- Email: [email protected]
Effects of time of progesterone supplementation on embryo development and interferon-τ production in the cow
The Veterinary Journal The Veterinary Journal 171 (2006) 500–503 www.elsevier.com/locate/tvjl
Effects of time of progesterone supplementation on embryo development and interferon-s production in the cow G.E. Mann a
a,*
, M.D. Fray b, G.E. Lamming
a
Division of Animal Physiology, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK b Institute for Animal Health, Compton, Newbury RG16 ONN, UK Accepted 2 December 2004
Abstract We have investigated the effects of the timing of progesterone supplementation on early embryo development in mature, nonlactating Holstein–Friesian cows. Animals were inseminated 72 h (day 1) and 96 h following prostaglandin injection and were either left as untreated controls (n = 6) or received progesterone supplementation from either days 5 to 9 (early; n = 6) or from days 12 to 16 (late; n = 6). Daily plasma samples were collected until day 16, when cows were slaughtered and reproductive tracts recovered and flushed to collect embryos and to measure interferon-s activity. Both early and later progesterone supplementation resulted in marked increases in plasma progesterone (P < 0.01). Early, but not late, progesterone supplementation resulted in a fourfold increase in trophoblast length (P < 0.01) and a sixfold increase in uterine concentration of interferon-s (P < 0.05). The results demonstrate that progesterone supplementation during the postovulatory rise, but not later in the luteal phase, increases embryo development and interferon-s production. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cow; Progesterone; Embryo; Pregnancy; Interferon
1. Introduction In the cow, the successful establishment of pregnancy depends on the embryo developing sufficiently well to produce adequate quantities of the anti-luteolytic protein, interferon-s (for reviews see Thatcher et al., 1995; Mann et al., 1999). One of the key hormones in the control of embryo development is progesterone, which stimulates the production of the endometrial secretions necessary for the successful development of the embryo (see Geisert et al., 1992, for review). Low progesterone has been linked to early pregnancy failure (see Mann et al., 1999, for review) and poor embryo development
*
Corresponding author. Tel.: +44 1159 516326; fax: +44 1159 516302. E-mail address: [email protected] (G.E. Mann). 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.12.005
(Walton et al., 1990) while supplementing cows with progesterone has been shown to enhance conceptus development (Garrett et al., 1988). Studies in which progesterone has been administered in an attempt to improve pregnancy rates have yielded variable results. In these studies, progesterone supplementation initiated at the time of onset of the postovulatory rise (between days 4 and 5) has resulted in consistent increases in pregnancy rate. However, when supplementation has been initiated later than this, consistent improvements have not been reported (for review see Mann and Lamming, 1999). Furthermore, in a recent study we found a close association between the specific pattern of maternal progesterone secretion and the development of the early embryo, poor embryo development being associated with both a delayed postovulatory progesterone rise and a low subsequent luteal phase concentrations (Mann and Lamming, 2001).
G.E. Mann et al. / The Veterinary Journal 171 (2006) 500–503
2. Materials and methods 2.1. Experimental protocol The study was performed in 18 mature non-lactating Holstein–Friesian cows. All cows had calved at least once and were obtained from the University of Nottingham commercial herd at the end of lactation. Throughout the study, cows were maintained on a diet of hay and concentrate pellets. All experimental procedures were carried out under an appropriate United Kingdom Home Office project licence. The oestrous cycles of all cows were synchronised by two injections of the prostaglandin (PG) F2a analogue, cloprostenol (Estrumate; Schering-Plough Animal Health) administered 11–13 days apart. All cows were inseminated 72 and 96 h following the second injection of cloprostenol by Genus technicians (Genus plc) using semen from beef bulls. Cows were then allocated to receive no progesterone supplementation (control, n = 6), progesterone supplementation from days 5 to 9 (early, n = 6) or progesterone supplementation from days 12 to 16 (late, n = 6). Progesterone supplementation was via an intravaginal CIDR-B device containing 0.95 g progesterone (InterAG, New Zealand) inserted at 0900 h on the day of treatment initiation and withdrawn at 0900 h on the day treatment ceased. Prior to the second PG injection all cows underwent jugular vein cannulation to allow collection of daily blood samples. On day 16, cows were slaughtered by captive bolt and exsanguination and the reproductive tract collected and transported to the laboratory. The uterine horns were dissected free from surrounding tissues and separated past the bifurcation. The horn adjacent to the corpus luteum was then flushed with 20 mL saline into a single dish and the embryo collected and its length measured. Uterine flushings were then frozen at 20 °C until subsequent analysis for interferon-s content. 2.2. Hormone assays Progesterone was measured in plasma by previously described radioimmunoassay (Law et al., 1992). Intraand inter-assay coefficients of variation were 6.3% and 9.8% and a sensitivity of the assay was 0.5 ng/mL. Inter-
feron-s activity of samples was measured by an Mx/ CAT reporter gene assay based on Madin Darby Bovine Kidney cells transfected with plasmid, p123-intron containing a chloramphenicol acetyltransferase (CAT) expression unit linked to a human MxA promoter (Fray et al., 2001). The potencies of the unknown samples were calculated relative to recombinant bovine IFNa1, the anti-viral activity of which had previously been determined in an anti-viral assay and compared against the first international reference preparation of human leucocyte IFN (69/019). CAT expression was determined using a commercial ELISA kit (Roche Molecular Biochemicals). Sensitivity was 0.5 anti-viral units (avu) interferon-s/mL and the inter assay coefficient of variation 9.5%. 2.3. Statistical analysis Plasma concentrations of progesterone were analysed by repeated sample analysis of variance. Differences between groups in embryo length and level of interferon-s were analysed by analysis of variance. The relationship between embryo length and interferon-s production was analysed by regression analysis.
3. Results During the periods when progesterone was not supplemented, plasma concentrations were similar between groups (Fig. 1). During supplementation, progesterone was elevated (P < 0.01) in the early group on days
12
10
Progesterone (ng/mL)
In the present study, we have investigated the importance of the time of progesterone supplementation on the development of the embryo in the cow by treating cows with exogenous progesterone at two stages of the luteal phase. The principle aim was to determine whether different effects on embryo development support reported differences in the effectiveness of different times of progesterone supplementation on pregnancy rates.
501
8
6
4
2
0 -3
-1
1
3
5
7 Day
9
11
13
15
17
Fig. 1. Means (±SEM) plasma concentration of progesterone in inseminated cows either untreated (control; n; n = 6) or supplemented with progesterone via an intravaginal CIDR from days 5 to 9 (early; s: n = 6) or from days 12 to 16 (late; h; n = 6).
502
G.E. Mann et al. / The Veterinary Journal 171 (2006) 500–503
6–9 and in the late group on days 13–16. During early supplementation (on days 6–9) mean plasma progesterone was elevated to 6.1 ± 0.5 compared with 3.4 ± 0.5 ng/ mL in the other two groups (P < 0.01). During late supplementation (on days 13–16) mean plasma, progesterone was elevated to 10.7 ± 0.3 compared with 7.4 ± 0.3 ng/mL in the other two groups (P < 0.01). The proportion of cows from which an embryo was recovered at slaughter was similar in the three groups (control, 4/6; early, 4/6; late, 3/6). Interferon-s was undetectable in the uterine flushes of all cows from which no embryo was recovered. Compared to the control and late progesterone groups, in which both embryo length and uterine interferon-s concentrations were similar (Fig. 2), the early progesterone supplemented group showed a significant increase in both embryo length (P < 0.01) and uterine interferon-s concentration (P < 0.05). Furthermore, there was a significant association between embryo length and uterine interferon-s concentration (R2 = 0.83; P < 0.001; n = 11).
4. Discussion The results of the present study demonstrate that progesterone supplementation early in the luteal phase, during the period of the postovulatory progesterone rise (days 5–9), but not later supplementation (days 12–16) can enhance both embryo development and secretion of anti-luteolytic interferon-s. We have previously demonstrated that the degree of embryo development and interferon-s secretion is associated with both the timing of the postovulatory progesterone rise and the concentration to which progesterone eventually rises (Mann b
4
90
c
Trophoblast length (cm)
60
2
a 1
30 a a
a
0
Interferon t (antiviral units/flush)
3
and Lamming, 2001). The results of the present study suggest that it is the timing and/or strength of the postovulatory progesterone rise that is critical to the development of the embryo rather than the final concentration of progesterone achieved later in the luteal phase. There have been numerous studies in which progesterone supplementation has been used in an attempt to increase pregnancy rates in the cow. In these studies, progesterone supplementation initiated at the time of onset of the postovulatory rise (between days 4 and 5) has proved successful while later supplementation has not resulted in consistent increases in pregnancy rate (Mann and Lamming, 1999). The finding in the present study that early, but not later, progesterone supplementation can advance embryo development provides an explanation for this observation. In the present study, there was a close relationship between trophoblast length and uterine interferon-s concentration. This suggests that the enhanced interferons production associated with increased progesterone may result from the increase in embryo size rather than any increase in the rate of interferon-s production per unit trophoblast. We have previously demonstrated that as trophoblast elongation occurs, between days 14 and 18, there is no increase in interferon-s mRNA level per unit trophoblast (Robinson et al., 1999); increased interferon-s production results from the dramatic increase in trophoblast size that occurs at this time. In conclusion, the present study has demonstrated that it is the concentration of progesterone during the postovulatory increase (rather than the level to which progesterone secretion eventually rises) that is the critical factor in the influence of progesterone on the development of the early embryo. This result provides an explanation for the fact that, in a number of studies, supplementation of progesterone around day 5 after mating has proven more successful than later supplementation.
Acknowledgements We are grateful to S.J. Mann for sample collection and analysis and to the staff of the Nottingham University Animal Research Unit for care of the animals. The CIDRs were supplied by InterAG, New Zealand. The work was funded by MAFF and MDC under the Link Sustainable Livestock Production Programme.
0 Control (n=4)
Late (n=3)
Early (n=4)
Fig. 2. Means (±SEM) trophoblast length (open bars) among embryos recovered on day 16 and means (±SEM) concentration of interferon-s per 20 mL uterine flush (solid bars) in cows inseminated and either untreated (control, n = 4) or supplemented with progesterone from days 5 to 9 (early, n = 4) or from days 12 to 16 (late, n = 3). (ab, P < 0.05; ac, P < 0.01).
References Fray, M.D., Mann, G.E., Charleston, B., 2001. Validation of an Mx/ CAT reporter gene assay for the quantification of bovine type-I interferon. Journal of Immunological Methods 249, 235–244.
G.E. Mann et al. / The Veterinary Journal 171 (2006) 500–503 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. Journal of Reproduction and Fertility 84, 437–446. Geisert, R.D., Morgan, G.L., Short, E.C., Zavy, M.T., 1992. Endocrine events associated with endometrial function and conceptus development in cattle. Reproduction Fertility and Development 4, 301–305. Law, A.S., Baxter, G., Logue, D.N., OÕshea, T., Webb, R., 1992. Evidence for the action of bovine follicular fluid factor(s) other than inhibin in suppressing follicular development and delaying estrus in heifers. Journal of Reproduction and Fertility 96, 603–616. Mann, G.E., Lamming, G.E., 1999. The influence of progesterone during early pregnancy in cattle. Reproduction in Domestic Animals 34, 269–274. Mann, G.E., Lamming, G.E., 2001. Relationship between the maternal endocrine environment, early embryo development and the inhibition of the luteolytic mechanism in the cow. Reproduction 121, 175–180.
503
Mann, G.E., Lamming, G.E., Robinson, R.S., Wathes, D.C., 1999. The regulation of interferon-tau production and uterine hormone receptors during early pregnancy. Journal of Reproduction and Fertility Supplement 54, 317–328. Robinson, R.S., Mann, G.E., Lamming, G.E., Wathes, D.C., 1999. Embryonic–endometrial interactions during early pregnancy in the cow. In: Proceedings of British Society of Animal Science meeting on Fertility in the High-Producing Dairy Cow. British Society for Animal Science Occasional Publication Number 26, pp. 36–37. Thatcher, W.W., Meyer, M.D., Danet-desnoyers, G., 1995. Maternal recognition of pregnancy. Journal of Reproduction and Fertility Supplement 49, 15–28. Walton, J.S., Halbert, G.W., Robinson, N.A., Leslie, K.E., 1990. 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. Canadian Journal of Veterinary Science 54, 305–308.
Effects of time of progesterone supplementation on embryo development and interferon-s production in the cow G.E. Mann a
a,*
, M.D. Fray b, G.E. Lamming
a
Division of Animal Physiology, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK b Institute for Animal Health, Compton, Newbury RG16 ONN, UK Accepted 2 December 2004
Abstract We have investigated the effects of the timing of progesterone supplementation on early embryo development in mature, nonlactating Holstein–Friesian cows. Animals were inseminated 72 h (day 1) and 96 h following prostaglandin injection and were either left as untreated controls (n = 6) or received progesterone supplementation from either days 5 to 9 (early; n = 6) or from days 12 to 16 (late; n = 6). Daily plasma samples were collected until day 16, when cows were slaughtered and reproductive tracts recovered and flushed to collect embryos and to measure interferon-s activity. Both early and later progesterone supplementation resulted in marked increases in plasma progesterone (P < 0.01). Early, but not late, progesterone supplementation resulted in a fourfold increase in trophoblast length (P < 0.01) and a sixfold increase in uterine concentration of interferon-s (P < 0.05). The results demonstrate that progesterone supplementation during the postovulatory rise, but not later in the luteal phase, increases embryo development and interferon-s production. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cow; Progesterone; Embryo; Pregnancy; Interferon
1. Introduction In the cow, the successful establishment of pregnancy depends on the embryo developing sufficiently well to produce adequate quantities of the anti-luteolytic protein, interferon-s (for reviews see Thatcher et al., 1995; Mann et al., 1999). One of the key hormones in the control of embryo development is progesterone, which stimulates the production of the endometrial secretions necessary for the successful development of the embryo (see Geisert et al., 1992, for review). Low progesterone has been linked to early pregnancy failure (see Mann et al., 1999, for review) and poor embryo development
*
Corresponding author. Tel.: +44 1159 516326; fax: +44 1159 516302. E-mail address: [email protected] (G.E. Mann). 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.12.005
(Walton et al., 1990) while supplementing cows with progesterone has been shown to enhance conceptus development (Garrett et al., 1988). Studies in which progesterone has been administered in an attempt to improve pregnancy rates have yielded variable results. In these studies, progesterone supplementation initiated at the time of onset of the postovulatory rise (between days 4 and 5) has resulted in consistent increases in pregnancy rate. However, when supplementation has been initiated later than this, consistent improvements have not been reported (for review see Mann and Lamming, 1999). Furthermore, in a recent study we found a close association between the specific pattern of maternal progesterone secretion and the development of the early embryo, poor embryo development being associated with both a delayed postovulatory progesterone rise and a low subsequent luteal phase concentrations (Mann and Lamming, 2001).
G.E. Mann et al. / The Veterinary Journal 171 (2006) 500–503
2. Materials and methods 2.1. Experimental protocol The study was performed in 18 mature non-lactating Holstein–Friesian cows. All cows had calved at least once and were obtained from the University of Nottingham commercial herd at the end of lactation. Throughout the study, cows were maintained on a diet of hay and concentrate pellets. All experimental procedures were carried out under an appropriate United Kingdom Home Office project licence. The oestrous cycles of all cows were synchronised by two injections of the prostaglandin (PG) F2a analogue, cloprostenol (Estrumate; Schering-Plough Animal Health) administered 11–13 days apart. All cows were inseminated 72 and 96 h following the second injection of cloprostenol by Genus technicians (Genus plc) using semen from beef bulls. Cows were then allocated to receive no progesterone supplementation (control, n = 6), progesterone supplementation from days 5 to 9 (early, n = 6) or progesterone supplementation from days 12 to 16 (late, n = 6). Progesterone supplementation was via an intravaginal CIDR-B device containing 0.95 g progesterone (InterAG, New Zealand) inserted at 0900 h on the day of treatment initiation and withdrawn at 0900 h on the day treatment ceased. Prior to the second PG injection all cows underwent jugular vein cannulation to allow collection of daily blood samples. On day 16, cows were slaughtered by captive bolt and exsanguination and the reproductive tract collected and transported to the laboratory. The uterine horns were dissected free from surrounding tissues and separated past the bifurcation. The horn adjacent to the corpus luteum was then flushed with 20 mL saline into a single dish and the embryo collected and its length measured. Uterine flushings were then frozen at 20 °C until subsequent analysis for interferon-s content. 2.2. Hormone assays Progesterone was measured in plasma by previously described radioimmunoassay (Law et al., 1992). Intraand inter-assay coefficients of variation were 6.3% and 9.8% and a sensitivity of the assay was 0.5 ng/mL. Inter-
feron-s activity of samples was measured by an Mx/ CAT reporter gene assay based on Madin Darby Bovine Kidney cells transfected with plasmid, p123-intron containing a chloramphenicol acetyltransferase (CAT) expression unit linked to a human MxA promoter (Fray et al., 2001). The potencies of the unknown samples were calculated relative to recombinant bovine IFNa1, the anti-viral activity of which had previously been determined in an anti-viral assay and compared against the first international reference preparation of human leucocyte IFN (69/019). CAT expression was determined using a commercial ELISA kit (Roche Molecular Biochemicals). Sensitivity was 0.5 anti-viral units (avu) interferon-s/mL and the inter assay coefficient of variation 9.5%. 2.3. Statistical analysis Plasma concentrations of progesterone were analysed by repeated sample analysis of variance. Differences between groups in embryo length and level of interferon-s were analysed by analysis of variance. The relationship between embryo length and interferon-s production was analysed by regression analysis.
3. Results During the periods when progesterone was not supplemented, plasma concentrations were similar between groups (Fig. 1). During supplementation, progesterone was elevated (P < 0.01) in the early group on days
12
10
Progesterone (ng/mL)
In the present study, we have investigated the importance of the time of progesterone supplementation on the development of the embryo in the cow by treating cows with exogenous progesterone at two stages of the luteal phase. The principle aim was to determine whether different effects on embryo development support reported differences in the effectiveness of different times of progesterone supplementation on pregnancy rates.
501
8
6
4
2
0 -3
-1
1
3
5
7 Day
9
11
13
15
17
Fig. 1. Means (±SEM) plasma concentration of progesterone in inseminated cows either untreated (control; n; n = 6) or supplemented with progesterone via an intravaginal CIDR from days 5 to 9 (early; s: n = 6) or from days 12 to 16 (late; h; n = 6).
502
G.E. Mann et al. / The Veterinary Journal 171 (2006) 500–503
6–9 and in the late group on days 13–16. During early supplementation (on days 6–9) mean plasma progesterone was elevated to 6.1 ± 0.5 compared with 3.4 ± 0.5 ng/ mL in the other two groups (P < 0.01). During late supplementation (on days 13–16) mean plasma, progesterone was elevated to 10.7 ± 0.3 compared with 7.4 ± 0.3 ng/mL in the other two groups (P < 0.01). The proportion of cows from which an embryo was recovered at slaughter was similar in the three groups (control, 4/6; early, 4/6; late, 3/6). Interferon-s was undetectable in the uterine flushes of all cows from which no embryo was recovered. Compared to the control and late progesterone groups, in which both embryo length and uterine interferon-s concentrations were similar (Fig. 2), the early progesterone supplemented group showed a significant increase in both embryo length (P < 0.01) and uterine interferon-s concentration (P < 0.05). Furthermore, there was a significant association between embryo length and uterine interferon-s concentration (R2 = 0.83; P < 0.001; n = 11).
4. Discussion The results of the present study demonstrate that progesterone supplementation early in the luteal phase, during the period of the postovulatory progesterone rise (days 5–9), but not later supplementation (days 12–16) can enhance both embryo development and secretion of anti-luteolytic interferon-s. We have previously demonstrated that the degree of embryo development and interferon-s secretion is associated with both the timing of the postovulatory progesterone rise and the concentration to which progesterone eventually rises (Mann b
4
90
c
Trophoblast length (cm)
60
2
a 1
30 a a
a
0
Interferon t (antiviral units/flush)
3
and Lamming, 2001). The results of the present study suggest that it is the timing and/or strength of the postovulatory progesterone rise that is critical to the development of the embryo rather than the final concentration of progesterone achieved later in the luteal phase. There have been numerous studies in which progesterone supplementation has been used in an attempt to increase pregnancy rates in the cow. In these studies, progesterone supplementation initiated at the time of onset of the postovulatory rise (between days 4 and 5) has proved successful while later supplementation has not resulted in consistent increases in pregnancy rate (Mann and Lamming, 1999). The finding in the present study that early, but not later, progesterone supplementation can advance embryo development provides an explanation for this observation. In the present study, there was a close relationship between trophoblast length and uterine interferon-s concentration. This suggests that the enhanced interferons production associated with increased progesterone may result from the increase in embryo size rather than any increase in the rate of interferon-s production per unit trophoblast. We have previously demonstrated that as trophoblast elongation occurs, between days 14 and 18, there is no increase in interferon-s mRNA level per unit trophoblast (Robinson et al., 1999); increased interferon-s production results from the dramatic increase in trophoblast size that occurs at this time. In conclusion, the present study has demonstrated that it is the concentration of progesterone during the postovulatory increase (rather than the level to which progesterone secretion eventually rises) that is the critical factor in the influence of progesterone on the development of the early embryo. This result provides an explanation for the fact that, in a number of studies, supplementation of progesterone around day 5 after mating has proven more successful than later supplementation.
Acknowledgements We are grateful to S.J. Mann for sample collection and analysis and to the staff of the Nottingham University Animal Research Unit for care of the animals. The CIDRs were supplied by InterAG, New Zealand. The work was funded by MAFF and MDC under the Link Sustainable Livestock Production Programme.
0 Control (n=4)
Late (n=3)
Early (n=4)
Fig. 2. Means (±SEM) trophoblast length (open bars) among embryos recovered on day 16 and means (±SEM) concentration of interferon-s per 20 mL uterine flush (solid bars) in cows inseminated and either untreated (control, n = 4) or supplemented with progesterone from days 5 to 9 (early, n = 4) or from days 12 to 16 (late, n = 3). (ab, P < 0.05; ac, P < 0.01).
References Fray, M.D., Mann, G.E., Charleston, B., 2001. Validation of an Mx/ CAT reporter gene assay for the quantification of bovine type-I interferon. Journal of Immunological Methods 249, 235–244.
G.E. Mann et al. / The Veterinary Journal 171 (2006) 500–503 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. Journal of Reproduction and Fertility 84, 437–446. Geisert, R.D., Morgan, G.L., Short, E.C., Zavy, M.T., 1992. Endocrine events associated with endometrial function and conceptus development in cattle. Reproduction Fertility and Development 4, 301–305. Law, A.S., Baxter, G., Logue, D.N., OÕshea, T., Webb, R., 1992. Evidence for the action of bovine follicular fluid factor(s) other than inhibin in suppressing follicular development and delaying estrus in heifers. Journal of Reproduction and Fertility 96, 603–616. Mann, G.E., Lamming, G.E., 1999. The influence of progesterone during early pregnancy in cattle. Reproduction in Domestic Animals 34, 269–274. Mann, G.E., Lamming, G.E., 2001. Relationship between the maternal endocrine environment, early embryo development and the inhibition of the luteolytic mechanism in the cow. Reproduction 121, 175–180.
503
Mann, G.E., Lamming, G.E., Robinson, R.S., Wathes, D.C., 1999. The regulation of interferon-tau production and uterine hormone receptors during early pregnancy. Journal of Reproduction and Fertility Supplement 54, 317–328. Robinson, R.S., Mann, G.E., Lamming, G.E., Wathes, D.C., 1999. Embryonic–endometrial interactions during early pregnancy in the cow. In: Proceedings of British Society of Animal Science meeting on Fertility in the High-Producing Dairy Cow. British Society for Animal Science Occasional Publication Number 26, pp. 36–37. Thatcher, W.W., Meyer, M.D., Danet-desnoyers, G., 1995. Maternal recognition of pregnancy. Journal of Reproduction and Fertility Supplement 49, 15–28. Walton, J.S., Halbert, G.W., Robinson, N.A., Leslie, K.E., 1990. 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. Canadian Journal of Veterinary Science 54, 305–308.