Br. vet.J. (1993). 149, 585

REPRODUCTIVE PATTERNS OF THE YAK. II. PROGESTERONE AND OESTRADIOL-17fl I JEVELS IN

PLASMA AND MILK JUST BEFORE THE BREEDING SEASON; ALSO DURING NORMAL AND SHORT

OESTROUS CYCLES

S.J. YU, Y. M. HUANG and B. X. CHEN

Department of Veterinary Medicine, Gansu Agricultural University Lanzhou, Gansu, Peoples Republic of China

SUMMARY

Concentrations of oestradiol-17fl and progesterone in plasma and milk of the yak before the first oestrus at the beginning of the breeding season and during normal and short oestrous cycles were measured by radio- immunoassay (RIA). Both oestradiol-17fl and progesterone were main- tained at stable, low levels before the first oestrus. However, oestradiol-17fl showed a peak, similar to that on the day of oestrus (P>0.05), about 6 days before the first oestrus and a small progesterone peak was seen 2 days before it. There were three peaks of oestradiol-17fl in plasma and milk on the day of oestrus [24.57 (sD 11.35) pg ml -~ and 97.84 (so 26.02) pg ml -~ respectively] , and on days 5 and 14 of the cycle. Progesterone levels in plasma and milk were low during oestrus but peaks were seen on day 15 [5.23 (SD 1.02) ng ml -~ and 20.44 (so 3.64) ng ml -t respectively]. The pat- terns of oestradiol-17fl and progesterone during the short cycle were simi- lar to those during the normal cycle (P>0.05), but the values were lower. The levels of oestradiol-17fl and progesterone in milk were about 3 or 4 times higher than those in plasma before the first oestrus, and about 4 or 5 times higher during the normal cycle.

INTRODUCTION

In their natural habitat, yaks are seasonally polyoestrous and mating occurs from July to October. The length of oestrus is 19-21 days, but with a wide variation (Yu et al., 1993). At the beginning of the breeding season but before normal oestrus, yaks usually show oestrus-like symptoms and some of them present a short cycle (Yu et al., 1993), but nothing is known about their endocrine bases. Therefore,

0007/1935/93/060585-09/$08.00/0 © 1993 Bailli&e Tindall

586 BRITISH VETERINARY JOURNAL, 149, 6

this investigation was undertaken to study progesterone and oestradiol-17fl levels just before the breeding season as well as during normal and short cycles.

MATERIAl ~ A N D M E T H O D S

Animals and detection of oestrus Nine yak cows, 5-11 years of age, and 2 yak bulls, 7 and 8 years of age, were kept

on high mountain grassland at an elevation of 2500-3000 m above sea-level all year round in Sunan Autonomous County of Gansu. Their nutrition was good, and there were no apparent disease conditions.

Herd monitoring was started before the beginning of the breeding season. The yak herd was followed and observed from 0900 h, when released from the pen, to midnight. The herd was penned at 1900 h. When a cow was followed closely by a bull or bulls, she was considered in heat.

Rectal palpation Three of the nine experimental yaks were palpated per rectum every other day

from the beginning of the breeding season until oestrus. It was found that two cows each developed a large follicle (0.8 cm), one at 8 days prior to oestrus and the other at 6 days. In all three subjects the follicles were present for 2-3 days before they disappeared. No distinct corpus luteum was found.

Sampling Blood and milk samples were taken daily from nine cows before the first

oestrus; six cows during normal oestrus cycles (lasting 19, 19, 20, 20, 20 and 20 days respectively) and three cows during short cycles (of 6, 7 and 9 days respectively). The samples were collected between 0800 and 0900, after milking. Blood was collected from the jugular vein into tubes containing 0.01% heparin; "after centrifugation (1000g, 10 min), the plasma was put into small bottles with 1% thiomersalate. Milk was taken from the bucket and put into small bottles con- taining 1% (v/v) saturated potassium bichromate for antisepsis. All the samples were stored at a low temperature (-25°C) until assay.

HoT.one assay Progesterone. Progesterone was measured using a modification of the radio-

immunoassay (RIA) method described by Furr (1973). The specific activity of (1,2,6,7)H)progesterone was 80 Ci mmol -~ (Shanghai Atomic Energy InStitute, Shanghai, China). Standard progesterone and antiserum were obtained from the Shanghai Endocrinology Institute. Dilution of the antibody (raised in the rabbit) was 1:4000. The standard curve ranged from 25 to 800 pg. The sensitivity was 4.12pg/assay tube and plasma blanks contained 7.2+2.6pg oestradiol-17fl equiv, ml -~. The recovery rate was 105.8%. The within- and between-assay CVs were 8.3% (n=10) and 11.74% (n=5) respectively. The percentage of cross-reac- tions of the antibody were: progesterone 100, 17fl-hydroxyprogesterone 2.5, cor- tisol 0.01, corticosterone 0.06, testosterone 0.02, oestrone<0.01, oestradiol<0.01 and oestriol<0.01. For each assay, 0.02 ml of plasma were extracted with 10 vol

REPRODUCTIVE PATTERNS OF THE YAK. II. 587

anhydrous ether. The percentage extracted was 93.3%. The dilution curve of the plasma was parallel to the curve of the standard oestradi.ol-17fl,

RIA of the milk progesterone was carried out by direct assay. This employed a slight modification of the milk progesterone assay described by Heap et al. (1976) and Pope et al. (1976). Dilution of the antibody was 1:4000. The standard curve ranged from 20-800 pg and the sensitivity was 4.81 pg/assay tube. The mean recovery rate was 101.3%. The within- and between-assay CVs were 4.7% (n=10) and 18.87% (n=14) respectively. Standards comprised 100/11 of a milk sample of oestrous cow diluted 1:1 by phosphate buffered saline (PBS); sample tubes that contained an equal volume of the milk sample under test were also diluted with PBS (1:1-1:2).

Oestradiol-17ft. Oestradiol-17fl was measured using a modification of the RIA method described by Exley et al. (1971). Dilution of the antibody (raised in the rabbit, obtained from Shanghai Institute of Endocrinology) was 1:15 000. The standard curve ranged from 5 to 160 pg and the sensitivity of the assay was 5.04 pg/assay tube. The within- and between-assay CVs were 5.51% (n=10) and 18.01% (n=5) respectively. The percentage of cross-reactions of the antibody were: oestradiol-17fl 100, oestriol 0.02, oestrone 0.03, progesterone<0.01, 17a- hydroxyprogesterone<0.01, testosterone<0.01 and cortisol<0.01. For each assay 0.5 ml of plasma were extracted with 2 vol anhydrous ether. The percentage of extraction was 88.2%.

RIA of the milk oestradiol-17fl was carried out by the method described by Zhang (1988). Dilution of the antibody was 1:15 000. The standard curve ranged from 4 to 160 pg and the sensitivity of the assay was 7.1 pg/assay tube. The recov- ery rate was 90.0%. The within- and between-assay CVs were 8.5% (n=10) and 19.2% (n=8) respectively. The percentage of extraction was 90.8%.

Calculation and statistical analysis Concentrations of the hormones in plasma and milk are expressed as the mean

+SD. Changes in the concentrations in milk and plasma were examined by Stud- ent's t-test to assess the significance between differences. The relationships between the concentrations of oestradiol-17fl and progesterone in milk and in plasma were examined by least squares linear regression. The significance of the correlation coefficient was assessed using Student's t-test.

RESULTS

Levels of oestradiol-17fl in plasma and milk Fifteen days prior to the first oestrus during the breeding season. The combined data

for nine cows are shown in Fig. 1. The baseline level of plasma oestradiol-17fl, 15 days (day-15) before the first oestrus was 12.33 (SD 4.63) pg ml-l; a peak of 26.76 (sD 8.31) pg ml -I occurred from day -8 to -6 which was similar to that of the day of oestrus (day 0, P>0.05); by day -2 this had dropped to 10.41 (SD 6.53) pg m1-1. The baseline level of milk oestradiol-17fl on day -15 was 41.61 (SD 12.83) pg ml -~. The concentration began to rise from day -10, peaking at 76.54 (SD 25.02) pg ml -~

588 BRITISH VETERINARY JOURNAL, 149, 6

130

120

110

100

90

80 7 ~ 7o

~ 60

~ 50 ,.2,'

0 ~ 40

~ 30 ~ ©

20

10

V - I t

5 + + 1%-1 16'-'2'5

Onset of oestrus Onset of oestrus Time {days)

Fig. 1 Concentrations of plasma and milk oestradiol-17fljust before breeding season (n=9) and during normal cycles (n=6). Milk oestradiol-17fl (O); plasma oestradiol-17fl (0).

on day -6. On day -2, the concent ra t ion d ropped to a low level of 52.17 (SD 16.49) pg ml -~. However, both the plasma and milk oestradiol-17fl increased on the day before the first oestrus, regaining the previous peak on the day of oestrus.

During the normal cycle. Mean values for oestradiol-17/3 from six cows are shown in Fig. 1. Concentra t ions in both plasma and milk reached a peak on the day of oestrus of 24.6 (SD 11.35) pg ml -~ and 97.84 (SD 26.02) pg ml -~, respectively. Sub- sequendy they decreased to the baseline levels of 6.97 (sD 5.76) pg m1-1 and 39.36 (SD 19.87) pg ml -l respectively on day 3. They then rose again and two peaks, 25.18 (SD 6.54) pg ml -~ and 18.8 (sD 9.09) pg ml -~, were found on days 5 and 15 in the plasma; two peaks, 108.13 (SD 22.89) pg ml -~ and 81.65 (SD 0.28) pg ml -~, on days 5 and 13 in the milk.

During the short cycle. The assay results of blood and milk samples of three cows (Fig. 2) showed that oestradiol-17fl of both the plasma and the milk presented a peak, 23.7 (SD 3.66) pg ml -j and 75.76 (SD 7.01) pg ml -j respectively, on the day of oestrus. They then decl ined to a baseline level of 7.84 (SD 0.70) pg rnl -~ and 33.55 (SD 6.14) pg m1-1 respectively, 3 days (day -3) before the next normal oestrus; but in one of the three cows which had a ra ther longer 'short ' cycle (9 days), a small peak appeared 4 days after oestrus. They increased markedly on day -2 (P<0.01)

R E P R O D U C T I V E PATTERNS OF T H E YAK. II. 589

i

100

90

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I l I I 1 t t I I I

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Fig. 2. Concentrations of plasma and milk oestradiol-17fl during short cycles in three yaks. O cow-52;/~ cow-53; Ocow-54.

and r eached a peak o f 28.76 (SD 1.43) pg ml -~ in the plasma and 87.4 (SD 10.75) p g m l -~ in the milk, on day 0 which was h igher than that o f the last oestrus (P<O.05).

Correlation between oestradiol-17fl levels in plasma and milk. Because the lengths of the short cycle o f the three cows were not equal, it was not possible to estimate average values of bo th plasma and milk oestradiol-17fl; the corre la t ion between the oestradiol-17fl levels dur ing the short cycle was the re fore also impossible. T h e concen t ra t ion in plasma (xl, x..,) was significantly cor re la ted to that in milk (yl, y.~) before the first oestrus and dur ing the normal cycle (r~=0.7219, rz=0.7629); y~= 0.9870x~+43.9406 and y2=2.8060x2+27.4824. Th e oestradiol-17fl levels in milk were

590 BRITISH VETERINARY JOURNAL, 149, 6

24~-

22

20

18

16

14

=o 12

o 8

6

- 1 6 - 1 4 - 1 2 -10 - 8 - 6 - 4 - 2 0 0

Onset ofoestrus

i - , i , , i , i i i i , f

2 4 6 8 10 12 14 1 6 - 2 0 t

Onset of oestrus

Time {days)

Fig. 3. Concentrations of plasma and milk progesterone just befi),'e the breeding season (n= 9) aqd during ,aormal cycles (n=6). Milk progeste,'one (O); plasma progesterone (Q).

about 4 and 5 times higher than those in plasma (calculated from the means of multiples on all the corresponding points).

Levels of progesterone in plasma and milk Fifteen days prior to the first oestrus in the breeding season. The baseline levels of the

plasma and milk progesterone in nine cows before the first oestrus were 0.22 (SD 0.09) ng ml -I and 0.30 (sD 0.18) ng ml -I. They rose gradually from 5 days before the first oestrus (day-5) , reaching a peak on day -2 of 1.23 (sD 0.11) ng ml -~ in the plasma and of 2.78 (SD 1.23) ng ml -~ in the milk; thereafter the concentrations dropped abruptly to 0.36 (st~ 0.08) ng ml -I in the plasma and 0.66 (sD 0.32) ng ml -~ in the milk on day -1 (P<0.01), similar to those on the day of oestrus (P> 0.05, Fig. 3).

During the normal cycle. The progesterone level of blood and milk samples in six cows (Fig. 3) were 0.19 (SD 0.08) ng ml -~ in the plasma and 0.41 (SD 0.19) ng ml -~ in the milk on the day of oestrus (day 0), and these levels were maintained for 3 to 4 days. They rose markedly to 1.14 (sD 0.31) ng ml -l and 4.01 (SD 0.97) ng ml -~ respectively on day 5 (P<0.05) and reached a peak value of 5.23 (SD 1.02) ng ml -~ in the plasma and 20.44 (sD 3.64) ng ml -j in the milk, on day 15 in either case. Concentrations dropped promptly from day 17 (day-3 in a 20 day cycle, P<0.001) down to 0.27 (SD 0.11) ng m1-1 and 0.45 (sD 0.06) ng m1-1 on day 0, similar to the concentrations at the time of the last oestrus (P>0.05).

R E P R O D U C T I V E P A T I ' E R N S O F T H E YAK. II . 591

16

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¢a,

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Time (days)

Fig. 4 Concentrations of plasma and milk progesterone during short cycles in three yaks. © cow-52; A cow-53; @ cow-54.

During the short cycle. Analysis of blood and milk samples in three cows (Fig. 4) indicated that the progesterone levels were 0.18 (st) 0.05) ng ml -t in the plasma and 0.67 (st) 0.07) ng ml -~ in the milk on the day of oestrus (day 0). These levels were mainta ined for several days, but rose markedly 3 or 4 days before the next oestrus (day -3 or -4, P<0.01), reaching a peak of 2.01 (st) 0.08) ng ml -~ in the plasma and 14.34 (so 1.34) ng ml -~ in the milk on d a y - 2 . Thereafter , concen- trations d ropped abruptly on d a y - 1 (P<0.01) to 0.24 (so 0.09) ng ml-' and 1.47 (st) 0.41) ng ml -] on day 0, close to those of the last oestrus (P~0.05).

Correlation between the progesterone levels in plasma and milk. Concentrat ions of progesterone in plasma (xl, x,2) and in milk (yl, y'_,) before the first oestrus and dur ing normal cycle were significantly correlated (r1=0.9956, rz=0.9873); yl-- 2.4675x]-0.1569 and y.,=3.9167x.2+0.0403. The equations indicated that the pro- gesterone levels in the milk were approximately 3 and 4 times higher than those in the plasma.

592 BRITISH VETERINARY JOURNAL, 149, 6

DISCUSSION

It has been reported that the pre-ovulation peak of oestradiol-17[3 in dairy cows occurs on the day before oestrus (Stabenfeldt et al., 1970; Wettlemann, 1972) or on the day of oestrus (Dobson, 1973; Glencross & Abeywardene, 1983). The results of the present study support the latter view. The second and the third peaks of oestradiol-17fl during the normal cycle in dairy cows were lower than the first one (Shemesh, 1972). However, the first and second peaks are similar in the yak. The profile of the oestradiol-17fl level during the normal cycle in the yak is similar to that of the dairy cow (Huang et al., 1987), but the peaks are distinctly higher.

The progesterone profiles in plasma and milk during the normal cycle are simi- lar to those of the dairy cow, and the time when they started to rise distinctly and when they dropped abruptly are also similar to observations in dairy cows (Echternkamp, 1973; Wan et al., 1985).

A progesterone elevation of short duration, comparable with that previously reported in the sheep (Berardinelli et al., 1980) and in heifers (Gonzalez-Padilla et al., 1975; Berardinelli et al., 1979), was observed before first oestrus in the breed- ing season in the yak. It also occurred during a short cycle. Evidence from short cycles after periods of anovulation in sheep indicated that this transient progester- one rise was produced after ovulation of an immature follicle which responded poorly to the preovulatory gonadotrophin surge (Hunter et al., 1986). Dodson et al. (1988) stated that the progesterone produced by this abnormal structure was sufficient to synchronize follicular development and ensure that the second pre- ovulatory gonadotrophin surge occurred at the optimal time in relation to preovu- latory follicle development.

In the present study, a peak of oestradiol-17fl occurred just before the pro- gesterone elevation at the beginning of the breeding season which indicated that some follicles must have been developing; this was confirmed in all three cows examined rectally. We also found that all yak cows showed some oestrus-like symp- toms and some of them presented a short cycle at the beginning of the breeding season before a normal cycle (Yu et al., 1993). It might be concluded that a tran- sition to the normal cyclic activity occurs at the beginning of the breeding season in all yak cows.

ACKNOWI.EDGEMENTS

This research was supported by the International Foundation for Science (Grant No. B/1424-1) and International Atomic Energy Agency (Research Contract No. 5102/RB). The authors thank Professor G. H. Arthur and Dr R. C. Harris for help with the preparation of the manuscript in English.

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REPRODUCTIVE PATTERNS OF THE YAK. II. 593

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(A ccepted for publication 2 April 1993)