Anda di halaman 1dari 10

Animal Reproduction Science 72 (2002) 73–82

Fetal growth and reproductive performance in ewes


administered GnRH agonist on day 12 post-mating
Mehmet Akif Cam a,∗ , Mehmet Kuran a ,
Sedat Yildiz b , Erdogan Selcuk a
a Ondokuz Mayis Universitesi, Ziraat Fakultesi, Kurupelit, 55139 Samsun, Turkey
b Kafkas Universitesi, Veteriner Fakultesi, Kars, Turkey

Received 8 October 2001; received in revised form 8 February 2002; accepted 3 April 2002

Abstract
Reproductive performance and fetal growth was determined in GnRH (4 ␮g synthetic GnRH
agonist, Receptal) administered (i.m.) to ewes on day 12 post-mating (n = 103) compared to
control ewes (n = 97) during the breeding season. Plasma progesterone and LH concentrations
were analyzed. A total of 13 ewes was slaughtered on day 45 of pregnancy (six from control,
seven from GnRH treated groups). GnRH administration on day 12 post-mating increased plasma
progesterone concentration (4.39 ± 0.25 ng/ml) compared to control group (3.43 ± 0.15 ng/ml) on
days 13–15 post-mating (P < 0.01). GnRH administration also increased plasma LH concentration
between 1 and 4 h after GnRH administration (P < 0.01). Pregnancy rate was higher in GnRH
treated group (84%) than control (66%) group (P < 0.05). The ewes in GnRH administered group
had more twins (P < 0.05) than those in control group. The ovarian weights (P < 0.05) and the
number of corpora lutea (CL) (P < 0.01) were greater in ewes slaughtered on day 45 of pregnancy
in GnRH treated group than those in control group. GnRH administration on day 12 post-mating did
not have any effect on products of conception at day 45 of pregnancy except on crown-rump length
(CRL) of fetuses and cotyledon weight. CRL of fetuses and cotyledon weight in GnRH treated group
was higher than those in control group (P < 0.05). In conclusion GnRH administration improved
reproductive performance of ewes when administered on day 12 post-mating probably through its
beneficial effect on embryo survival by enhancing luteal function, but not through stimulating fetal
growth. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Sheep-endocrinology; GnRH; Reproductive performance; Fetal growth

∗ Corresponding author. Tel.: +90-362-4576020x1364; fax: +90-362-4576034.

E-mail address: makifcam@omu.edu.tr (M.A. Cam).

0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 ( 0 2 ) 0 0 0 7 1 - 4
74 M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82

1. Introduction

Successful development and survival of an embryo depends on an integrated sequence


of biological events involving the ovary, embryo, oviduct and uterus. A perturbation of the
system can lead to reduced embryo survival (Thatcher et al., 1994). Preimplantation embry-
onic loss is the major limiting factor for obtaining an optimum reproductive performance
in farm animals. In sheep, during the first 3 weeks of pregnancy, 30–40% of fertilized eggs
are lost (Bolet, 1986; Nancarrow, 1994; Michels et al., 1998). Of this total loss, 70–80%
occurs between days 8 and 16 after insemination (Sreenan et al., 1996).
One of the major causes of embryonic loss is thought to be the inadequate luteal func-
tion (Wilmut et al., 1986; Ashworth et al., 1989; Nancarrow, 1994). In attempts to reduce
embryonic mortality and hence to improve reproductive performance, progesterone sup-
plementation during early pregnancy has been employed in sheep and cattle (reviewed by
Thatcher et al., 1994; Sreenan et al., 1996). There are studies showing that progesterone
supplementation after breeding improves not only pregnancy rate (McMillan et al., 1986;
Davies and Beck, 1992) but also stimulates subsequent fetal growth (Garrett et al., 1988;
Kleemann et al., 1994). Progesterone supplementation after breeding, to compensate for
possible luteal insufficiency or to stimulate embryo development in order to amplify the em-
bryonic signal, has beneficial effect but with variation between studies (Sreenan et al., 1996).
Another approach in reducing embryonic loss during early pregnancy has been the admin-
istration of hCG or GnRH which results in a significant increase in systemic progesterone
(Sreenan et al., 1996). Much of this rise in progesterone is due to the induction of accessory
corpora lutea (CL) (Mann and Picton, 1995; Beck et al., 1996). There are conflicting reports
on the effect of GnRH on pregnancy rate in cattle. GnRH treatment post-mating in some
studies has been shown to increase pregnancy rate (Macmillan et al., 1986; Sheldon and
Dobson, 1993; Drew and Peters, 1994) while some studies have failed to demonstrate any
beneficial effect (Jubb et al., 1990; Ryan et al., 1994). Administration of GnRH on days 10,
11, 12 or 13 post-mating has been shown to improve early embryo survival (Beck et al.,
1994) and pregnancy rate (McMillan et al., 1986) in sheep.
GnRH administration on day 12 post-mating may stimulate subsequent fetal growth in
uterus through its effect on progesterone production which is stimulatory for fetal growth.
Therefore, the objective of the present study was to investigate the effects of GnRH ad-
ministration on day 12 post-mating on subsequent fetal growth caused by enhanced luteal
function and on the reproductive performance of ewes.

2. Material and methods

A total of 200 ewes (149 Karayaka and 51 Karayaka × Sakiz F2 crossbreeds) between 2
and 6 years were used in the experiment that was conducted at Ondokuz Mayis University,
Samsun field station (4◦ 35 N) during the breeding season. The individually identifiable
ewes in natural estrous were randomly allocated into two treatment groups. Both groups
were mated during September to nine fertile (six Karayaka and three Karayaka × Sakiz
F2 crossbreeds) rams. Estrous was detected by teaser rams. Ewes in estrous were mated
naturally. On day 12 post-mating (day of estrous = day 0), the ewes in one group were
M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82 75

given an i.m. injection of 4 ␮g of synthetic GnRH agonist (Receptal, Topkim, Istanbul,


Turkey) and the other group was left as control. Ewes returning to service were determined
using a teaser ram to calculate non-return rate. The ewes were maintained on pasture and
supplemented with hay and concentrates during pregnancy. At lambing, the date of birth,
number and weight of each lamb were recorded.
Blood samples were collected into heparinized vacutainer tubes by jugular venipuncture
from days 12 to 15 post-mating (prior to and 1–3 days after GnRH administration). Plasma
was isolated and stored (−20 ◦ C) until analyzed. Plasma progesterone concentrations of ran-
domly chosen six ewes that lambed from each treatment group were determined by the meth-
ods of Prakash et al. (1987) by enzyme immunoassay. Briefly, in duplicate, 10 ␮l unknown
plasma samples were analyzed against standards ranging from 0.13 to 16 ␮g progesterone/l.
The sensitivity of the assay was 0.20 ␮g/l and the intra- and inter-assay coefficients of vari-
ation were 8.4 and 15.0%, respectively. Plasma collected from ewes prior to and 1, 4, 8 and
12 h after GnRH administration and from control ewes were analyzed for LH concentrations.
A sensitive competitive enzyme immunoassay method developed by Mutayoba et al. (1990)
was used to analyze ovine LH. d-Biotinyl-ε-aminocaproic acid N-hydroxy-succimidine es-
ter (Biotin-X-NHS, Sigma–Aldrich, Taufkirchen, Germany) was used for labeling oLH
(NIDDK-oLH-I-4 (AFP-8614B)). Affinity purified goat IgG anti-rabbit IgG was attached
to the solid phase and labeled and non-labeled (sample) oLH were competed against the
anti-oLH raised in rabbit (NIDDK-anti-oLH-1 (AFP-192279)). A two-dimensional titer
determination for the optimum dilution of biotinyl-LH and the antiserum was carried out
and optimum dilutions were found to be 1:5000 for biotinyl-LH and 1:3200000 for oLH
antiserum. In duplicate, 20 ␮l unknown plasma samples or standards ranging from 0.78 to
50 ␮g oLH/l plus 100 ␮l diluted antibody 1:3200 in the assay buffer were pipetted into all
wells except the NSB (non-specific binding) tubes. Plates were incubated at 4 ◦ C for 24 h
with gentle agitation followed by decantation and addition of 100 ␮l biotinyl oLH (2 ␮l
biotinyl oLH/10 ml assay). Plates were further incubated at 4 ◦ C for 2 h, decanted, 20 ng
streptavidin-peroxidase in 100 ␮l assay buffer was added and incubated at 4 ◦ C for 15 min.
Plates were decanted, washed three times with Tween 80 and 150 ␮l substrate was added into
the wells (substrate buffer: 100 mmol/l CH3 COONa, pH 5.5, with citric acid; substrate solu-
tion: 25 ml substrate buffer plus 100 ␮l 1% H2 O2 plus 0.6% 3,3 ,5,5 -tetramethylbenzidine
in dimethyl sulfoxide). The plates were incubated at 22 ◦ C for 40 min and the reaction was
stopped by the addition of 50 ␮l 2 mol/l H2 SO4 . The color was measured at 450 nm with
an eight-channel microtitration plate photometer (Tecan, Grödig, Austria) and results were
evaluated using EasyWin Kinetics software supplied by Tecan. Intra- and inter-assay coef-
ficients of variation were 9.2 and 16.7%, respectively. The sensitivity limit was 0.95 ng/ml.
A total of 13 ewes was slaughtered on day 45 of pregnancy (six from control group, seven
from GnRH treated group). The number of CL was recorded post-slaughter, and ovarian
and individual CL weights were determined after isolation using scissors and forceps.
Curved crown-rump length (CRL) and weights of gravid uterus, fetuses, chorioallantois,
caruncle and cotyledon were determined. Placental weight was calculated using the weights
of caruncle and cotyledon. Weight of fetal fluid was calculated from gravid uterus, fetus,
chorioallantois, uterus and caruncle weights. The number of fetuses were also recorded
post-slaughter. Plasma progesterone concentrations in blood samples collected at slaughter
were determined as explained earlier.
76 M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82

Differences between treatment groups were analyzed with χ 2 -analysis for non-return rate,
pregnancy rate and twinnings, and analysis of variance for gestation length, birth weight,
ovarian weight, numbers and weights of CL, products of conception, plasma progesterone
and LH concentrations. Data for plasma progesterone and LH concentrations were log10
transformed prior to statistical analysis and untransformed means (±S.E.M.) are presented.

3. Results

3.1. Plasma hormone concentrations

Plasma progesterone concentrations of ewes in control and GnRH treatment groups are
presented in Fig. 1. GnRH administration on day 12 post-mating increased plasma proges-
terone concentration (4.39 ± 0.25 ng/ml) compared to control group (3.43 ± 0.15 ng/ml)
on days 13–15 post-mating (P < 0.01). GnRH administration increased plasma LH con-
centration compared to control group (P < 0.01). Plasma LH concentrations (Fig. 2) were
reached a peak level at about 1–4 h after GnRH administration (P < 0.01).

3.2. Lambing performance

The number of ewes returning to estrous, non-return rate and number of lambing in control
and GnRH treatment (on day 12 post-mating) groups are presented in Table 1. There was
a tendency for a higher non-return rate and a lower number of ewes returned to estrous in
the GnRH treated ewes, but the difference between groups was not significant (P > 0.05).
GnRH treatment on day 12 post-mating increased lambing rate when compared to control
group (P < 0.05).
The number of ewes lambing to first estrous, number of lambs born and litter size
for ewes in control and GnRH treatment groups are presented in Table 2. The ewes in
GnRH administered group had more twins (P < 0.05) and consequently they had a higher

Fig. 1. Plasma progesterone concentration of ewes in control (䊉) and GnRH (䉱) treatment (on day 12 post-mating)
groups.
M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82 77

Fig. 2. Plasma LH concentration (ng/ml) of ewes in control (䊉) and GnRH (䉱) treatment (on day 12 post-mating)
groups.

Table 1
Number of ewes returning to estrous, non-return rate and number of lambing in control and GnRH treatment (on
day 12 post-mating) groups
No. of ewes Non-return rate No. of lambing (%)
Mated/treated Returning to estrous

Control
97 17 0.82 64 (66.0) a
GnRH
103 12 0.88 84 (81.6) b
Values within column with different letters differ significantly (P < 0.05).

total number of lambs born and a larger litter size than those in control group. There was
no difference between control and GnRH administered ewes in terms of gestation length
(Table 3). GnRH administration on day 12 post-mating did not affect lamb birth weight
(P > 0.05).

Table 2
Number of ewes lambing to first estrous and litter size for ewes in control and GnRH treatment (on day 12
post-mating) groups
No. of lambing No. of lambs Total lambs Litter size

Singles Twins Triplets

Control
59 45 28 a 0 73 1.24
GnRH
77 46 60 b 3 109 1.42
Values within column with different letters differ significantly (P < 0.05).
78 M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82

Table 3
Mean (±S.E.M.) gestation length and birth weight of ewes in control and GnRH treatment groups
Gestation length (days) Birth weight (kg)

Singles Twins Singles Twins

Control
149.0 ± 0.4 148.4 ± 0.5 3.94 ± 0.08 3.87 ± 0.10
GnRH
148.1 ± 0.8 148.7 ± 0.4 3.98 ± 0.10 3.66 ± 0.08

Table 4
Number of viable fetuses and litter size distribution of ewes at slaughter on day 45 post-mating from control and
GnRH administered group
n No. ewes pregnant No. of viable fetuses Total no. of fetuses Litter size

Singles Twins Triplets

Control
6 5 4 2a 0 6 1.20
GnRH
7 7 2 8b 3 13 1.86
Values within column with different letters differ significantly (P < 0.05).

3.3. Litter size, ovarian characteristics and products of conception at slaughter on day 45

The number of viable fetuses and litter size distribution in ewes slaughtered on day 45
post-mating from control and GnRH administered groups are presented in Table 4. A total
of 13 ewes were slaughtered on day 45 post-mating. One ewe was not pregnant at slaughter
in control group and all ewes were pregnant in GnRH administered group. More ewes had
twins in GnRH administered group (P < 0.05) and consequently they had a higher total
number of viable fetuses and a larger litter size than those in control group.
Total ovarian weight was higher (P < 0.05) in GnRH administered ewes on day 45
post-mating than those in control group (Table 5). The number of CL was 2.6 times higher
in GnRH administered ewes than those in control group (P < 0.01), but the weights of
individual CL were greater in control group than GnRH administered ewes (P < 0.01).

Table 5
The weights of ovarian and CL, the number of CL, and plasma progesterone concentration at day 45 of pregnancy
in control and GnRH administered ewes
Ovarian weight (g) No. of CL Plasma progesterone (ng/ml) CL weight (g)

Control
2.27 ± 0.46 a 1.33 ± 0.21 c 4.52 ± 0.41 0.67 ± 0.05 c
GnRH
3.81 ± 0.15 b 3.50 ± 0.20 d 6.33 ± 0.75 0.46 ± 0.03 d
Values within columns with different letters differ significantly (a, b; P < 0.05) (c, d; P < 0.01).
M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82 79

Table 6
Means (±S.E.M.) for products of conception at day 45 of pregnancy for fetuses from control or GnRH administered
ewes on day 12 post-mating
Control GnRH

Gravid uterus (g) 312.2 ± 70.1 445.1 ± 60.5


Fetal weight (g) 6.90 ± 1.34 8.71 ± 0.37
Fetal fluids (g) 113.4 ± 38.4 197.2 ± 37.0
Chorioallantois (g) 66.9 ± 17.3 87.2 ± 12.5
Fetal CRL (mm) 64.7 ± 5.1 a 73.1 ± 0.8 b
Total caruncles weight (g) 14.8 ± 4.0 19.1 ± 1.8
Total cotyledon weight (g) 15.0 ± 4.0 a 31.8 ± 4.6 b
Placental weight (g) 29.8 ± 7.6 50.9 ± 6.1
Values within rows with different letters differ significantly (P < 0.05).

There was tendency for a higher plasma progesterone concentration on day 45 post-mating
in ewes administered GnRH on day 12 post-mating compared to those in control group but
the difference between groups was not significant.
GnRH administration on day 12 post-mating did not have any effect on products of
conception at day 45 of pregnancy (Table 6) except on CRL of fetuses and cotyledon
weight. CRL of fetuses and cotyledon weight in GnRH treated group was higher than those in
control group (P < 0.05). Fetal weights did not differ between treatment groups. There was
a tendency for higher products of conception in GnRH treated group but difference between
groups was not significant. However litter size had a significant effect on all products of
conception (P < 0.05).

4. Discussion

The results of the present study showed that GnRH agonist administration on day 12
post-mating improves pregnancy rate and number of lambs born. This is in agreement with
the previous findings of McMillan et al. (1986) and Beck et al. (1994) who also observed
beneficial effect of GnRH administration on embryo survival. Lambing and twinning rates
were 16% higher in GnRH administered ewes which resulted in an improved reproductive
performance in ewes given GnRH on day 12 post-mating compared to control group. These
results indicate that GnRH administration improves pregnancy rate and the number of lambs
born by improving embryo survival.
Luteal function is one of the critical factors affecting embryo survival (Wilmut et al.,
1986; Ashworth et al., 1989). GnRH administration in the present study increased plasma
progesterone concentration between days 13 and 15 post-mating. This effect of GnRH
was still evident on day 45 post-mating although the difference between groups was not
significant. Therefore, it may be suggested that GnRH administration improved lambing
rate by increasing embryo survival through enhanced luteal function. This effect of GnRH
on plasma progesterone concentration is in agreement with the previous findings in cattle
(Mann and Picton, 1995) but contrary to the findings of Beck et al. (1996) who were not able
to detect any stimulatory effect of GnRH on plasma progesterone concentration in sheep.
80 M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82

This effect of GnRH may occur through GnRH-stimulated LH surge (see Fig. 2) stim-
ulating production of progesterone by CL and/or causing ovulation and the formation of
accessory CLs. The results of the present study provide evidence that GnRH causes ovula-
tion and the formation of accessory CLs, since higher number of CL was observed in GnRH
given ewes at slaughter on day 45 of pregnancy. This result is agreement with the findings
of Mann and Picton (1995) and Beck et al. (1996) that GnRH administration results in an
increased number of CL.
The beneficial effect of GnRH administration on embryo survival may also be through
the stimulatory effect of progesterone on fetal growth, because it has been shown that proges-
terone supplementation increases subsequent fetal growth (Garrett et al., 1988;
Kleemann et al., 1994). The results observed in the present study do not support their
results because we were not able to detect any difference in lamb birth weight between
treatment groups. There were also no difference in products of conceptuses on day 45
of pregnancy except CRL of fetuses and cotyledon weight which were higher in GnRH
administered groups. Although GnRH administered group had higher products of concep-
tuses, the difference between groups were not significant. These observations may suggest
that the stimulatory effect of GnRH, if any, on fetal growth disappeared by the time of
lambing.
Days 12 and 13 post-mating are the critical period for maternal recognition of pregnancy
which coincides with the beginning of regression of the CL in the natural estrous cycle
(Bazer et al., 1998). GnRH induced progesterone production after on day 12 may increase
interferon-␶ production (Thatcher et al., 1995; Mann et al., 1998) which in turn might
prevent luteolysis by preventing PGF2␣ secretion (Bazer et al., 1998). Luteolysis may also
be prevented by GnRH administration through its effect on any developing follicle on day
12 post-mating. GnRH administration will ovulate or lutenize any developing follicle and
hence decrease estradiol production by the follicles. The decrease in estradiol production
(Beck et al., 1996) and an increase in progesterone production may block expression of
oxytocin receptor in uterus and hence prevent synthesis of PGF2␣ (Khan et al., 2001).
This would prevent luteolysis and allow the development of embryos that would otherwise
degenerate.

5. Conclusion

The results of the present study show that GnRH agonist administration improves repro-
ductive performance of ewes when administered on day 12 post-mating probably through
its beneficial effect on embryo survival by enhancing luteal function, but not through stim-
ulating fetal growth.

Acknowledgements

We are grateful to Prof. Dr. D.F.M. van de Wiel for providing progesterone antibody and
to Dr. A.F. Parlow and NIDDK for the provision of LH antibody and antigen. This study
was financially supported by Ondokuz Mayis University Research Fund.
M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82 81

References

Ashworth, C.J., Sales, D.I., Wilmut, I., 1989. Evidence of an association between the survival of embryos and
periovulatory plasma progesterone concentration in the ewe. J. Reprod. Fertil. 87, 23–32.
Bazer, F.W., Ott, T.L., Spencer, T.E., 1998. Maternal recognition of pregnancy: comparative aspects—a review.
Trophoplast Res. 12, 375–386.
Beck, N.F.G., Peters, A.R., Williams, S.P., 1994. The effect of GnRH agonist (buserelin) treatment on day 12
post-mating on the reproductive performance of ewes. Anim. Prod. 58, 243–247.
Beck, N.F.G., Jones, M., Davies, B., Mann, G.E., Peters, A.R., 1996. The effect of GnRH analogue (buserelin)
treatment on day 12 post-mating on ovarian structure and plasma progesterone and estradiol concentration in
ewes. Anim. Sci. 63, 407–412.
Bolet, G., 1986. Timing and extent of embryonic mortality in pigs, sheep and goats: genetic variability. In: Sreenan,
J.M., Diskin, M.G. (Eds.), Embryonic Mortality in Farm Animals. Martinus Nijhoff, The Hague, pp. 13–
43.
Davies, M.C.G., Beck, N.F.G., 1992. Plasma hormone profiles and fertility in ewe lambs given progestagen
supplementation after mating. Theriogenology 38, 513–526.
Drew, S.B., Peters, A.R., 1994. Effect of buserelin on pregnancy rates in dairy cows. Vet. Rec. 134, 267–269.
Garrett, J.E., Geisert, R.D., Zavy, M.T., Morgan, G.L., 1988. Evidence for maternal regulation of early conceptus
growth and development in beef cattle. J. Reprod. Fertil. 84, 437–446.
Jubb, J.P., Abhayaratne, D., Malmo, J., Anderson, G.A., 1990. Failure of an intramucular injection of gonadotrophin
releasing hormone 11–13 days after insemination to increase pregnancy rates in dairy cattle. Aust. Vet. J. 67,
359–361.
Khan, T.H., Beck, N.F.G., Khalid, M., Mann, G.E., 2001. Effect of post-mating GnRH analogue buserelin treatment
on prostaglandin F2␣ release in sheep. Reprod. Abstr. Ser. 27 (47) (abstract).
Kleemann, D.O., Walker, S.K., Seamark, R.F., 1994. Enhanced fetal growth in sheep administered progesterone
during the first 3 days of pregnancy. J. Reprod. Fertil. 102, 411–417.
McMillan, W., Knight, T.W., Macmillan, K.L., 1986. Effects of gonadotrophin releasing hormone (buserelin) on
sheep fertility. Proc. New Zealand Soc. Anim. Prod. 46, 161–163.
Macmillan, K.L., Taufa, V.K., Day, A.M., 1986. Effects of an agonist of gonadotrophin releasing hormone
(buserelin) in cattle. III. Pregnancy rates after a post-insemination injection during metoestrus or dioestrus.
Anim. Reprod. Sci. 11, 1–10.
Mann, G.E., Picton, H.M., 1995. Ovarian and uterine effects of a single buserelin injection on day 12 of the estrous
cycle in the cow. J. Reprod. Fertil. Abstr. Ser. 15 (61) (abstract).
Mann, G.E., Lamming, G.E., Fisher, P.A., 1998. Progesterone control of embryonic interferon-␶ production during
early pregnancy in the cow. J. Reprod. Fertil. Abstr. Ser. 21 (20) (abstract).
Michels, H., Vanmontfort, D., Dewil, E., Decuypere, E., 1998. Genetic variation of prenatal survival in relation to
ovulation rate in sheep: a review. Small Rum. Res. 29, 129–142.
Mutayoba, B.M., Meyer, H.D.D., Schams, D., Schallenberger, E., 1990. Development of a sensitive enzyme
immunoassay for LH determination in bovine plasma using the streptavidin–biotin technique. Acta Endocrinol.
(Copenhagen) 122, 227–232.
Nancarrow, C.D., 1994. Embryonic mortality in the ewe and doe. In: Zavy, M.T., Geisart, R.D. (Eds.), Embryonic
Mortality in Domestic Species. CRC Press, London, pp. 79–97.
Prakash, B.S., Meyer, H.D.D., Schallenberger, E., Van de Wiel, D.F.M., 1987. Development of sensitive EIA for
progesterone determination. J. Steroid Biochem. 28, 623–627.
Ryan, D.P., Snijders, S., Condon, T., Grealy, M., Sreenan, J., O’Farrell, K.J., 1994. Endocrine and ovarian
responses and pregnancy rates in dairy cows following the administration of a gonadotrophin releasing hormone
analog at the time of artificial insemination or at mid-cycle post-insemination. Anim. Reprod. Sci. 34, 179–
191.
Sheldon, I.M., Dobson, H., 1993. Effects of gonadotrophin releasing hormone administered 11 days after
insemination on the pregnancy rates of cattle to the first and later services. Vet. Rec. 133, 160–163.
Sreenan, J.M., Diskin, M.G., Dunne, L., 1996. Embryonic mortality: the major cause of reproductive wastage in
cattle. In: Proceedings of the 47th Annual Meeting of the European Association of Animal Production, August
1996. Lillihammer.
82 M.A. Cam et al. / Animal Reproduction Science 72 (2002) 73–82

Thatcher, W.W., Staples, C.R., Danet-Desnoyers, G., Oldick, B., Schmitt, E.P., 1994. Embryo health and mortality
in sheep and cattle. J. Anim. Sci. 72 (Suppl. 3), 16–30.
Thatcher, W.W., Meyer, M.D., Danet-Desnoyers, G., 1995. Maternal recognition of pregnancy. J. Reprod. Fertil.
Suppl. 49, 15–28.
Wilmut, I., Sales, D.I., Ashworth, C.J., 1986. Maternal and embryonic factors associated with prenatal loss in
mammals. J. Reprod. Fertil. 76, 851–864.