckling in beef cows prolongs the anoestrous period (Carruthers et al., 1980) but there is evidence to indicate that season of calving may be more important than suckling (Peters et al., 1980). Prepartum and post partum energy intake affect the interval from parturition to first ovulation and an energy deficit affects primiparous cows more severely than multiparous animals (Wiltbank et al., 1964). In the buffalo, suckling significantly increases the interval from parturition to first oestrus and ovulation. Resumption of ovarian cyclicity occurred earlier in milked than suckled buffaloes and ovarian activity was restored in only 38% suckled buffaloes within 3 months of parturition (E1-Fouly et al., 1976). These findings agree with our data in which 32% of suckled buffaloes were cycling at an average of 96 days post partum. Buffaloes suckling calves lost body weight between days 60 and 130 post partum, regaining it thereafter. Though these changes in body weight may reflect suckling intensity of the growing calf, the possibility also exists that
189
confinement for oestrus detection deprived the animals of night grazing. Since cattle are infertile while losing body weight (McClure, 1965) and as the length of interval between calving and conception is inversely related to feed input and suckling intensity (Wiltbank et al., 1964), it may be that the long calving to conception interval of 198 days in the suckled swamp buffalo (Jainudeen, 1977) is due to the adverse effects of suckling and a nutritional deficiency acting either separately or in combination. Perhaps this could be corrected in the future by feeding a concentrate supplement during the appropriate period. Thirty-three percent of suckling buffalo cows, though exhibiting an ovarian cycle, were n o t detected as in oestrus by the vasectomized bull. This condition can be treated with cioprostenol, a synthetic analogue of prostaglandin F2a (Jainudeen, 1976). The possibility of short-term calf removal at the end of a short-term progestagen treatment needs to be investigated for treatment of post partum anoestrus, as this treatment has successfully induced oestrus and ovulation in anoestrous post partum beef cows (Smith et al., 1979). In conclusion, studies need to be conducted to establish the patterns of gonadotrophic and steroid hormone concentrations in the blood of suckled buffaloes and to determine the influence of weaning, suckling intensity, age of dam, nutrition, season or a combination of these factors. It may then be possible to define the circumstances in which an early resumption of ovarian cyclicity is achieved in the swamp buffalo. ACKNOWLEDGEMENTS We thank the Universiti Pertanian Malaysia and the International Atomic Energy Agency, Vienna, Austria (Research Contract No. 2436/RB) for financial support; Mr. Yap Keng Chee for excellent technical assistance in RIA; Mr. Abu Bakar Dahari for blood collection, oestrus observations and artificial insemination; Prof. L.E. Edqvist (Sweden) for progesterone antiserum; and the farm staff for their invaluable assistance. REFERENCES
Boyd, H. and Munro, C.D., 1979. Progesterone a~ays and rectal palpation in pre-service management of a dairy herd. Vet. Rec., 104: 841--343. Bulman, C.D. and Lamming, G.E., 1978. Milk progesterone levels in relation to conception, repeat breeding and factors influencing acyelicity in dairy cows. J. Reprod. Ferfil.,54: 447--458.
Carruthers,T.D., Convey, E.M., Keaner, J.S.,Hafs, H.D. and Chen& K.W., 1980. The hypo~h-i-mo-pituitary gonadotrophic axis of suckled and nonJuckled dairy cows post partum. 51: 949--957.
Carter, M.L., Diersehke, D.J.,Rutledp, J.J. and Hauesr, E.R., 1980. Effect of gonadotrophin-rele~t~ hormone and calf removal on pituitary ovarian function and reproductive performance in post partum beef cows. J. Anita. Sci., 51: 903-910. Dawson, F.L.M., 1975. Accuracy of rectal palpation in the diagnosis of ovarian function in the cow. Vet. Rec., 96: 218--220.
190 EI-Fouly, M.A., Kotby, E.A. and E1-Sobhy, 1976. Post partum ovarian activity in suckled and milked buffaloes. Theriogenology, 5: 69--79. El-Sheikh, A.S. and Mohamed, A.A., 1977. Uterine involution in the Egyptian buffalo. Indian J. Anim. Sci., 47: 165--169. Erb, R.E., Surve, A.H., Callahan, C.J., Randel, R.D. and Garverick, H.A., 1971. Reproductive steroids in the bovine. VH. Changes post partum. J. Anim. Sci., 33: 1060--1071. Haresign, W., Foster, J.P., Haynes, N.B., Crighton, D.B. and Lamming, G.E., 1975. Progesterone levels following treatment of seasonally anoestrous ewes with synthetic LH-releasing hormone. J. Reprod. Fertil., 43: 269--279. Jainudeen, M.R., 1976. Induction of oestrns and ovulation in buffalo (Bubalus bubalis) using a prostaglandin F2a analogue (ICI 80996). Proc. 8th Int. Congr. Anita. Reprod. and A.I., Krakow, Vol. HI, pp. 470--472. Jainudeen, M.R., 1977. Reproduction in the Malaysian swamp buffalo (Bubalus bubalis) Proc. 1st. Joint Conf. Health and Prod. Austr. and Local Cattle in S.E. Asia. Ministry of Agriculture, Malaysia, Bull., 146: 162--169. Jainudeen, M.R., Bongso, T.A., Bashir Ahmad, F. and Sharifuddin, W., 1982. A laparoscopic technique for in vivo observation of ovaries in the water buffalo (Bubalus bubalis). Vet. Rec., 111: 32--35. Kamonpatana, M. Kunawongkrit, A., Bodhipaksha, P., and Luvira, Y., 1979. Effect of PGF-2~ on serum progesterone levels in the swamp buffalo (Bubalus bubalis). J. Reprod. Fertll., 56: 445--449. McClure, T.J., 1965. A nutritional cause of low non-return rates in dairy cattle. Aust. Vet. J., 41: 119--122. Morrow, D.A., Roberts, S.J. and McEntee, K., 1969. Post partum ovarian activity of the uterus and cervix in dairy cattle. 1. Ovarian activity. Cornell Vet., 59: 173--190. Perera, B.M.O.A., Pathiraja, N., Buvanendran, V., Abeywardena, S.A. and Piyasena, R.D., 1978. Plasma progesterone levels during natural and prostaglandin~ynchronized oestrous cycles in buffaloes. Ceylon Vet. J., 26: 29--34. Peters, A.R., Riley, G., Rhodes, J. and Lamming, G.E., 1980. Milk progesterone profiles and oestrous activity in post partum beef cows. Proc. 9th Int. Congr. Anim. Reprod. and A.I., Madrid, Vol. HI, p. 28. Radford, H.M., Nancarrow, C.D. and Mattner, P.E., 1978. Ovarian function in suckling and non~uckling beef cows post partum. J. Reprod. Fertil., 54: 49--56. Rawlings, N.C., Weir, L., Todd, B., Manns, J. and Hyland, J.H., 1980. Some endocrine changes associated with the post partum period of the suckling beef cow. J. Reprod. Fertil., 60: 301--308. Roy, D.J. and Luktuke, S.N., 1962. Studies on involution of the uterus in buffalo. Indian J. Vet. Sci., 32: 208--213. Smith, M.F., Burrell, W.C., Shipp, L.D., Songster, W.R. and Wiltbank, J.N., 1979. Hormone treatments and use of calf removal in post partum beef cows. J. Anita. Sci., 48 : 1285--1294. Wagner, W.C. and Hansel, W., 1969. Reproductive physiology of the post partum cow. 1. Clinical and histological findings. J. Reprod. Fertll., 18: 493---500. Watson, E.D. and Munro, C.D., 1980. A re-assessment of the technique of rectal palpation of corpora lutea in cows. Br. Vet. J., 136: 555---560. Webb, R., Lamming, G.E., Haynes, N.B., Hafs, H.D. and Manns, J.G., 1977. Response of cyclic and post partum suckled cows to injections of synthetic LH-RH. J. Reprod. Fertll., 50: 203--210. Webb, R., Lamming, G.E., Haynes, N.B. and Foxcroft, G.R., 1980. Plasma progesterone and gonadotrophin concentrations and ovarian activity in post partum dairy cows. J. Reprod. Fertil., 59: 133--143. Wettemann, R.P. and Turman, E.J., Wyatt, R.D. and Totusek, R., 1978. Influence of suckling intensity on reproductive performance of range cows. J. Anim. Sci., 47 : 342--346. Wiltbank, J.N., Rowden, W.W., Engalls, J.E. and Zimmermann, D.R., 1974. Influence of post partum energy level on reproductive performance of Hereford cows restricted in energy intake prior to calving. J. Anim. Sci., 23: 1049--1053.
181
Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
POST PARTUM OVARIAN ACTIVITY AND UTERINE INVOLUTION IN THE SUCKLED SWAMP BUFFALO (BUBALUS BUBALIS)
M.R. JAINUDEEN, T~A. BONGSO and H.S. TAN
Department of Veterinary Clinical Studies, Universiti Pertanian Malaysia, Serdang, Selangor (Malaysia) (Accepted 11 October 1982)
ABSTRACT Jainudeen, M.R., Bongso, T.A. and Tan, H.S., 1983. Post partum ovarian activity and uterine involution in the suckled swamp buffalo (Bubalus bubalis). Anita. Reprod. Sci., 5: 181--190. Ovarian activity and uterine involution were monitored b y rectal palpation, oestrus detection and plasma progesterone analysis from 3 to 4 days to approx|m~tely 150 days post p a r t u m in 38 suckled swamp buffaloes (Bubalus bubalis). The intervals from parturition t o regression of the corpus luteum (CL) o f pregnancy and involution o f the uterus were 10 and 28 ± 6 (S.D.) days respectively. First detected oestrus and first elevation of plasma progesterone (> 0.7 ng/ml) occurred at 88 -+ 26 and 96 -+ 22 days in 8 and 12 buffaloes respectively. During the f'n'st 150 days post partum, 26 o f 38 suckling buffaloes (88°£) were acyclic (anoestrus) and o f 12 animals (32%) exhibiting ovarian cycles, 4 were n o t detected in oestrus. The tentative diagnosis, based on rectal palpation, that CL were present between days 30 and 90 after parturition (without concurrent luteal levels o f progesterone in plasma) suggests that confirmation should be by laparoscopy. It is concluded that a delay in the resumption of ovarian cyclicity post partum represents an imp o r t a n t factor contributing to the prolonged calving to conception interval in the suckled swamp buffalo.
INTRODUCTION
Problems of oestrus detection and long calving intervals are among the major constraints that impede genetic prowess and productivity of the swamp buffalo (Bubalus bubalis). The calving to conception interval of 190--200 days in the suckled swamp buffalo (Jainudeen, 1977) may be related to a delay in the resumption of post partum ovarian activity, abnormalities of oestrus or the oestrous cycle, and low conception rates. Uterine involution and ovarian activity have been investigated in cattle by rectal palpation (Morrow et al., 1969; Wagner and Hansel, 1969) and plasma or milk progesterone profiles have been determined in post partum dairy (Bulman and Lamming, 1978; Boyd and Munro, 1979) and beef (Radford et al., 1978; Rawlings et al., 1980) cows. Hitherto, no similar studies have been conducted in the swamp buffalo. 0378-4320183/0000--0000/$03.00
© 1983 Elsevier Scientific Publishing Company
182 Since swamp buffaloes suckle their calves for 6- to 9-month periods, a knowledge of the events that lead to the resumption of post partum ovarian cyclicity in beef cattle may be useful in studying the endocrinology of the post partum swamp buffalo. This information could assist us in reducing the contribution made by post partum anoestrus to the long calving interval in this species. As a first step in investigating this problem, a study was undertaken to determine the time of uterine involution and the resumption of post partum ovarian cyclicity in the suckled swamp buffalo. MATERIALS AND METHODS The experimental animals were 38 suckled swamp buffaloes (Bubalus bubalis), 4--9 years old, which calved normally between October 1979 and October 1980 and were managed in paddocks with access to salt blocks containing phosphorus and trace minerals. Each buffalo was weighed, the uterus and ovaries palpated per rectum and jugular blood collected in heparinized evacuated tubes at weekly intervals, commencing 3--4 days after parturition and continuing until 90--150 days post partum. The position of the uterus in the body cavity and the diameter at the external bifurcation of each uterine horn were recorded. Uterine involution was complete when both uterine horns reached normal nongravid size and position. The ovaries were palpated to estimate size and to record palpable structures (follicles or CL). The day of ovulation was estimated to be 4 days before the time a CL was first palpable. Buffaloes were penned and tested daily for oestrus between 20.00 and 22.00 h using a vasectomized buffalo fitted with a chinball marking device. The male remained with the cows until the next morning. A female was judged as in oestrus (day 0) if mating was observed or she was marked by the male. Rectal palpation of the ovaries and collection of heparinized blood samples were performed daily from four suckled buffaloes during six oestrous cycles.
Progesterone assays Blood samples were centrifuged and the plasma stored at --20°C until assayed. The progesterone levels in the plasma were determined by the radioimmunoassay technique as adapted for the buffalo (Perera et al., 1978; Kamonpatana et al., 1979). Progesterone was extracted from 500 #l plasma with petroleum ether and assayed directly using antiserum against 1 la-hydroxyprogesterone hemisuccinate-BSA-conjugate. All determinations were performed in duplicate and corrected for procedural losses and volume of plasma used. The assay standard curve ranged from 25 to 1000 pg per assay tube with an assay sensitivity of 0.20 ng/ml plasma, i.e. the lowest concentration of unlabelled progesterone that significantly displaced labelled progesterone (P < 0.05). The average extraction efficiency of labelled progesterone was
183
80 ± 8.3% (S.D., n = 67). The intra-assay coefficients of variation were 9% (n = 30) at 1.07 + 0.10 ng/ml and 12% (n =30) at 1.78 + 0.22 ng/ml. The inter-assay coefficients of variation were 10% (n = 5) at 1.09 + 0.11 ng/ml and 19% (n = 16} at 1.82 + 0.34 ng/ml. RESULTS
The mean -+ S.D. (n = 38) liveweight (kg) of the buffaloes immediately after calving was 419 + 65. Changes in body weight during the post partum period expressed as a percentage of the body weight at calving (Fig. 1) showed that animals lost weight between days 60 and 130 post partum (P < 0.05) and regained it thereafter. Uterine involution. The uterine horns were palpable cranial and ventral to the pelvic brim from day 7 to 10 post partum. Thereafter, as the uterus gradually decreased in size, luminal distension decreased, caruncles were not palpable, the horns became clearly demarcated and the uterus attained its normal shape and position. Uterine lochia was recognized in 42% of the animals as a white mucoid discharge until day 18 post partum; it became clear and ceased by day 28 post partum. The cervix which was 4--5 cm in diameter and soft during the first week post partum, gradually reduced in size to 2--3 cm diameter, became firm and its outline could be defined as involution progressed. Involution was completed by day 16 to 39 (mean + S.D. = day 28 + 6) post partum (Table I and Fig. 1). Ovarian activity The ovaries of the swamp buffalo were ovoid and firm. On rectal palpaTABLE I Uterine involution and ovarian activity in post partum swamp buffaloes Item
No. o f observations
Mean (-+ S.D.) (days)
Post partum interval to: Uterine involution Detected oestrus Elevation o f plasma progesterone (> 0.7 ng/ml) for more than I week
38 8
28 ± 6 88 -+ 26
12
96
Ovarian activity 1 (day 3 to 150 post partum) Cycling with detected oestrus Cycling with non-detected oestrus Anoestrus (non-functioning ovaries)
8 (21%) 4 (II%) 26 (68%)
~Based on plasma progesterone profiles.
±
22
184 A
f -10 .C
-15
r-
LP •
P
¢-
a
O O
14
28
42
56
70
84
98
1 ?2
126
140
154
Days post partum Fig. 1. Temporal changes in body weight and occurrence (mean -+ S.D.) o f ovarian activity and uterine involution in 38 suckled swamp buffaloes. Shaded area (mean + S.D.) represents increase (+) or decrease (--) in body weight as a percent o f weight at day 3 or 4 post partum. UI = uterine involution; E = 8 animals with detected oestrus; LP = 12 animals with luteal levels of progesterone (> 0.7 ng/ml).
tion, they measured 2--3 cm in length, 1--1.5 cm from surface to surface and 1--2 cm from the attached to the free border, and were located within the pelvic cavity, caudal and lateral to the uterine horns.
Oestrous cycle. The mean plasma progesterone levels in six complete oestrous cycles, that ranged from 19 to 22 days in length, are presented in Fig. 2. During oestrus (day 0), a follicle (10 mm in diameter) was palpable as a turgid area on the ovarian surface and the progesterone level in plasma was 0.24 + 0.12 ng/ml (S.D.). Following ovulation, the progesterone level increased gradually to 0.42 + 0.21 ng/ml (days 4 to 6) when a CL was palpable as a soft protrusion. With further growth, the CL was clearly demarcated from the rest of the ovary as a firm projection measuring 5--10 mm in diameter (days 8 to 16) and the plasma levels of progesterone increased rapidly to reach a peak concentration o f 1.51 + 0.35 ng/ml (day 14). On or about day 17, the CL decreased in size, became hard and nodular and was accompanied by a sharp decline in plasma progesterone level to reach 0.25 + 0.18 ng/ml at the next oestrus.
Post partum period. The CL of pregnancy (corpus albicans) regressed very rapidly following parturition and by day 10 post partum was palpable as a hard small protuberance (< 3 mm diameter) on the ovarian surface. During the first m o n t h post partum, the ovaries were less than 1 cm in length, smooth and were devoid of either follicles or CL. The plasma concentrations of progesterone were below 0.5 ng/ml. Between days 29 and 56 (day 40 + 7) post partum, 24 of 38 buffaloes possessed mature ovarian follicles, marked uterine tone and a discharge of cervical mucus w i t h o u t estrus. However, ovarian structures palpated as CL were n o t associated with luteal phase progesterone levels.
185 1.8
1.6
E
i-4
4) c-
1.2
o I,=.
e U} 1.0 0
,,t-.
@ ~, 0.8
= 06
,:
l
0"4
0.2
'
-4
-2
'
I
I
I
I
0
2
4
5
8 10 12 14 16 18 20 22 24
|
I
I
I
I
I
i
I
I
26
Days of o e s t r o u s cycle Fig. 2. Mean (+- S.D.) peripheral blood plasma levels of progesterone during six oestrous cycles (19--22 days) from four suckled sw~m'lp buffaloes. E = oes~'us.
Regular ovarian cyclicity, characterized by palpable CL and plasma progesterone levels of 0.7 ng/ml or more on at least two successive samplings (7-day-interval), was initiated at a mean time of 96 +- 22 days (Table I) in 12 of 38 buffaloes. Of the 12 cycling buffaloes, eight were detected in oestrus with an average post partum interval to first oestrus of 88 + 26 days (Table I). The remaining 26 buffaloes (68%) were acyclic (anoestrus), had smooth ovaries with no palpable ovarian structures and plasma concentrations of progesterone were less than 0.5 ng/ml. Wide variations existed in the patterns of pl_~sma progesterone levels in the post partum swamp buffalo (Fig. 3). Over a 90
186
2L 'L
Buffalo 8 0 2 8
1
i
30
40
50
60
70
80
90
100
110
Buffalo 8027
1
•.
. . . . . . . . . . .
~.
.
.
.
"- ' - ' L - --- ' -- ~
.
.*'/"
"~.-,"-,
i
30
E O) cO
:f
40
50
60
70
Buffalo 3007
80
90
100
110
•
~1
•
./'~
.....
•,_..
...!!:./
20
40
60
80
100
120
140
40
80
80
100
120
140
Buffllo 8037
0
E {e O.
I
20
Buffalo 3034
,~
..,....~,~,.. e _ _ 20
iL:
~,,.'-.\
40
,u,,.,o 3oo,
100
:\
110
60
,
120
: .,.,
80
100
~ j > j , . *. / . , 130
.;'.,..f\. " "'\
i / Il . , .41 . / % / ""
120
..... 140
""\ / . .""/\ . / \ .: 140
150
180
Days post partum
Fig. 3. Plasma progesterone profiles of suckled swamp buffaloes to illustrate post partum anoestrus (buffaloes 8028 and 8027), resumption of the oestrous cycle and luteal activity (buffaloes 3007, 3034 and 3002). Note palpable CL with low levels of progesterone (< 0.5 ng/ml) before first oestrus (buffalo 3007 and 3034). • = palpable CL; E = detected oestrus; I = artificial insemination.
A CL was palpated on days 43, 52 and 65 in buffalo 8037 but this could n o t be confirmed by plasma concentrations of progesterone which were below 0.25 ng/ml. At the first oestrus detected on day 73 the animal was inseminated but progesterone levels persisted at basal levels until day 83. This indicated failure of ovulation. A peak of progesterone was recorded on day 85 but the animal returned to service on day 90 after a 17
187 observed only after first oestrus. The animal conceived to insemination at this oestrus. Buffalo 3002 showed a small peak of progesterone (0.5 ng/ml) before oestrus and 21 days after insemination the level of progesterone was 1.4 ng/ml which persisted thereafter. The animal was confirmed pregnant by rectal palpation. DISCUSSION The present study employed both rectal palpation of the ovaries and patterns of plasma concentrations of progesterone to diagnose cyclicity and acyclicity during the post partum period in the suckled buffalo. Our data confirm that plasma concentrations of progesterone during the oestrous cycle are considerably lower in buffalo (Perera et al., 1978; Kamonpatana et al., 1979) than in cattle (Boyd and Munro, 1979; Webb et al., 1980). Plasma concentrations of progesterone during the post partum period remained at baseline values in anoestrous buffaloes whereas they fluctuated -- periods of low progesterone (~ 0.25 ng/ml) alternating with periods of high progesterone (~> 0.7 ng/ml) -- in cycling animals. The present study shows that plasma progesterone analysis is a convenient technique for monitoring the resumption of ovarian activity in the suckled buffalo. Rectal findings accorded well with plasma concentrations of progesterone in the diagnosis of anoestrus. There was also a close agreement between the palpation of a CL and plasma concentrations of progesterone in those buffaloes which were detected in oestrus or had luteal phase concentrations of progesterone beyond 60 days post partum. However, between days 30 and 60 post partum, rectal palpation revealed follicular activity and apparently normal CL development in 24 of 38 suckled buffaloes, but subsequent luteal function, as indicated by plasma concentrations of progesterone, did n o t reach normal luteal phase values of cycling buffaloes. This situation is comparable to that observed after LH-RH treatment for anoestrus in ewes (Haresign et al., 1975) and suckled beef cows (Webb et al., 1977). At least two possibilities exist for the apparent discrepancy between progesterone concentrations and rectal findings during the early post partum period in the buffalo. One possibility is that an error in diagnosing a CL per rectum could have occurred. This could n o t be verified as the accuracy of rectal palpation in the diagnosis of ovarian structures in the buffalo has, as yet, n o t been determined. But in cattle, rectal palpation of the ovaries was 89% accurate in diagnosing a CL (Dawson, 1975) or 77--85% accurate in diagnosing functional luteal tissue based on plasma or milk progesterone concentrations (Boyd and Munro, 1979; Watson and Munro, 1980). A further possibility is that these early CL failed to secrete progesterone. In cattle, the first two successive ovulation post partum are followed by the development of abnormally short-lived CL, undersized and with subnormal production of progesterone (Erb et al., 1971; Webb et ah, 1980). Our results indicate that a similar but longer sequence may occur frequently in the buffalo but that setting a
188 discrete level of plasma progesterone (> 0.7 ng/ml) for the presence of a CL introduced a potential error. Studies are, therefore, needed to observe ovarian contents of the buffalo by laparotomy or laparoscopy (Jainudeen et al., 1982) during the first 3 months post partum. This may provide information on the accuracy of rectal palpation in the assessment of luteal function in the buffalo. The resumption of ovarian cyclicity in this study was based on functional activity (progesterone concentrations) rather than on the morphological presence (rectal palpation) of a CL. The interval from parturition to first ovulation was 96 +- 22 days in 32% of buffaloes and, in most cases, ovulation was preceded by oestrus. Over 68% of suckled buffaloes were in anoestrus by 150 days post partum. This period of anoestrus is not due to a maintenance of the CL of pregnancy or a delay in uterine involution. The CL of pregnancy regressed rapidly and progesterone levels remained at basal levels during the first 30 days post partum. Uterine involution was also completed by 28 days post partum as compared to 39--41 days in dairy buffaloes (Roy and Luktuke, 1962; El-Sheikh and Mohamed, 1977). Post partum anoestrus is an important cause of infertility in the suckled buffalo but whether it is due to inactive ovaries or those that are cycling but not detected in oestrus has not been established. Progesterone profiles in this study provide convincing evidence that post partum anoestrus is due to a failure in the resumption of ovarian cyclicity in the suckled buffalo. Once cyclicity is re-established, buffaloes are able to conceive readily to natural service at the first or second oestrus (unpublished data), although only 2 of 8 buffaloes conceived to insemination with frozen semen in this study. Several factors contribute to anoestrus in suckled beef cows. Suckling regulates the resumption of post partum ovarian activity and the intervals from parturition to first oestrus, ovulation and conception are shorter in non-suckled than suckled cows (Radford et al., 1978; Wettemann et al., 1978; Carter et al., 1980). Increasing the frequency of milking or suckling in beef cows prolongs the anoestrous period (Carruthers et al., 1980) but there is evidence to indicate that season of calving may be more important than suckling (Peters et al., 1980). Prepartum and post partum energy intake affect the interval from parturition to first ovulation and an energy deficit affects primiparous cows more severely than multiparous animals (Wiltbank et al., 1964). In the buffalo, suckling significantly increases the interval from parturition to first oestrus and ovulation. Resumption of ovarian cyclicity occurred earlier in milked than suckled buffaloes and ovarian activity was restored in only 38% suckled buffaloes within 3 months of parturition (E1-Fouly et al., 1976). These findings agree with our data in which 32% of suckled buffaloes were cycling at an average of 96 days post partum. Buffaloes suckling calves lost body weight between days 60 and 130 post partum, regaining it thereafter. Though these changes in body weight may reflect suckling intensity of the growing calf, the possibility also exists that
189
confinement for oestrus detection deprived the animals of night grazing. Since cattle are infertile while losing body weight (McClure, 1965) and as the length of interval between calving and conception is inversely related to feed input and suckling intensity (Wiltbank et al., 1964), it may be that the long calving to conception interval of 198 days in the suckled swamp buffalo (Jainudeen, 1977) is due to the adverse effects of suckling and a nutritional deficiency acting either separately or in combination. Perhaps this could be corrected in the future by feeding a concentrate supplement during the appropriate period. Thirty-three percent of suckling buffalo cows, though exhibiting an ovarian cycle, were n o t detected as in oestrus by the vasectomized bull. This condition can be treated with cioprostenol, a synthetic analogue of prostaglandin F2a (Jainudeen, 1976). The possibility of short-term calf removal at the end of a short-term progestagen treatment needs to be investigated for treatment of post partum anoestrus, as this treatment has successfully induced oestrus and ovulation in anoestrous post partum beef cows (Smith et al., 1979). In conclusion, studies need to be conducted to establish the patterns of gonadotrophic and steroid hormone concentrations in the blood of suckled buffaloes and to determine the influence of weaning, suckling intensity, age of dam, nutrition, season or a combination of these factors. It may then be possible to define the circumstances in which an early resumption of ovarian cyclicity is achieved in the swamp buffalo. ACKNOWLEDGEMENTS We thank the Universiti Pertanian Malaysia and the International Atomic Energy Agency, Vienna, Austria (Research Contract No. 2436/RB) for financial support; Mr. Yap Keng Chee for excellent technical assistance in RIA; Mr. Abu Bakar Dahari for blood collection, oestrus observations and artificial insemination; Prof. L.E. Edqvist (Sweden) for progesterone antiserum; and the farm staff for their invaluable assistance. REFERENCES
Boyd, H. and Munro, C.D., 1979. Progesterone a~ays and rectal palpation in pre-service management of a dairy herd. Vet. Rec., 104: 841--343. Bulman, C.D. and Lamming, G.E., 1978. Milk progesterone levels in relation to conception, repeat breeding and factors influencing acyelicity in dairy cows. J. Reprod. Ferfil.,54: 447--458.
Carruthers,T.D., Convey, E.M., Keaner, J.S.,Hafs, H.D. and Chen& K.W., 1980. The hypo~h-i-mo-pituitary gonadotrophic axis of suckled and nonJuckled dairy cows post partum. 51: 949--957.
Carter, M.L., Diersehke, D.J.,Rutledp, J.J. and Hauesr, E.R., 1980. Effect of gonadotrophin-rele~t~ hormone and calf removal on pituitary ovarian function and reproductive performance in post partum beef cows. J. Anita. Sci., 51: 903-910. Dawson, F.L.M., 1975. Accuracy of rectal palpation in the diagnosis of ovarian function in the cow. Vet. Rec., 96: 218--220.
190 EI-Fouly, M.A., Kotby, E.A. and E1-Sobhy, 1976. Post partum ovarian activity in suckled and milked buffaloes. Theriogenology, 5: 69--79. El-Sheikh, A.S. and Mohamed, A.A., 1977. Uterine involution in the Egyptian buffalo. Indian J. Anim. Sci., 47: 165--169. Erb, R.E., Surve, A.H., Callahan, C.J., Randel, R.D. and Garverick, H.A., 1971. Reproductive steroids in the bovine. VH. Changes post partum. J. Anim. Sci., 33: 1060--1071. Haresign, W., Foster, J.P., Haynes, N.B., Crighton, D.B. and Lamming, G.E., 1975. Progesterone levels following treatment of seasonally anoestrous ewes with synthetic LH-releasing hormone. J. Reprod. Fertil., 43: 269--279. Jainudeen, M.R., 1976. Induction of oestrns and ovulation in buffalo (Bubalus bubalis) using a prostaglandin F2a analogue (ICI 80996). Proc. 8th Int. Congr. Anita. Reprod. and A.I., Krakow, Vol. HI, pp. 470--472. Jainudeen, M.R., 1977. Reproduction in the Malaysian swamp buffalo (Bubalus bubalis) Proc. 1st. Joint Conf. Health and Prod. Austr. and Local Cattle in S.E. Asia. Ministry of Agriculture, Malaysia, Bull., 146: 162--169. Jainudeen, M.R., Bongso, T.A., Bashir Ahmad, F. and Sharifuddin, W., 1982. A laparoscopic technique for in vivo observation of ovaries in the water buffalo (Bubalus bubalis). Vet. Rec., 111: 32--35. Kamonpatana, M. Kunawongkrit, A., Bodhipaksha, P., and Luvira, Y., 1979. Effect of PGF-2~ on serum progesterone levels in the swamp buffalo (Bubalus bubalis). J. Reprod. Fertll., 56: 445--449. McClure, T.J., 1965. A nutritional cause of low non-return rates in dairy cattle. Aust. Vet. J., 41: 119--122. Morrow, D.A., Roberts, S.J. and McEntee, K., 1969. Post partum ovarian activity of the uterus and cervix in dairy cattle. 1. Ovarian activity. Cornell Vet., 59: 173--190. Perera, B.M.O.A., Pathiraja, N., Buvanendran, V., Abeywardena, S.A. and Piyasena, R.D., 1978. Plasma progesterone levels during natural and prostaglandin~ynchronized oestrous cycles in buffaloes. Ceylon Vet. J., 26: 29--34. Peters, A.R., Riley, G., Rhodes, J. and Lamming, G.E., 1980. Milk progesterone profiles and oestrous activity in post partum beef cows. Proc. 9th Int. Congr. Anim. Reprod. and A.I., Madrid, Vol. HI, p. 28. Radford, H.M., Nancarrow, C.D. and Mattner, P.E., 1978. Ovarian function in suckling and non~uckling beef cows post partum. J. Reprod. Fertil., 54: 49--56. Rawlings, N.C., Weir, L., Todd, B., Manns, J. and Hyland, J.H., 1980. Some endocrine changes associated with the post partum period of the suckling beef cow. J. Reprod. Fertil., 60: 301--308. Roy, D.J. and Luktuke, S.N., 1962. Studies on involution of the uterus in buffalo. Indian J. Vet. Sci., 32: 208--213. Smith, M.F., Burrell, W.C., Shipp, L.D., Songster, W.R. and Wiltbank, J.N., 1979. Hormone treatments and use of calf removal in post partum beef cows. J. Anita. Sci., 48 : 1285--1294. Wagner, W.C. and Hansel, W., 1969. Reproductive physiology of the post partum cow. 1. Clinical and histological findings. J. Reprod. Fertll., 18: 493---500. Watson, E.D. and Munro, C.D., 1980. A re-assessment of the technique of rectal palpation of corpora lutea in cows. Br. Vet. J., 136: 555---560. Webb, R., Lamming, G.E., Haynes, N.B., Hafs, H.D. and Manns, J.G., 1977. Response of cyclic and post partum suckled cows to injections of synthetic LH-RH. J. Reprod. Fertll., 50: 203--210. Webb, R., Lamming, G.E., Haynes, N.B. and Foxcroft, G.R., 1980. Plasma progesterone and gonadotrophin concentrations and ovarian activity in post partum dairy cows. J. Reprod. Fertil., 59: 133--143. Wettemann, R.P. and Turman, E.J., Wyatt, R.D. and Totusek, R., 1978. Influence of suckling intensity on reproductive performance of range cows. J. Anim. Sci., 47 : 342--346. Wiltbank, J.N., Rowden, W.W., Engalls, J.E. and Zimmermann, D.R., 1974. Influence of post partum energy level on reproductive performance of Hereford cows restricted in energy intake prior to calving. J. Anim. Sci., 23: 1049--1053.