ELSEVIER

RELATIONSHIP BETWEEN MATERNAL PLASMA PROGESTERONE

CONCENTRATION AND INTERFERON-TAU SYNTHESIS BY THE CONCEPTUS IN
CATTLE

T.L. Kerbler,’ M.M. Buhr,’ L.T. Jordan,’ K.E. Leslie* and J.S. Walton’

Departments of ‘Animal and Poultry Science, ‘Veterinary Microbiology and Immunology, and
3Population Medicine, University of Guelph
Guelph, Ontario, Canada N 1G 2 W 1

Received for publication: September 11, 1995

Accepted: SepLember i9, i996

ABSTRACT

The objective of this study was to determine the relationship between maternal progesterone
concentration and conceptus synthesis of interferon-r as an index of conceptus viability at the time
of maternal recognition of pregnancy. Heifers of mixed beef breeds were randomly assigned to
receive 1 of 2 treatments: 1) intramuscular injection of 1500 IU hCG on Day 5 after artificial
insemination (AI; n=12) or 2) intramuscular injection of saline on Day 5 after AI (n=17). Ovaries
were scanned daily by transrectal real-time ultrasonography. Progesterone concentrations were
determined from daily blood samples collected l?om the jugular vein. Heifers were slaughtered on
Day 18 afler AI and conceptus tissues were collected. These were incubated individually at 37°C
in RPMI medium, and supernatant collected after 24 h. Conceptus secretory products in the
supematant were analyzed for interferon concentration by antiviral assay using vesicular stomatitis
virus.

Transrectal ultrasonography showed all heifers that received hCG had at least 1 extra corpus
luteum (CL) in addition to the spontaneous CL formed from the previous ovulation (10 with 2 CL,
2 with 3 CL). A significant increase in plasma progesterone concentration was detected in pregnant
heifers treated with hCG (n=9) vs pregnant control heifers (n=l 1; P < 0.001). There was a tendency
for an increase (P=O.O59) in synthesis of interferon-r by conceptuses horn hCG-treated heifers
compared to control heifers. Maternal plasma progesterone concentrations were correlated with
interferon-r production by the conceptuses (r = 0.593 P < 0.006), suggesting that higher maternal
progesterone may provide a more suitable environment for the developing conceptus.
0 1997 by Elsevw Scxnce Inc

Key words: human chorionic gonadotrophin, progesterone, pregnancy, interferon-z

Acknowledgments
This study was supported by the Natural Sciences and Engineering Research Council of Canada
and the Ontario Ministry of Agriculture, Food, and Rural Affairs. The authors are indebted to
Michelle Goodwin for technical assistance and to W. Szkotnicki for statistical support. Charlie
Watson and the staff at the Elora Beef Research Station are thanked for providing animal care
and assistance, and the Meat Technology Laboratory of the Department of Animal and Poultry
Science is gratefully acknowledged for slaughtering the animals and providing tissues.
Theriogenology 47:703-714. 1997 0093~691x/97/$17.00
0 1997 by Elsevier Science Inc. PII s0093-691x(97)00028-9
704 Theriogenology

INTRODUCTION

High rates of embryo mortality continue to be a major cause of reproductive failure in cattle
(25). It has already been established that progesterone is required to maintain a suitable uterine
environment for the healthy development of the conceptus (1829). A number of approaches
increasing progesterone levels have been studied. The use of progesterone-releasing intravaginal
devices (PRID; 42,45) has proven successll in increasing progesterone levels and pregnancy
rate. The PRIDs, however, are expensive, disturb vaginal microflora and drainage (4), and are
subject to societal concerns about the use of exogenous steroid hormones. Increasing
progesterone levels endogenously by the induction of accessory CL has proven to be a less
expensive and potentially more attractive alternative. An injection of 1000 to 1500 IU hCG 5 d
post breed& causes the ovulation of one or more large follicles leading to supplementary CL and
a consistent increase in progesterone concentrations over a sustained period (19,38,39,45).

Trophoblast protein-l, also known as interferon-r (IFN-z) (41), is a complex of

glycoproteins secreted in large quantities by the conceptus between Days 17 to 22 of gestation
(2,13,14) that act in an antiluteolytic manner to either inhibit or alter the pattern of endometrial
release of prostaglandin F-2a to remove its luteolytic effects (15,20,2 1). In addition, IFN-t also
possesses potent antiviral and anti-proliferative activities (36,37,40) which assist maintenance of
pregnancy. Since it has been shown that conceptuses at different stages of development produce
different amounts of IFN-z (14,22), the production of IFN-t, expressed as anti-viral activity,
can, therefore, be used as a measure of conceptus development and viability.

The primary objective of this research was to determine if increasing maternal plasma
progesterone concentrations via a single injection of hCG would provide a more suitable
environment for the development of the conceptus as demonstrated by an increase in IFN-r
synthesis at the time of maternal recognition of pregnancy.

MATERIALS AND METHODS

Animals

Twenty-nine crossbred beef heii housed at the Elora Beef Research Station were treated
with 2 intramuscular injections of cloprostenol(5OOpg; Estrumate, Cooper’s Agropharm Inc.,
Ajax, ON, Canada) 11 d apart, and were observed 3 d later for signs of estrus. The heifers were
artiiicially inseminated with previously frozen semen from a single ejaculate of a Shorthorn bull
(Strathmore’s Extra Edition, United Breeders Inc., Guelph, ON, Canada) 72 h a&r the last
cloprostenol injection.

Progesterone and Ovarian Ultrasonography

Beginning at the second cloprostenol injection, blood samples were collected daily by
jugular venipuncture into hep arinized tubes, and cooled on ice. Plasma was separated within 3
h and stored at -20°C for progesterone analysis. Plasma progesterone concentrations were
Theriogenology 705

determined using the solid phase radioimmunoassay (RIA) previously described by Robinson et
al. (42). The intra- and inter-assay coefficients of variation were 8.8 and 11So?, respectively.

Daily transrectal ultrasonography commenced 2 d following insemination. Ultrasound

examinations of the ovaries were performed using a linear array real-time ultrasound device
(Aloka Echo Camera Model SSD-210-DX, Aloka Co., Tokyo, Japan) equipped with a 5 MHZ
rectal probe (Aloka Probe Model LIST-58 13-5). The ovaries were scanned in several planes to
identify all follicles greater than 1 mm in diameter, and to provide an image of the CL with its
greatest cross sectional area. A hard copy was made of all appropriate images with a video
printer (Mitsubishi Video Copy Processor, Tokyo, Japan), and growth of follicles and CLs were
charted over time.

Conceptus Isolation and Culture

Eighteen days after insemination the heifers were slaughtered by captive bolt pistol and
exsanguination, and the reproductive organs immediately removed. The ovaries were removed,
and CLs dissected and diameters measured. The vagina was severed at the external OSof the
cervix and both uterine horns were simultaneously flushed with 35 ml PBS introduced into the
oviducts. The flushings were collected into petri dishes. Conceptuses were transferred
individually into petri dishes containing 15 ml RPMI medium 1640, with L-glutamine and without
L-leucine (Gibco BRL, Life Technologies Inc., Burlington, ON, Canada) supplemented with 2%
penicillin and streptomycin (G&co) and were incubated (Model 3860, Forma Scientific Inc..
Marietta, OH, USA) for 24 hat 37°C in 5% CO, / 95% air (35).

After culture, the medium was collected, divided into 5-ml aliquots and stored at -20°C
until analyzed for anti-viral activity using an interferon anti-viral assay by plaque reduction
adapted f?om Loewen and Derbyshire (27). Inoculum of culture supematant (0.4 ml) was placed
on 1-d-old monolayers of Madin-Darby bovine kidney celIs (MDBK)(VMI, OVC, Guelph, ON,
Canada) and incubated in 5% CO, / 95% air, at 37°C for 18 h. After this IFN pretreatment, the
plates were washed and 40 to 60 plaque-forming units of vesicular stomatitis virus (vsv) were
added to each well. The plates were incubated under the previous conditions for 1 h, then the vsv
was removed and the plates washed with PBS (0.1 M phosphate, 9% sodium chloride, 0.1%
sodium azide). One milhhtre of 0.9% tragacanth overlay (VMI, OVC, Guelph, ON, Canada) was
added to each plate and plates were incubated for 48 h. A 10% formalin solution in PBS was
used to fix each well, and crystal violet stain was used for plaque observation. Antiviral titre was
determined by counting plaques and recording the dilution at which there was at least a 50%
reduction in the number of plaques relative to the virus control wells. Antiviral titres were related
to a laboratory porcine leucocyte IFN standard and a human recombinant IFN-a,, (Roferon,
Hofhnan-La Roche, Mississauga ON, Canada). Each culture supematant was tested in duplicate
at 2 or more dilutions, with an intra-assay coefficient of variation of 15%.

Statistical Analysis

To determine the et&t of hCG treatment on progesterone concentrations, the progesterone

profiles were analysed by calculating the arca under the curve using the trapezoid rule. The areas
706 Theriogenology

were then subjected to the General Linear Model procedure of the Statistical Analysis System
(SAS; 43) to test for treatment efkcts. DiEerences in CL diameter were analyzed using Student’s
t-tests. The IFN-r data were normalized using a natural log transformation and analyzed using
the General Linear Model procedure of SAS to determine differences between treatments. The
regression procedure of SAS was used to determine the regression of IFN-r synthesis by
conceptuses with area under the maternal progesterone profiles.

RESULTS

Ovarian Changes After Treatment

Injection of hCG on Day 5 induced ovulation of the large dominant follicle which grew after
insemination, as evidenced by the diippearance of the follicle, followed by the appearance of an
additional CL observed by ultrasound on Days 10 to 11, and present at slaughter on Day 18 in
all treated cows.

Ten of the 12 heiters treated with hCG had 2 CL, with the remaining 2 heifers treated with
hCG having 3 CL (Table 1). Measurements using real-time ultrasonographic images detected a
difference (PcO.05) in CL diameter Gram Day 10 until slaughter between the induced CL and the
spontaneous CL in pregnant hCG-treated heifers (Figure 1). The diameters of the spontaneous
CL in hCG-treated heifers and control heifers were similar from Day 10 until slaughter.
Measurements taken on Day 18 confirmed differences between the induced CL (2.4 * 0.15 cm)
and the spontaneous CL (2.7 f 0.13 cm) in pregnant hCG-treated heifers. The dieters of the
spontaneous CL of the hCG-treated (2.7 f 0.13 cm) and the control heifers (2.4 f 0.08 cm) were
Shlih.

Table 1. Pregnancy rates and luteal status of heifers on Day 18 a&r treatment with 1500 IU hCG
or saline on Day 5 after insemination

Observations Treatment
hCG Saline

Pregnant 9 (75%) 11 (64.7%)

Non Pregnant 3 (25%) 6 (35.3%)

Mean no. of CL 2.2 + 0.11 1

No. with 1 CL 0 # Pregnant 17
No. with 2 CL 10 7 (70%) 0
No. contralateral 4 3 (75%)
No. ipsilateral 6 4 (67%)
No. with 3 CL 2 2 (100%)
Theriogenology 707

3-

2.5 -

2-

1.5 -

********
l-
*p<o.o5
I

0 I I I I I I I I I I I , I I I, I

0 2 4 6 8 10 12 14 16
Day of Estrous Cycle

Figure 1. Mean CL diameter (cm) for pregnant heifers treated with 1500 IU hCG (n=9) or saline
(n=l 1) on Day 5. *Induced CL (hCG[I]) di&rs t?om spontaneous CL (hCG[S]).

Plasma Progesterone

Plasma progesterone concentrations were simihtr from Day 1 to Day 5 (Figure 2), and there
was a significant increase in heifers treated with hCG compared with that of controls (P<O.OOl) by
Day 8. This increase in plasma progesterone concentration was maintained through Days 16 and
17 (Figure 2) with a maximum of 15.5 f 0.25 ng/ml and 10.5 f 0.25 ng/ml on Day 17 for hCG-
treated heifers and control heifers, respectively.

Pregnancy

Pregnancy rates of 75% (9/12) for heii treated with hCG and 64.7% (1 l/l 7) for the control
heifers were observed. There was a similar distribution of contralateral (4) and ipsiIateraI (6)
accessory CL in the hCG-treated heifers, with pregnancy rates of 75 and 67%, respectively (Table
1).
 Theriogenology

20

15
t

1.
10

0
12 3 4 5 6 7 8 g 10 11 12 13 14 15 16 17

Day After Insemination

Figure 2. Plasma progesterone profiles fbr pregnant heiibrs treated with 1500IU hCG (n=9) or saline
(n=ll) on Day 5. *Difkrs from control (P<O.OOl).

Conceptus Culture

All conceptuses produced ink&-o&r. There was a tendency for an increase (P=O.O59; Figure
3) in synthesis of interferon-r by conceptuses from heifers treated with hCG. When the dam were
lez&yz&withoutregardto tmatmem the correlation(r) between interkron-z synthesis and maternal
plasma progesterone concentrations was 0.593 (PcO.006; Figure 4).

DISCUSSION

This study cordirmed that the fkst dominant follicle of the cycle can be readily forced to ovulate
by treatment with hCG (38,39,45). In this study, hCG treatment caused the formation of 1, and
occasionally 2, accessory CL and increases in circulating concentrations of progesterone. This
increase in progesterone concentration was detected by Day 8 and was sustained through Day 18,
extendiig over the period of maternal recognition of pregnancy .

The near equal diibution of contralateral (4) and ipsilateral(6) accessory CL was also
reported by Price and Webb (38), who de&m&xl that the existing CL has no sign&ant efkct upon
the orientation or distribution of follicles capable of responding to hCG. This observation is also
consistent with the fkquency of bilateral and unilateral double ovulations observed in a herd of cattle
selected for high twinning rate (3 1).
Theriogenology 709

16 I

...............
.......
..........
I:.:::::.-‘-

Treatment

Figure 3. l[nte&ron Tau synthesized after 24 hour culture of Day 18 bovine conceptuses from
heikrs treated with hCG (1500 IU; n=9) or saline (sl 1) on Day 5 after insemination.

ril i i

A *

;5- A
=
r= 0.593
4.8 -
A P < 0.006
-+
0 $ I I I I I I i
0 a0 100 120 140 160 180 200

Area Under Progesterone Curve Day 5 To 18

(arbitrary units)

Figure 4. Positive correlation between maternal plasma progesterone concentration and interferon
Tau synthesis by Day 18 conceptuses (n=20) after 24-hour culture.
710 Theriogenology

The development of the accessory CL was similar to that of the spontaneous CL (10). This
induced luteal structure appears to develop normally, since no dh%mnces have been detected between
the CL of hCG-treated cows and control cows in luteal weights, DNA, RNA or protein
concentrations (10). Price and Webb (38) found the hCG-induced CL to be smaller than the
spontaneous CL, attributing the size difference to the difference in the ages of the CL. In contrast,
Rajamahendran and Sianangama (39) found treatment with hCG leads to significant increases in total
diameter of the CL, with the induced CL becoming difficult to distinguish from the spontaneous CL.
The induced CL in this study , however, grew to a smaller diameter than the spontaneous CL
confirming the work of Price and Webb (38). The reduced size of the induced CL could be due to
a smaller follicle being induced to ovulate by hCG. With normal ovulation, the preovulatory follicle
has 2 or 3 additional days for growth as it progresses to ovulation. According to ultrasound
observations (not shown), the dominant follicle was still increasing in size at the time of hCG
injection. This was followed by a sudden disappearance of the follicle, followed 2 to 3 days later by
the appearance of a rapidly growing but smaller CL at the same location.

A significant increase in plasma progesterone concentrations occurred between Days 8 and 20

post insemination after hCG treatment on Day 5. The effect of hCG was not apparent until around
Day 8 post estrus, by which time accessory CL are formed and produce supplementary progesterone
(10,45). In this experiment the accessory CL were not evident by ultrasonography until Day 10 to 11,
but increased progesterone concentrations were detectable by Day 8. The latter may have been due
to an effect of hCG on the spontaneous CL. Since, ovulation occurs 20 to 25 h after the LH surge
(6), it does not seem likely that the increase in progesterone concentration detected by Day 8 is the
result of the luteal structure formed by hCG administration on Day 5. It has been reported that
increases in plasma progesterone (8,11,26) after hCG administration were due to a direct effect of
hCG on hypertrophy (8) and or to increased blood flow to the CL (34). Farin et al. (8) found that
treatment with hCG can also alter the population of large and small luteal cells, since small luteal cells
may differentiate into large luteal cells. As luteinization of both CL continues, progesterone
production by the small luteal cells is responsive to LH (17,23); any diirentiation of small to large
luteal cells plus the additional large cell population in the induced CL leads to larger amounts of
progesterone being secreted by LH-independent mechanisms (48) and the increased plasma
progesterone concentrations observed.
Interferon-r synthesis tended to be greater in conceptuses from heifers treated with hCG
(P<O.O59). At this level of replication, an increased probability would be expected. Although the
antiviral assay used was very sensitive, requiring serial dilutions, the variation among conceptuses was
quite large. To add rigor to the statistical approach, regression analysis was used to determine
whether a correlation between the area under the progesterone profiles and interferon-r synthesis
could be found. A strong relationship was obtained between progesterone concentration and IFN-r
synthesis by the conceptus when data for both treatments were combined. This positive correlation
between progesterone concentration and synthesis of interferon-r by the conceptus would suggest
that increasing progesterone concentrations in the cow is beneficial to embryonic development.

A delay in conceptus development can result in embryonic loss (30,33,46). This embryonic loss
may be due to an inability of the conceptus to signal its presence to the maternal system in time to
prevent luteolysis. It has also been demonstrated that a uterine environment advanced by treatment
with exogenous progesterone accelerated conceptus development (46,47,49). In ewes, administration
Theriogenology 711

of progesterone advanced uterine secretions so that a Day 6 uterine environment was suitable for
development of a Day 10 embryo (26,44). Exogenous progesterone also induces similar
advancements in uterine environment and conceptus development in cattle (12). In the current study,
interferon-z measurement was on a conceptus basis and it is uncertain whether the effect of
progesterone was due to increased interferon-r synthesis or advanced conceptus development.
Conceptuses were cultured immediately to avoid compromising their viability. There were, however,
no obvious differences between treatments in the size or development of the conceptuses which
would suggest the effect is on synthetic ability.

Nephew et al. (32) recently also found conceptuses fiorn ewes treated with hCG secreted more
IFN-r than conceptuses from control ewes. A precedent for this relationship between progesterone
concentration and synthesis of interferon-r by the conceptus has also been provided by observations
that pregnant heifers have higher progesterone concentrations than non-pregnant heifers during early
pregnancy (5,7,16,28). This may indicate the importance of progesterone to conceptus survival or
vice versa. The amount of interferon-r synthesized by the conceptus is influenced by the development
status of the embryo, and may be used as an objective indicator of embryo quality since it provides
a direct measure of trophoblast biosynthetic activity (1,22).

It is also possible that hCG may have a direct influence on endometrial function and, therefore,
indirectly on conceptus development. The recent observation of the presence of endometrial
receptors for hCG/LH in the bovine endometrium early in the estrous cycle (9) indicates that such a
possibility cannot be excluded. Nevertheless, the strengthened relationship resulting from the
association of progesterone with interferon-z without concern for hCG treatment may indicate that
the indirect effect through progesterone is more important than any hCG effects per se.

In this and other studies (39,45), the effect of hCG treatment on pregnancy rates has generahy
been equivocal. There is generally a small increase in pregnancy rate after hCG, but this is not
signilicant perhaps because of inadequate sample sizes, The data from this study are encouraging in
demonstrating that maternal plasma progesterone concentrations are strongly associated with the
abii of the conceptus to synthesize a secretory product known to be important for the recognition
of pregnancy. Further studies will be necessary to determine if these observations are consistent with
the use of hCG treatment to increase pregnancy rates.

In conclusion, these results show that the large early luteal-phase follicle is capable of
responding to hCG to produce an accessory CL, which allows for an endogenous increase in plasma
progesterone concentration in the cow. This increase in progesterone concentration would appear
to be beneficial to the development of the conceptus as seen by the strong correlation between
progesterone concentration and interferon-s synthesis by the conceptus.

REFERENCES

1. Ashworth CJ, Bazer FW. Changes in ovine conceptus and endometrial function following
asynchronous embryo transfer or administration of progesterone. Biol Reprod 1989;40: 425
434.
712 Theriogenology

2. Bartol FF, Roberts RM, Bazer FW, Lewis GS, Godkin JD, Thatcher WW. Characterization
of proteins produced in vitro by periattachment bovine conceptuses. Biol Reprod 1985;32:681-
693.
3. Bazer FW. Mediators of maternal recognition of pregnancy in mammals. Proc Sot Exp Biol
Med 1992;199:373-384.
4. Bulman DC, M&&bin PE, Appleyard WT, Lamming GE. Effect of a progesterone-releasing
intravaginal device on the milk progesterone levels, vaginal flora, milk yield and fertility of
cyclic and noncyclic dairy cows. J Reprod Fertil1976;53:289-296.
5. Bulman DC, Lamming GE. Milk progesterone levels in relation to conception, repeat breeding
and influencing acyclicity in dairy cow. J Reprod Fertil 1978;54:447-458.
6. Chenault JR, Thatcher WW, Kalra PS, Abrams RM, Wilcox CJ. Transitory changes in plasma
progestins, estradiol, and luteinizing hormone approaching ovulation in the bovine. J Dairy Sci
1975;58:709-717.
7. Erb RE, Garverick HA, Randel RD, Brown BL, Callahan CJ. Profiles of reproductive
hormones associated with fertile and nonfertile inseminations of dairy cows. Theriogenology
1976;5:227-242.
8. Farin CE, Moeller CL, Mayan H, Gamboni F, Sawyer I-IR, Niswender GD. Effect of
luteinizing hormone and human chorionic gonadotrophin on cell populations in the ovine
corpus luteum. Biol Reprod 1988;38:413-421.
9. Freidman S, Gurevich M, Shemesh M. Bovine cyclic endometrium contains high aI%@
luteinizing hormone&nnan chorionic gonadotrophin binding sites. Biol Reprod 1995;52: 1020-
1026.
10. Fricke PM Reynolds LP, Redmer DA. E&ct of human chorionic gonadotrophin administered
early in the estrous cycle on ovulation and subsequent luteal function in cows. J Anim Sci
1993;71:1242-1246.
11. Gamboni F, Fitz TA, Hazer PB, Wise ME, Mayan MH, Niswender GD. Effect of human
chorionic gonadotrophin on induced ovine corpora lutea during the anestrous season. Dom
Anim Endocrinol 1984;1:79-88.
12. Garrett JE, Geisert RD, Zavy MT, Morgan GL. Evidence for maternal regulation of early
conceptus growth and development in the bovine. J Reprod Fertil1988;84:437-446.
13. Geisert RD, Zavy MT, Biggers BG, Garret JE, Wettemann RP. Characterization of the uterine
environment during early conceptus expansion in the bovine. Anim Reprod Sci 1988;16: 1l-25.
14. Godkin JD, Lifsey BJ, Gillespie BE. Characterization of bovine conceptus proteins produced
during the peri- and postattachement periods of early pregnancy. Biol Reprod 1988;38:703-
712.
15. Gross TS, Plante C, Thatcher WW, Hansen PJ, Helmer SD, Putney DJ. Secretory proteins of
the bovine conceptus alter endometrial prostaglanclm and protein secretion in vitro. Biol
Reprod 1988;39:977-987.
16. Harper HJ, Walton JS. Plasma concentrations of progesterone and luteinizing hormone during
the estrous cycle and early pregnancy in Holstein heifers. Proc 10th Int Congr Anim Reprod
ArtifInsem 1984;2:87.
17. Harrison LM, Kenny N, Niswender GD. Progesterone production, LH receptors, and oxytocin
secretion by ovine luteal cells on Days 6, 10 and 15 of the estrous cycle and Day 25 of
pregnancy. J Reprod Fertil 1987;79: 539-548.
Theriogenology 713

18. Heap RB. Role of hormones in pregnancy. In: Austin CR, Short RV (ed), Reproduction in
Mammals. Book 3, Hormones in Reproduction. Cambridge: University Press, 1972;73- 105.
19. Hehner SD, Britt JH. Fertility of dairy cattle treated with human chorionic gonadotrophin
(hCG) to stimulate progesterone secretion. Theriogenology 1986;26:683-695.
20. Helmer SD, Gross TS, Newton GR, Hansen PJ. Thatcher WW. Bovine trophoblast protein- 1
complex alters endometrial protein and prostaglandin secretion and induces an intracellular
inhibitor of prostaglandm synthesis in vitro. J Reprod Fertil1989;87:421-430.
21. Helmer SD, Hansen PJ, Thatcher WW, Johnson JW, Baser FW. Intrauterine infusion of highly
enriched bovine trophoblast protein-l complex exerts an antiluteolytic eITect to extend corpus
luteum lifespan in cyclic cattle. J Reprod Fertil 1989:87:89-101.
22. Hernandez-Ledezma JJ, Mathialagan M, Villanueva C, Sikes JD, Roberts RM. Expression of
bovine trophoblast interferons by in vitro-derived blastocysts is correlated with their
morphological quality and stage of development. Mol Reprod Dev 1993;36: l-6.
23. Hoyer PB, Niswender GD. The regulation of steroidogenesis in the two types of ovine luteal
cells. Can J Physiol Pharmacol 1985;63:240-248.
24. Kittok RJ, Stellllug JN, Lowry SR. Enhanced progesterone and pregnancy rate tier
gonadotrophin administration in lactating ewes. J Anim Sci 1983;56:652-655.
25. Lamming GE, Darwash AO, Back HL. Corpus luteum function in dairy cows and embryo
mortality. J Reprod Fertil 1989;37(Suppl):245-252.
26. Lawson RAS, Cahill LP, Modification of the embryo maternal relationship in ewes by
progesterone treatment early in the oestrous cycle. J Reprod Fertil 1983;67: 473-475.
27. Loewen KG, Derbyshire JB. Interferon induction with polyinosinic: polycytidylic acid in the
newborn piglet. Can J Vet Res 1986;50:232-237.
28. Lukaszewska J, Hansel W. Corpus luteum maintenance during early pregnancy in the cow.
J Reprod Fertil 1980;59:485-493.
29. McDonald LE, McNutt SH, Nichols RE. On the essentiality of the bovine corpus luteum of
pregnancy. Am J Vet Res 1953;14:539-541.
30. Moore NW, Shelton JN. Egg transfer in sheep. Effect of degree of synchronization between
donor and recipient, age of egg and site of transfer on survival of transferred eggs. J Reprod
Fertil 1964;7:145-152.
31. Morris CA, Day AM, Peterson AJ. Effects of ovulation status and stage of oestrous cycle on
plasma progesterone concentrations in cattle with or without a history of twin calvings. Anim
Prod 1987;45:205-209.
32. Nephew KP, Cardenas H, McClure KE, Ott TL, Bazer FW, Pope WF. Effects of
*.
admnmtmtion of human chorionic gonadotrophin or progesterone before maternal recognition
of pregnancy on blastocyst development and pregnancy in sheep. J Anim Sci 1993;72:453-458.
33. Newcomb R, Rowson LEA. Conception rate after transfer of cow eggs in relation to
synchronization of oestrus and age of egg. J Reprod Fertil1975;43:539-541.
34. Niswender GD, Reimers TJ, Diekman MA, Nett TM. Blood flow: a mediation of ovarian
function. Biol Reprod 1976;14:64-81.35.
35. Plante C, Hansen PJ, Thatcher WW, Johnson JW, Pollard JW, Mirando MA, Bazer FW.
Purification of bovine trophoblast protein-l complex and quantitication of its
microheterogenous variants as a&ted by culture conditions. J Reprod Immunol 1990;18:27 l-
291.
714 Theriogenology

36. Pontzer CH, Bazer FW Johnson HM. Antiproliferative activity of a pregnancy recognition
hormone, ovine trophoblast protein-l. Cancer Res 1991;51:5304-5307.
37. Pontzer CH, Tortes BT, Vallet JL, Bazer FW, Johnson HM. Antiviral activity of the
pregnancy recognition hormone ovine trophoblast protein-l. Biochem Biophys Res Commun
1988;152:801-807.
38. Price CA, Webb R. Ovarian response to hCG treatment during the oestrous cycle in heifers.
J Reprod Fertil 1989;86:303-308.
39. Rajamahendran R, Sianangama PC. Effect of human chorionic gonadotrophin on dominant
follicles in cows: Formation of accessory corpora lutea, progesterone production and
pregnancy rates. J Reprod Fertil 1992;95:577-584.
40. Roberts RM. A novel group of interlisrons associated with the early ovine and bovine embryo.
J Interferon Res 1989;9: 175-183.
41. Roberts RM, Cross JC, Leaman DW. Interferons as hormones of pregnancy. Endocrine
Reviews 1992;13 1432-452.
42. Robinson NA, Leslie KE, Walton JS. E&ct of treatment with progesterone on pregnancy rate
and plasma concentrations of progesterone in Holstein cows. J Dairy Sci 1989;72:202-207.
43. SAS. System for elementary statistical analysis. Gary NC: SAS Institute Inc. 1987.
44. Vincent DL, Meredith S, Inskeep EK. Advancement of uterine secretion of prostaglandin E,
by treatment with progesterone and transfer of asynchronous embryos. Endocrinology
1986;119:527-529.
45. Walton JS, Halbert GW, Robion NA, Leslie KE. Effects of progesterone and human
chorionic gonadotrophin administration five days post insemination on plasma and milk
concentrations of progesterone and pregnancy rates of normal and repeat breeder dairy cows.
Can J Vet Res 1990;54:305-308.
46. Wilmut I, Sales DI. Effect of an asynchronous environment on embryonic development in
sheep. J Reprod Fertll 198 1;6 1: 179- 184.
47. Wilmut I, Sales DI, Ashworth CJ. The influence of variation in embryo state and maternal
hormone profiles on embryo survival in farm animals. Theriogenology 1985;23: 107- 119.
48. Wiltbank MC, Niswender GD. Functional aspects of differentiation and degeneration of the
steroidogenic cells of the corpus luteum in domestic ruminants. Anim Reprod Sci
1992;28:103-110.
49. Wiitenberger-Torres S, Rubin L, Gerard M, Solari A, Cornu C. Effect of progesterone and
ovarian steroids on egg segmentation in the ewe. Ann Biol Anim Biochem Biophys
1967:7:391-406.