Theriogenology 40:41 l-425,1993

ASSESSMENT OF FETAL NUMBER, AND FETAL AND PLACENTAL VIABILITY THROUGHOUT PREGNANCY IN CATTLE

H. Dobson,' T. G. Rowan, I. S. Kippaxa and P. Humblotb 1 Department of Veterinary Clinical Science and Animal Husbandry

University of Liverpool, Leahurst, Neston, South Wirral, L64 7TE, U.K.

Received for publication: December 4, 19li; Accepted : May 6, 1993

ABSTRACl

Production of identical twin calves by embryo demisection requires a reliable system for continual monitoring of pregnancy. Both halves of bisected embryos were replaced nonsurgically into one uterine horn on Day 7 in 80 recipients. Monthly blood sampling began on Day 22 of pregnancy and transrectal echography took place between 50 and 80 days. Fifty-four recipients had elevated plasma progesterone concen- trations on Day 22 (67% pregnancy rate). Of 21 pregnancies diagnosed as twins by echography, 15 live sets were born, 3 singles accompanied by a stillbirth, and 3 complete abortions. Twenty-five singleton pregnancies resulted in 23 live calves, 1 still-birth and 1 abortion. It was not possible to monitor echographically the remaining 8 pregnancies.

At all stages of pregnancy studied, mean concentrations of bovine pregnancy-specific protein B (bPSPB) and estrone sulphate were higher (P<O.Ol) in twin (n = 17) than in single (n = 26) pregnancies, but the high individual variation obviated any predictive value for fetal number. Although bPSPB and estrone sulphate concentrations were positively correlated at most stages of normal pregnancies after Day 100, divergence was observed in the unsuccessful pregnancies between the concentrations of all 3 hormones, suggesting synthesis/ release is under independent control. Measurement of bPSPB may be useful for prediction of fetal well-being, whereas estrone sulphate may reflect placental viability.

Key words: PSPB, estrone sulphate, progesterone, ecnography, twinning, abortion

Acknowledgements

Tom Houghton and Nigel Jones gave invaluable assistance with the cattle; we are also indebted to Jean Tebble, Hilary Pursell, Mary Enston and Nadine Jeanguyot for technical assistance; to Sheila Nugent for typing the manuscript: and to R. G. Sasser for bPSPB assay reagents. TGR was partly supported by the Perry Foundation.

1 Address far correspondence.

iGenus Embryo Transfer, Tarporley, Cheshire, U.K. Laboratoire d'hormonologie, UNCEIA, BP65, 94073-Maisons-Alfort, France.

Copyright0 1993 Butterworth-Heinemann

412 Theriogenology

INTRODUCTION

In a program to produce identical twin calves by embryo demi- section a reliable system for continual monitoring of pregnancy was required. It was hoped that such a method would give an estimate of the number of fetuses, which would assist calculations for correct nutritional support for the cows, especially in late pregnancy. Observations made during such a study might also allow consideration of the stage(s) of pregnancy, and the reasons, for embryo losses (which occur at a very high frequency; l-3), and hence a greater understanding of feto-placental function in the cow.

Transrectal echography has been reliably used to detect pregnancy in cattle from 28 to 30 days after insemination (4-6), although few attempts have been made to predict the number of fetuses at that time. After approximately 100 days of pregnancy it is difficult to monitor the continued viability of more than one fetus using ultrasound and hence alternative methods were necessary. To monitor the feeding of the potentially twin-bearing dams, 3 beta hydroxybutyrate was chosen to give an indication of nutritional compromise (7).

At Days 21 to 24 after insemination progesterone measurements were made to give a reliable indication of the failure to establish pregnancy (8). Continued progesterone support is essential for the maintenance of a full-term pregnancy in cattle (9). In the cow, luteal functioin may or may not be prolonged after embryonic death or removal of conceptus products (10,ll).

A bovine pregnancy specific protein (bPS?B) secreted by trophoblast binucleate cells has been isolated from cow placental membranes (12). Development of a radioimmunoassay has shown that bPSPB is secreted continuously throughout gestation, with concentrations increasing from approximately Days 24 to 28 to the end ‘of pregnancy (11,13). The half-life of bovine PSPB is about 8 days (12).

Estrone sulphate is another hormone of feto-placental origin; androstenedione is converted to estrone by placental membranes and the endometrium contains sulphatase enzymes (14). Concentrations of estrone sulphate increase in maternal peripheral circulation from as early as Day 72 and increase steadily throughout the remaining days of pregnancy (15). A positive correlation between maternal plasma concentrations and the number of calves has been reported (16), although no correlations have yet been made between estrone sulphate and bPSPB concentrations.

In the present study 54 pregnancies, established by twin embryo transfer, were monitored by echography and maternal plasma measurements of 3 beta hydroxybutyrate, progesterone, bPSPB and estrone sulphate. This provided the opportunity to examine changes in these measurements associated with 43 normal pregnancies (17 twin, 26 single), 5 preg- nancies leading to stillbirths and 6 spontaneous abortions before Day 230.

Theriogenolog y 413

MATERIALS AND METHODS

Animals

A total of 80 Hereford-cross recipient heifers was used in this study as part of an embryo transfer program to produce homozygous twins by bisection (3). Briefly, donor cows were superovulated with FSH and artificially inseminated at estrus with frozen semen from 1 of 2 bulls. (There were no differences in any of the results attributable to a bull effect). Embryos were collected nonsurgically on Days 6 or 7 after AI. The inner-cell mass of Grade 1 and 2 embryos were bisected under a dissecting microscope and the 2 halves of each embryo (with or without zona pellucida) were replaced non- surgically into the uterine horn, ipsilateral to the corpus luteum, in 1 recipient heifer on the same day of a synchronized estrous cycle.

Blood samples were collected once from each recipient on Days 22 to 24 (15 to 17 days after transfer) for progesterone estimation of pregnancy. Forty-six (85%) of the heifers thus estimated to be pregnant were later subjected to echography (Scanner: Concept 1, Dynamic Imaging Ltd., Edinburgh, Scotland) and an estimate was made of the number of fetuses present at 50 to 80 days of pregnancy. The presence of twins was determined by observation of 2 single fetuses on 2 screen-scans in different positions within 1 uterus, or simultaneously 2 anatomically distinct fetuses present on the screen at one time.

Blood samples were taken into heparinized vacutainers and were centrifuged at 1000 g within 30 minutes. The plasma was then stored at -15OC. Blood samples were collected approximately every 30 days in 2 main groups: on Day 60 of pregnancy in Group I and on Day 80 in Group II.

Plasma Analyses: 3 beta-hydroxybutyrate

A calorimetric method was used (17). Values of >0.7 mmol/l were considered indicative of nutritional compromise (7).

Progesterone

For this radioimmunoassay (18) the contemporary inter-assay coefficient of variation was 12.8%, and the minimum detectable amount was 0.015 ng/ml. Animals were considered to be pregnant at Days 22 to 24, when concentrations were >0.5 ng/ml.

PSPB

The homologous direct radioimmunoassay used was modified from that of Sasser (11, 13). Briefly, plasma samples were diluted with assay buffer according to time in pregnancy, viz; Days 22 to 59 neat plasma; Days 60 to 89 diluted 1:5; Days 90 to 140 diluted 1:lO; and Days 141 to 260 diluted 1:20. One hundred microliters of the

414 Theriogenology

appropriate saBple or standard (R37 bPSPB) dilution was incubated for 48 hours at 15 C with rabbit anti-bovine PSPB (RGS 38T$5 initial dilution 1:100,000) and approximately,~~,OOO dpm of I labelled bPSPB (R37). The bound and unbound I -bPSPB was separated by centrifugation after 24 hours incubation with anti-rabbit gamma globulin and the addition of 4% polyethylene glycol just before centrifugation. The contemporary intra- and inter-assay coefficients of variation were 9.9 and 20.4%, respectively, with a minimum detectable value of 0.13 ng per assay tube.

Estrone sulphate

The direct radioimmunoassay used was modified from that of Holdsworth (19, 20). Briefly, plasma samples were diluted with assay buffer according to the stage of pregnancy, viz; Days 60 to 147 neat; Days 148 to 229 diluted 1:5; Days 230 to 260 diluted 1:lO; One hundred microliters of the appropriate sample or standard (Sigma, Poole, UK), dilution was incubated for 1 hour at 37'C with 50 ul 2, 4, 6, 7 3H-estrone sulphate (8000 dpm; 3.63 TBq/mmol; Radiochemical Centre, Amersham, UK) and 50 microliter anti estrone-3-glucuronide (initiab dilution 1:12,000). After a further 30 minutes of incubation at 4 C, the unbound estrone sulphate was extracted by vigorous shaking with 2.5 ml xylene-based scintillant (O.C.S., Radiochemical Centre, Amersham, UK). After 5 minutes of extraction the aqueous phase was separated from scintillant by dipping the assay tubes in ethanol at -45 C. The contemporary intra- and inter-assay coefficients of variation were 9.6% and 20%, respectively, and the minimum detectable concentration 0.01 ng/ml.

Statistical Analysis

Because of the timing of the different blood sampling regimens, it was necessary to batch results for different periods throughout pregnancy (i.e., Days 20, 60, 80 to 90, 110 to 120, 140 to 150, 160 to 170, 190 to 200, 220 to 230, 240 to 250 and 255 to 270. This provided sufficient but variable amounts of data per period for statistical analysis.

The effects of twinning and stage of pregnancy and their interaction on bPSPB and estrone sulphate concentrations were analyzed by ANOVA (21) with the following fixed effects model:

Yijk = u + ai + bj t a_bij t e..

1Jk Where a. was the effect of the stage of pregnancy, b. the effect of

the number bf fetuses, a-b.. the interaction between thdse 2 factors and, e.. consid&&d significant.

the error term akaociated with yijk. Values of P co.05 were

At each stage of gestation, differences between the mean concen- trations observed for single and twin pregnancies and the homogeneity of variances of the 2 groups were subsequently analyzed using Student's t- test (22).

Theriogenology 415

RESULTS

Twenty-six of the 80 heifers had progesterone values ~0.5 ng/ml at Days 22 to 24, suggesting failure to establish pregnancy. In the remaining 54 heifers pregnancy was established as revealed by either echography or bPSPB concentrations. A total of 43 heifers calved, 5 had stillbirths, and 6 aborted.

Echography

From the 21 pregnancies diagnosed as twins by echography, 15 live sets were born. In addition, 3 pregnancies each resulted in the birth of 1 live calf along with 1 fresh stillbirth, and 3 heifers aborted: one of them with fresh dead twins after a ruptured uterus (Table 1).

Twenty-five dams were diagnosed to have single pregnancies, of which 23 had single live calves and 1 had a stillbirth. The remaining animal Dam 352 was scanned positive at 57 days, but the fetus was lost before Day 90.

The echography machine was not available to scan 8 of the heifers. However, pregnancy was assumed from subsequent bPSPB concentrations and/or calvings.

In summary, echography resulted in a confirmed correct diagnosis of 24 out of 25 (96%) single pregnancies, and 19 out of 21 (90%) twin pregnancies. (For 2 of the 3 aborted twin diagnoses it was not possible to confirm the number of fetuses lost.)

Table 1. Outcome of twin embryo recipients diagnosed pregnant at Days 22 to 24 by progesterone concentrations >0.5 ng/ml. Ecography was carried out between Days 50 & 80 of pregnancy

Echography diagnosis Twin Single

No echography Total

Total 21 25 8 54

Births: Twins 15 0 2 17

Single 0 23 3 26

Stillbirths 3a Aborted 3c

1 lb 5 1 2 6

a One of each pair of twins born alive, the others were born stillborn.

b Born mummified. c One heifer (c) had a ruptured uterus on day 223 with freshly dead

twins.

416 Theriogenology

In view of the apparent accuracy of echography, the results for blood constituents were grouped into single or twin pregnancies according to echographical diagnosis. The remaining pregnancies, undiagnosed by echography, were subsequently grouped according to calving data.

3 beta hydroxybutyrate

Only 3 cows had values of this metabolite >0.7 mmol/l, indic- ating nutritional compromise (7). One cow was in the last month of a single pregnancy, one in the last two months carrying twins, and one, on Day 118, having lost twins within the preceding month.

Hormones in Normal Pregnancies

Progesterone. Throughout pregnancy there were no differences in progesterone concentrations between single or twin pregnancies; all values were within the range 1.8 to 8.5 ng/ml.

bPSP6. The ANOVA performed on concentrations revealed highly signmnt main effects of stage of pregnancy (P <0.001) and of twinning (P <O.OOl). There was a tendency for the effect of twinning to be greater at the end of pregnancy (between 150 days and 250 days) than at the beginning (Figure la; STAGE OF PREGNANCY X TWINNING interaction : P = 0.07). Crude mean concentrations and estimates were constantly higher in twin than in single pregnancies.

On Days 22 to 24 in the twin pregnancies only 2 of 7 samples contained bPSPB (1.04 and 3.79 ng/ml); 5 of 13 samples from single pregnancies had measurable concentrations of bPSPB (0.6 + 0.5, range 0.2 to 1.65 ng/ml). Estimates of mean values (very close to least squares means) at this stage (including animals with undetectable concen- trations) were not different: 0.22 t 0.12 ng/ml for single pregnancies (n = 13) and 0.80 + 0.6 ng/ml for twin pregnancies (n = 6).

From Day 60 to the end of pregnancy all samples from normal twin and single pregnancies had measurable PSPB values, and the heifers with twins had higher mean bPSPB concentrations than those with single calves (Figure la), but the range of individual values overlapped considerably.

At each stage between 150 days and 250 days of pregnancy, estimates of bPSPB concentrations in twins were almost double those of single pregnancies (Figure la). These higher concentrations were also associated with a higher variability of individual values, as reflected in the differences in the variances of the 2 groups (P ~0.05).

Estrone Sulphate The ANOVA revealed an effect of stage of pregnancy (P <O.OOl) and of twinning (P ~0.05) on estrone sulphate concentrations. The interaction between these 2 factors was not significant. As shown for PSPB, estimates of estrone sulphate concentrations in twin pregnancies were higher than in single pregnancies (Figure lb).

Theriogenology 417

b)

8,

6_

4_

2,

0 _-_.__+J

0 50 100 150 200 250 300

DAYS of PREGNANCY

Figure 1. Mean (kSEM) plasma concentrations of a) bPSPB (nglml) and b) estrone sulfate (ng/ml) at various stages of pregnancy in heifers bearing twin (a,~) or single (A,.) calves.

418 Theriogenology

On Days 80 to 90 estrone sulphate values were too low to be measured in 4 of the twin pregnant cows, but for the other 5 cows a mean (t SEM) of 0.17 t 0.13 (range 0.01 to 0.38) ng/ml was observed. In single pregnancies, values were below the limit of detection for 9 of 20 samples. Mean concentrations (+ SEM) for the remaining animals were 0.08 + 0.08 (range 0.01 to 0.22) @/ml. -

From Day 140 to the end of pregnancy, estimates of estrone suphate concentrations were higher in twin pregnancies than single pregnancies (Figure lb); however, again the ranges of values overlapped.

Concentrations of estrone sulphate and bPSPB were positively correlated (Pearson's procedure; 22) in normal pregnancies after Day 100 (Table 2).

Table 2. Correlation coefficients (r) between plasma estrone sulphate and bPSPB concentrations at different stages of pregnancy (twins and singles)

Days of pregnancy n Correlation Significance (p) coefficient (r)

110 160 z:

0.43 <O.Ol 0.61 <O.OOl

230 0.35 <0.05 250 ;3u 0.73 <O.OOl

Hormones in Abnormal Pregnancies

Abortions. None of these following results have been included in the foregoing hormone assessments. Six of the pregnancies monitored aborted - 5 for unknown reasons. One heifer (276) which was not scanned by echography had a measurable concentration of bPSPB (3.3 and 1.1 ng/ml) on Days 60 and 90, respectively, although the progesterone concentration had decreased to 0.3 ng/ml. The estrone sulphate concentration was 0.17 ng/ml on Day 90.

Another female (Heifer 352) had a single pregnancy detected by echography on Day 57 and a bPSPB value of 3.71 ng/ml on Day 60. However, by Day 90 the bPSPB concentration had decreased to 1.5 ng/ml and the simultaneous progesterone value was <O.Ol ng/ml. Estrone sulphate remained undetectable throughout.

A third female (Heifer 270; Figure 2a) was scanned with twins on Day 57. The bPSPB concentration increased from 1.4 ng/ml on Day 22 to 9.1 and 25.7 ng/ml on Days 60 and 90, respectively, but was 2.8 and 0.7 on Days 118 and 147. Progesterone concentrations remained about 7 ng/ml until Day 118 when 0.1 ng/ml was measured. Estrone sulphate remained undetectable throughout.

Theriogenology 419

- 100

- 80

”

-60 s s

ii

-40 &

%

-0

b) r 100

- 80

1 & :I;

f8’ : \ Abortion

-20 4 I I I

L I ’ I 0 0 50 100 150 200 250 300

DAYS of PREGNANCY

Figure 2. Plasma concentrations of bPSPB (A), estrone sulfate (0) and progesterone (D; all ng/ml) throughout pregnancy in a) Heifer 270 which aborted between days 90 and 118; b) Heifer 264 which expelled a fetus on day 197.

420 Theriogenology

A fourth female (Heifer 266) scanned with twins at 75 days had low bPSPB values between 1.7 and 3.0 ng/ml from Day 111 to 250; progesterone values remained elevated (22.5 ng/ml) throughout, except for 0.14 ng/ml on day 191. Estrone sulphate remained undetectable throughout and the heifer did not calve.

The fifth female (Heifer 264; Figure 2b) was not scanned by echography but had normal elevated concentrations of bPSPB only until Day 111, whereas estrone sulphate continued to increase up to Day 140. Progesterone values remained elevated throughout. No further samples were taken before this heifer expelled a single calf of undetermined age on Day 197.

One female (Heifer 384) aborted and suffered a ruptured uterus on Day 223; on post mortem examination there was no other evidence of physical trauma. Both twins were freshly dead. Hormone concentrations were within the normal ranges up to Day 202.

Stillbirths. There were 5 pregnancies which resulted in the birth of at least 1 dead calf. Two females (Heifers 260 and 382) each gave birth to 1 freshly dead twin at 260 (clinical case of hydrops) and 275 days, respectively, while another (Heifer 344) delivered a dead single calf on day 271. This latter heifer had low bPSPB values until Day 200; the other 4 animals had bPSPB concen- trations well in the upper quartile of the range of normal pregnancies. In contrast, there were 2 heifers with low bPSPB values throughout pregnancy; Heifer 154 delivered 1 freshly dead twin along with 1 normal calf, and Heifer 241 gave birth to ? normal calf and 1 mummified fetus.

Estrone sulphate concentrations for all these pregnancies were within the normal range, although the case of hydrops (Heifer 260) had values in the upper part of the range from Day 118 onwards, and the highest recorded value, 16.6 ng/ml, 10 days before birth. The progesterone concentrations for all these 5 pregnancies were within 2 to 9 ng/ml, except for Heifer 334, which had values cl.2 ng/ml on Days 200, 230 and 250 (Figure 3).

DISCUSSION

Echography at 50 to 80 days proved to be an accurate method for predicting the number of fetuses being carried in late pregnancy. The concentration of bPSPB was measurable earlier in pregnancy than estrone sulphate. However, the earliest time at which bPSPB or estrone sulphate was detected was not correlated with the number of calves scanned or born. It has been established (11) that measurement of bPSPB at Days 26 to 30 was a more accurate test for confirmation of pregnancy than the measurement of progesterone at Days 22 to 24. In the present study at Days 22 to 24, there was no difference in the concentration of bPSP6 between pregnant cows carrying twins and those which lost 1 embryo before 50 days of pregnancy.

Theriogenology

A 8’

,’ ,’

,’ 8’

8’ ,’

,’ J ,’ ,’

.*

-20 i(

421

- 100

- 80

0 50 100 150 200 250 300

DAYS of PREGNANCY

Figure 3. Plasma concentrations of bPSPB (A), estrone sulfate (0) and progesterone (m ; all ng/ml) throughout pregnancy in Heifer 334

which had very low progesterone values for the last month of pregnancy and gave birth to a single dead calf.

Both bPSPB and estrone sulphate were higher in twin than in single pregnancies, but there was too much individual variation for these measurements to be used predictively. Either the total tissue from an individual placenta from a twin pregnancy did not produce as much hormone as a single placenta, or the catabolic capacity to remove these hormones is underutilized in single pregnancies. In the goat, sheep and cow, mammary gland tissue actively synthesises estrone sulphate (23) and this source may mask any difference in estrone sulphate concentrations due to the differing amounts of placental tissue present.

The functions of bPSPB and estrone sulphate in bovine pregnancy are unknown; bPSPB can only be detected in peripheral plasma approx- imately IO days after the prevention of luteolysis. In addition, progesterone and bPSPB concentrations are not correlated, suggesting that progesterone production is neither stimulated by bPSPB nor vice versa (11). In the present study bPSPB and estrone sulphate concen- trations were positively correlated in normal pregnancies. However, in

422 Theriogenology

3 of the unsuccessful pregnancies bPSPB had increased, but oestrone sulphate was undetectable. In addition, there were 2 cases in which bPSP6 had decreased to very low concentrations, while estrone sulphate values continued to increase. It is suggested that the synthesis and/or release of these hormones is under independant control.

Measurement of bPSPB appeared to give a better assessment of fetal viability, whereas estrone sulphate monitored placental well- being. Indeed high concentrations of estrone sulphate occur for several days during placental retention following abortions induced by infectious agents (24).

In 3 of the cows aborting between 60 and 111 days, estrone sulphate was not measurable but the elevated bPSPB and progesterone concentrations then decreased simultaneously. With the infrequent sampling regime it was not possible to say if the demise of one led to that of the other. However, in a fourth case (Heifer 264) bPSPB concentrations declined, whereas progesterone concentrations remained elevated. This spontaneous situation resembled that induced by intrauterine infection with Actinomyces pyogenes (25). Prolonged corpus luteum maintenance for 25 to 37 days has been achieved in the cow by intrauterine implantation of trophoblast vesicles on Day 12 of the cycle; however, bPSPB concentration was not measured (1).

The increasing concentrations of estrone sulphate with simul- taneous decreased bPSPB values found in Heifer 264 would again suggest that bPSPB may be a better indicator of fetal viability. Throughout pregnancy in the cow binucleate cells migrate from trophoblast tissue into the uterine epithelium (26). If this is an active process requiring healthy trophoblast (and embryonic) tissue but ceases in a failing trophoblast, the source of bPSPB would fail to be maintained. The situation was slightly confused by the very high concentrations of bPSPB found in dams having at least 1 stillbirth. However, these calvings were early in the study and were unassisted and hence prolonged; this was probably the reason for the stillbirths rather than placental insufficiency. Latent placental infection with Leptospira hardjo may also be a cause of the stillbirths (unfortunately, neither spirochetes nor antibody titres were measured), but it is not known why this should coincide with increased bPSPB concentrations. Circumstantial evidence has been provided for reduced estrone sulphate concentrations in cows exposed to Leptospira hardjo infections (27).

In conclusion, echography between 50 to 80 days of pregnancy appeared to be the most useful monitor of the number of fetuses established after twin demi-embryo transfers. Most of the embryo failures occurred before this time, i.e., 26 of 80 (32%) heifers had lost both transferred demi-embryos, and 25 out of 80 (31%) had lost one demi-embryo leaving a single pregnancy. This concurs with previous work (1). The present study could give no indication of the reason for these losses. Nutritional compromise did not appear to be a major problem from the 3 beta hydroxybutyrate results.

Theriogenolog y 423

Monitoring of later pregnancy with progesterone, bPSPB and estrone sulphate did not allow a subsequent alternative prediction of the number of fetuses; however, it was possible to give an estimate of the time of fetal loss in some cases. Abortions occurred in 3 pregnancies between 60 and 111 days (2 singles and 1 twin pregnancy). An unscanned pregnancy was lost between 118 and 140 days. Unfort- unately, with the frequency of sampling it was not possible to say which decreased first, bPSPB or progesterone concentration. Later in pregnancy 1 set of twins was lost during uterine rupture at 223 days, and for 4 heifers 1 twin was stillborn or mummified at term, whereas only 1 singleton calf was stillborn. This would suggest that twin pregnancies were at greater risk in late pregnancy than single pregnancies.

Overall, in this initial part of the embryo transfer twinning program 33% transfers failed to establish a pregnancy, 32% transfers resulted in single pregnancies, 14% pregnancies were either lost or were reduced to produce one live calf, and 21% transfers resulted in the birth of twins. More recent results from the same on-going program indicate that typically 25 to 30% of transfers resulted in the birth of twins. To date, echography at 60 to 80 days proved to be the most cost effective way of monitoring progress.

REFERENCES

1. Heyman, Y. Factors affecting the survival of whole and half- embryos transferred in cattle. Theriogenology 23:63-75 (1985). -

2. McEvoy, T.G. and Sreenan, J.M. Effect of embryo quality and stage of development on the survival of zona pellucida-free cattle demi-embryos. Theriogenology 33:1245-1255 (1990). -

3. Kippax, I.S., Christie, W.B. & Rowan, T.G. Effects of method of splitting, stage of development and presence or absence of zona pellucida on foetal survival in commercial bovine embryo transfer of bisected embryos. Theriogenology 35:25-35 (1991). -

4. Reeves, J.J., Rantanen, N.W. & Houser, M. Trans rectal real-time scanning of the cow reproductive tract. Theriogenology 21:485-494 (1984).

-

5. Pierson, R.A. and Ginther, 0-J. Ultrasonography for detection of pregnancy and study of embryonic development in heifers. Theriogenology 22:225-233 (1984). -

6. White, I.R., Russel, A.J.F., Wright, I.A. & Whyte, T.K. Real-time ultrasonic scanning in the diagnosis of pregnancy and the estimation of gestational age in cattle. Vet. Rec. 117:5-8 (1985).

424 Theriogenology

7.

8.

9.

IO.

11.

12.

13.

14.

15.

16.

17.

18.

Kelly, J.M. Changes in serum B hydroxybutyrate concentrations in dairy cows kept under commercial farm conditions. Vet. Rec. 101: 499-502 (1977).

Dobson, H. and Fitzpatrick, R.J. Clinical application of the progesterone in milk test. Brit. Vet. J. 132:538-542 (1976). -

Shemesh, M. Production and regulation of progesterone in bovine corpus luteum and placenta in mid and late gestation - a personal review. Reprod. Fert. Dev. L:129-135 (1990).

Humblot, P. and Oalla Porta, M.A. Effect of conceptus removal and intra uterine administration of conceptus tissue on luteal function in the cow. Reprod. Nut. Dev. 24:529-541 (1984). -

Humblot, P., Camous, S., Martal, J., Charlery, J., Jeanguyot, N., Thibier, M. and Sasser, G. Pregnancy specific protein B, progesterone concentrations and embryonic mortality during early pregnancy in dairy cows. J. Reprod. Fert. 83:215-223 (1988). -

Sasser, R.G. and Ruder, C-A. Detection of early pregnancy in domestic ruminants. J. Reprod. Fert. 34(Suppl.):261-271 (1987).

Sasser, R.G., Ruder, C.A., Ivani, K-A., Butter, J.E. and Hamilton, W.C. Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol. Reprod. 35:936-942 (1986). -

Gadsby, J.E. Burton, R.D. and Heap, R.B. Oestrogen production by blastocyst and early embryonic tissue of various species. J. Reprod. Fertil. 60:409-417 (1980). -

Robertson, H.A. & King, G.J. Conjugated and unconjugated oestrogen in fetal-maternal fluids of the cow throughout pregnancy. J. Reprod. Fertil. 55:453-470 (1979). -

Worsfold, A.L., Williams, G. and Williams, D.O. Oestrone sulphate measurement in bovine serum during late pregnancy and its relationship with the number of calves born. Brit. Vet. J. 145:46-49 (1989).

Williamson, D-H., Mellanby, J. and Krebs, H.A. Enzymatic determination of D(-) beta hydroxybutyric acid and acetoacetic acid in blood. Biochem. J. 82:90-96 (1962). -

Kanchev, L.N., Dobson, H., Ward, W.R. and Fitzpatrick, R.J. Concentration of steroids in bovine peripheral plasma during the oestrous cycle and the effect of betamethasone treatment. J. Reprod. Fertil. 48:341-345 (1976). -

Theriogenology 425

19. Holdsworth, R.J., Heap, R.B., Booth, J.M. and Hamon, M. A rapid direct radioimmunoassay for the measurement of oestrone sulphate in the milk of dairy cows and its use in pregnancy diagnosis. J. Endocrinol. 95:7-12 (1982). -

20. Humblot, P., Jeanguyot, N., Hegde, G. and Thibier, M. Diagnostic tardif de gestation chez les bovins a partir d'echantiIlons de lait, plasma ou sang. Elev. Insem. 206:17-24 (1985).

21. Kobilinsky, A. Software for the treatment of the linear model on a mini computer. Procedures in Computational Statistics (Compstat 1980) Physica, Vienna, pp. 167-173 (1980).

22. SAS User's Guide: Statistics. SAS Inst., Cary, NC, (1985).

23. Heap, R-B., Haman, M. and Fleet, 1-R. Factors affecting oestrane sulphate concentrations in milk. Brit. Vet. J. 139:79-88 (1983).

24. Mohamed, A-R., Noakes, D.E., Booth, J.M. and Chaplin, V. Plasma oestrone sulphate and progesterone concentrations in cows and ewes associated with fetal death and abortion. Brit. Vet. J. 143:238-245 (1987).

25. Semambo, D.K.N., Eckersall, P.D., Sasser, R.G. and Ayliffe, T.R. Pregnancy specific protein B and progesterone in monitoring viability of the embryo in early pregnancy in the cow after experimental infection with Actinomyces pyogenes. Fertil. Abs. Series No. 5: p. 30 (1990).

J. Reprod.

26. Wooding, F.B.P. and Wathes, D.C. the bovine placentome.

Binucleate cell migration in J. Reprod. Fertil. 59:425-430 (1980). -

27. Hewitt, T-L., McCaughey, W.J. and Dobson, Hilary. Concentrations of oestrone sulphate and progesterone in cows' milk in association with an outbreak of Leptospira hardjo infection. Irish Vet. J. 3:96-98 (1990).