Milk Progesterone Profiles in Various Reproductive States in Dairy Buffaloes under Field Conditions

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Proc. Natl. Sci. Counc. ROC(B)

Vol. 24, No. 2, 2000. pp. 70-75

–70–

I. Introduction

Progesterone is a steroid hormone, the first biologi-

cally active compound in the steroid biosynthesis path-

way. This hormone was discovered by Loewe and Lange

in 1926 and isolated by Corner and Allen (1929). Its con-

centration in an animal’s body reflects the stage of the

reproductive cycle, pregnancy and ovarian disorders.

Radioimmunoassay (RIA) was developed to measure

progesterone in milk, and the ability to collect a milk sam-

ple rather than a blood sample made it possible to monitor

the ovarian activity of dairy animals using milk proges-

terone RIA (Heap, 1974). Milk is preferred over plasma

for progesterone assay under field conditions because col-

lection of blood samples from dairy animals is more inva-

sive and disliked by farmers while in milk sampling, no

problem of this nature is faced. Lamming and Darwash

(1998) concluded that milk progesterone monitoring

offers an objective and accurate method for assessing typi-

cal and atypical ovarian function in postpartum cows. In

buffaloes, progesterone profiles have been studied in post-

partum periods (Perera et al., 1981; Kamonpatana et al.,

1983; Jainudeen et al., 1983). Following the first postpar-

tum ovulation, plasma progesterone levels were found to

increase and remain above 0.7 ng/ml for about 10 days;

then, they declined to below 0.25 ng/ml at the next estrus.

Progesterone assay was successfully used in estrus detec-

tion (Qureshi et al., 1989) and in the assessment of ovari-

an status (Qureshi et al., 1992) in Nili-Ravi buffaloes.

Usmani et al. (1990) found that 86% of Nili-Ravi water

buffaloes showed at least 1 short luteal phase (8 to 13

days) before the first estrus. It was reported (Jain and

Pandey, 1985) that in buffalo heifers, plasma progesterone

concentrations were significantly affected by the season

Milk Progesterone Profiles in Various Reproductive States in

Dairy Buffaloes under Field Conditions

UHAMMAD

UBHAN

QURESHI

, G

HULAM

HABIB

, G

NAWAB

***

UHAMMAD

OHSIN

SIDDIWQUI

, N

AZIR

AHMAD

****

AND

AFIZ

BDUS

SAMAD

****

Veterinary Research Institute

Peshawar, Pakistan

NWFP Agricultural University

Peshawar, Pakistan

***

Institute of Radiotherapy and Nuclear Medicine

Peshawar, Pakistan

****

University of Agriculture

Faisalabad, Pakistan

(Received December 22, 1998; Accepted August 16, 1999)

ABSTRACT

Fifty-one dairy buffaloes in the last two months of gestation were selected at seven private peri-urban farms

in the Peshawar district. Observations were recorded in buffaloes during normal (NBS, August to January) and

low breeding seasons (LBS, February to July). After parturition, rectal examination of reproductive organs was

carried out. Estrus detection was made through visual observation and the use of intact bul1. Postpartum ovula-

tion was confirmed by ovarian palpation per rectum and milk progesterone levels (MPL), determined through

radio-immunoassay. MPL was higher (p < 0.01) at various intervals in NBS calves (1.97 ± 0.30 ng/ml) as com-

pared to LBS calves (0.68 ± 0.08 ng/ml). During LBS, MPL remained < 0.30 ng/ml up to the third fortnight and

started rising later, reaching a peak of 1.27 ng/ml during the sixth fortnight. During NBS, there was a sharp rise in

MPL during the second fortnight. reaching 3.64 ng/ml during the sixth fortnight. MPL was significantly different

on different experimental farms (p < 0.01). MPL reached the lowest levels on the day of estrus (0.10 ng/ml),

reached it’s peak on day 7 and started declining on day 17 of estrus. MPL showed two postpartum elevations. In

true anestrus buffaloes, MPL remained consistently low. However, in the anestrus period, silent ovu1ations were

also noted, as reflected by increasing MPL without estrus signs. In pregnant buffaloes, MPL remained > 1 ng/ml.

Results of the study showed that the low postpartum reproductive performance in dairy buffaloes during LBS was

primarily due to inadequate functioning of the corpus luteum in secreting optimum concentrations of progesterone.

The higher incidence of silent estrus during LBS indicated improved management for the detection of estrus.

Key Words: buffalo, dairy, Pakistan, reproduction, hormone, progesterone

Page 2

–71–

and weaning, and varied significantly between the puberty

and neonatal periods. The present study was conducted to

determine milk progesterone profiles under various repro-

ductive states in dairy buffaloes under field conditions.

II. Materials and Methods

Fifty-one dairy buffaloes in their last two months of

gestation in the normal (NBS, August to January) and low

breeding seasons (LBS, February to July) were selected at

seven private farms located within a radius of 70 ki1ome-

ters of Peshawar city.

The buffaloes were ear-tagged and kept under con-

ventional management at the respective farms. Restricted

calf suckling at the time of milking was allowed.

After parturition, rectal examination of the reproduc-

tive organs was carried out on days 14 and 21, and then

fortnightly until the occurrence of the first estrus was

detected as described by Usmani et al. (1985). The posi-

tions of the reproductive organs were recorded, and the

size of the graafian follicles and corpora lutea or corpora

albicancia on the ovaries was measured with the help of

fingers. Estrus detection was conducted twice daily, com-

mencing 15 days postpartum. In addition to visual signs

of vulvar mucus, frequent micturition and bellowing, an

intact bull was also used for the detection of estrus.

Standing heat was used as a criterion for estrus confirma-

tion. Postpartum ovulation was confirmed based on the

ovarian palpation per rectum and milk progesterone levels

(MPL).

Evening milk samples were collected once a week.

After removing the fat layer by means of centrifugation

(3000 rpm), 100 µl of 0.1% sodium azide was added to 5

ml of milk sample as a preservative. Samples were stored

at –20C until they were analyzed for MPL, using RIA.

The procedure suggested by the FAO/IAEA (1993) was

adopted. Milk samples and other assay components were

brou-ght to room temperature before the assay was started.

Antibody coated polypropylene tubes were labelled for

standard, quality control and samples, according to the

protocols. Non-coated normal tubes were used for total

count. 100 µl of standard, quality control or sample were

pipetted into the bottom of the corresponding tube. Then,

1 ml of

125

I-progesterone was pipetted into each tube. The

tubes were covered with parafilm and incubated at 4C

over-night. The next morning, the tubes were decanted

vigorously except for those used for total count. The

radioactivity was counted using a gamma counter (Vega-

calc (c) NE Technology Limited). The data were analyzed

using the Vegacalc software program. The intra- and inter-

assay coefficients of variation were 3.54% and 9.21%,

respectively. The sensitivity (detection limit) of the assay

was 0.09 ng/ml.

The data were analyzed using analysis of variance

and correlation analysis procedures (Steel and Torrie,

1980).

III. Results and Discussion

1. Seasonal Effect on Milk Progesterone Profiles

MPL was higher (p < 0.01) in buffaloes during calv-

ing NBS (1.97 ± 0.30 ng/ml) than LBS (0.68 ± 0.08

ng/ml, Table 1). The postpartum levels of milk proges-

terone followed different patterns during the two breeding

Fig. 1. Milk progesterone levels at various postpartum intervals in nor-

mal (NBS) and low breeding season calvers (LBS) (n = 384,

mean ± SEM = 1.37 ± 0.17 ng/ml).

Milk Progesterone Profiles in Dairy Buffaloes

Table 1. Mean Values ± Standard Error (SE) and Number of Observa-

tions (n) of Milk Progesterone Concentrations (ng/ml) during

Various Seasons and Reproductive States in Buffaloes

Group

Mean

± SE

Seasons

Spring

3.00 ± 0.12

Winter

1.77 ± 0.32

150

Autumn

0.84 ± 0.72

101

Summer

0.25 ± 0.04

Significance level

p < 0.01

Calving periods

Autumn-Winter (NBS)

1.97 ± 0.30

207

Spring-Summer (LBS)

0.68 ± 0.08

178

Significance level

p < 0.01

Postpartum intervals (days)

Up to 15

0.26 ± 0.06

16 to 30

0.55 ± 0.08

127

31 to 60

1.78 ± 0.26

100

61 to 90

1.84 ± 0.53

91 to 120

1.87 ± 0.35

121 to 150

2.55 ± 0.59

Significance level

Non significant

Stage of estrus cycle

During estrus

0.30 ± 0.98

Developing corpus luteum

1.43 ± 0.85

Developed corpus luteum

3.29 ± 0.84

Regressing corpus luteum

0.88 ± 0.15

Significance level

p < 0.01

Means in the same column with different superscripts differ from each

other.

__ NBS _ _ LBS

Milk progesterone levels (ng/ml)

Day postpartum

98 112 126 140

Page 3

–72–

seasons as shown in Fig. 1. During NBS, MPL remained

at the lowest level until day 28 postpartum, followed by a

sharp increase to a maximum concentration of 3.64 ng/ml

on day 84. During LBS, the changes in MPL were less

marked and were characterized by a minimum level of

0.30 ng/ml until day 42, followed by a peak level of 1.27

ng/ml on day 84. The earlier rise in MPL during NBS

suggested earlier resumption of estrus activity. In other

studies (Bahga and Ganwar, 1988), the progesterone lev-

els remained at basal levels from day 5 to day 30 postpar-

tum and started rising thereafter. An increase in proges-

terone levels beyond day 30 was correlated with luteal

function and cyclic activity (Madan, 1984).

The results of the present study suggest that during

LBS, MPL did not reach the optimum levels until day 84

postpartum, indicating that the corpus luteum did not

function efficiently so as to maintain sufficiently high

progesterone levels to maintain reproductive cyclicity.

This confirms the findings of Madan (1984), who attrib-

uted the low reproductive efficiency of buffaloes in sum-

mer to 1ow luteal activities, indicated by low progesterone

levels. You and Chen (1992) reported that a cow experi-

enced four estrus cycles, three of which were behavioral,

during a 2-months period. The missing cycle and the low

values of plasma and fecal progesterone during the luteal

phase from May to June implied a weak seasonal effect in

Taiwan. In the present study, fodder scarcity during May

to July of LBS, which was associated with a minimum

body condition score of 2.2 and low energy intake, could

have adversely affected ovarian activity through nutrition-

al deficiency (Qureshi, 1998).

2. Milk Progesterone Levels during Postpartum In-

tervals

The milk progesterone concentration varied during

various postpartum months (p < 0.08). The lowest con-

centrations were recorded 15 days postpartum, followed

M.S. Qureshi et al.

by the second fortnight and the second, third, fourth and

fifth months postpartum (0.26, 0.55, 1.78, 1.84, 1.87, 2.55

ng/ml, Table 1). The present findings are in agreement

with those of Perera et al. (1981) and Jainudeen et al.

(1983), who reported that elevated progesterone levels in

the serum of buffaloes declined rapidly following parturi-

tion to undetectable levels by day 3 or 4 postpartum and

remained low thereafter for different periods until ovarian

cyclicity was restored. In this study, MPL correlated neg-

atively with suckling duration (r = –0.13, p < 0.05) and

was significantly different on different experimental farms

(p < 0.01).

3. Estrous Cycle Pattern

The changes in the ovarian structure and concurrent

MPL are presented in Table 1. MPL varied during various

stages of the estrus cycle. Observations of twenty-four

first postpartum estrus cycles showed an average length of

21.0 days. The estrus cycle pattern of MPL is shown in

Fig. 2. MPL started falling eight days prior to the com-

mencement of the estrus cycle and reached the lowest lev-

els on day 0 (the day of estrus, 0.10 ng/ml). After ovula-

tion, MPL increased, indicating a developing corpus lu-

teum. MPL remained > 1.5 ng/ml from day 4 to 14 of the

estrus cycle, followed by a rapid decrease, indicating

developed and regressing luteal tissues, respectively.

In previous studies (Perera et al., 1981; Jainudeen et

al., 1983), following the first postpartum ovulation, the

plasma progesterone level increased and remained above

0.70 ng/ml for about 10 days, and then declined to below

0.25 ng/ml at the next estrus. In agreement with the find-

ings of this study, Kaur and Arora (1984) reported that the

plasma progesterone level was low on the day of oestrus

and then increased progressively, reaching a peak value

between days 14 and 18, depending upon the estrous cycle

length. Similarly, in Surti buffaloes, serum progesterone

levels were higher (p < 0.05) at the diestrus phase, fol-

lowed by the proestrus and estrus stages (2.78, 2.19 and

0.64 ng/ml) (Sarvaiya and Pathak, 1992). Kamonpatana

(1982) found that progesterone levels declined on days

19-21 of the estrous cycle in swamp buffaloes.

In the present study, MPL showed two postpartum

elevations. The first rise was noted at a postpartum inter-

val of 34.3 days and the second rise at 64.0 days. In pre-

vious studies, based on rectal palpation, the interval from

parturition to first ovulation was found to be 38 days in

milked river buffaloes in India (Singh et al., 1979) and to

be 69 days in suckled buffaloes in Egypt (El-Fouly et al.,

1976).

4. Anestrus and Silent Ovulation

MPL remained consistently lower (< 0.1 ng/ml) in

Fig. 2. Milk progesterone levels on different days of the estrus cycle in

Nili-Ravi buffaloes (n = 61, mean ± SEM = 1.48 ± 0.71 ng/ml).

Milk progesterone levels (ng/ml)

Day of estrous cycle

-8

-3

10 14 17

21 22 25

2.5

1.5

0.5

Page 4

–73–

neither estrus signs nor elevated milk progesterone con-

centrations. However, in the remaining four buffaloes,

during the anestrous period, silent ovulations were also

noted, reflected by increasing progesterone levels. Silent

ovulations (SO) were indicated by SO. Interestingly, the

incidence of silent ovulation in the buffaloes was higher in

LBS than in NBS (70.6 vs 29.4%, respectively). Similar-

ly, in a study on 17 complete postpartum periods in Mur-

rah buffaloes in Sri Lanka, plasma progesterone concen-

trations remained basal (< 0.25 ng/ml) for a period rang-

ing from 92-210 days (Perera et al., 1984). In Swamp

buffaloes (Perera, 1982), it was found that postpartum

ane-strus was due to a failure in the resumption of ovarian

cyclicity in the suckled buffaloes. Kaur and Arora (1984)

concluded that malnutrition coupled with high environ-

mental temperature stress was responsible for long ane-

strous periods in buffaloes.

Lamming and Darwash (1998) found that 10.94% of

dairy cows showed delayed type I ovulation (P

levels < 3

ng/ml for > 45 days postpartum), and 12.85% showed

type II (P

levels < 3 ng/ml for > 12 days between two

luteal phases). 31.7% of the animals had at least one atyp-

ical ovarian pattern before insemination that contributed to

delayed conception, a higher number of services per con-

ception, and a lower first service conception rate.

In pregnant buffaloes, MPL remained > 1 ng/ml (1.1

to 30.69 ng/ml). In a previous study (McCool et al.,

1987), plasma progesterone levels were found to be > 1

ng/ml for pregnant, 0.4-4.0 ng/ml for cycling and < 1

ng/ml for anestrous Swamp buffaloes.

Results of the present study lead us to the conclusion

that low postpartum reproductive performance in dairy

buffaloes during LBS was primarily due to inadequate

functioning of the corpus luteum in secreting optimum

concentrations of progesterone. The higher incidence of

silent estrus during LBS indicated improved management

for the detection of estrus.

Acknowledgment

Financial sponsorship by the National Coordinated Buffalo

Research Program/ARP-II/PARC, Islamabad, and the donation of Prog-

esterone RIA kits by the Animal Production and Health Section, the Joint

FAO/IAEA Division of Nuclear Techniques in Food and Agriculture,

IAEA Vienna, Austria, are acknowledged.

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Milk Progesterone Profiles in Dairy Buffaloes

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Milk Progesterone Profiles in Dairy Buffaloes

UHAMMAD

UBHAN

QURESHI

, G

HULAM

HABIB

, G

NAWAB

***

UHAMMAD

OHSIN

SIDDIWQUI

, N

AZIR

AHMAD

****

AND

AFIZ

BDUS

SAMAD

****

Veterinary Research Institute

Peshawar, Pakistan

NWFP Agricultural University

Peshawar, Pakistan

***

Institute of Radiotherapy and Nuclear Medicine

Peshawar, Pakistan

****

University of Agriculture

Faisalabad, Pakistan

Peshawar

(NBS, 8

1 )

(LBS, 2

7 )

(MPL)

NBS

LBS

MPL

(1.97 ± 0.30 VS. 0.68 ± 0.08 ng/ml)

LBS

MPL

< 0.30 ng/ml

1.27 ng/ml

NBS

MPL

3.64 ng/ml

MPL

(p < 0.01) MPL

(0.10 ng/ml)

MPL

> 1 ng/ml

LBS

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