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Pregnancy rates in lactating dairy cattle following supplementation of progesterone after artificial insemination
Animal Reproduction Science 102 (2007) 172–179
Short communication
Pregnancy rates in lactating dairy cattle following supplementation of progesterone after artificial insemination Sandra F. Larson ∗ , W.R. Butler, W. Bruce Currie Department of Animal Science, Cornell University, Ithaca, NY 14853, USA Received 21 November 2006; accepted 27 February 2007 Available online 3 March 2007
Abstract Poor conception rates in highly productive lactating cattle is especially prevalent in large, intensivelymanaged commercial herds. One of the causative factors is sub-optimal pre-implantation embryonic development which appears to result from inadequate circulating concentrations of progesterone. In the present study, the efficacy of very modest progesterone supplementation, between Days 3.5 and 10 post-AI, on pregnancy rates was determined in a commercial herd where bovine somatotropin (bST) was used as a management tool. All lactating cattle that were deemed to be in estrus and inseminated over a 4-week period were randomly assigned to either a control group (no treatment) or CIDR-1.9 g (previously used for estrous synchronization) treatment from Day 3.5 to Day 10 post-AI. Milk samples were collected four times: on the day of AI, at Day 2 or 3, at Day 4 and at Day 22 post-AI and were analyzed for progesterone content. Data from a total of 130 breedings were used in the final analysis. The CIDR treatment increased circulating concentrations of progesterone in treated animals over those of control animals on Day 4 by 0.7 ng/ml (P < 0.05) and increased pregnancy rate from 35% (22/63) to 48% (32/67) (P = 0.068). The effect of treatment was greater in first and second lactation cows, where pregnancy rates were 33% (18/55) in controls and 51% (31/61) in treated animals (P = 0.03). The results of this study indicate that the timing of onset of the progesterone influence is important for successful pregnancy outcome, particularly in first and second lactation cows. © 2007 Elsevier B.V. All rights reserved. Keywords: Progesterone; Pregnancy; Dairy cattle
∗ Corresponding author. Current address: Department of Biology, Furman University, Greenville, SC 29613, USA. Tel.: +1 864 294 2306; fax: +1 864 294 2058. E-mail address: [email protected] (S.F. Larson).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.02.023
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1. Introduction One of the factors implicated in low fertility in cattle is low circulating concentrations of progesterone during the pre-implantation phase of embryonic development (reviewed in Mann and Lamming, 1999). Several observational studies of lactating cattle have reported lower postinsemination progesterone in non-pregnant cattle compared to pregnant cattle (Butler et al., 1996; Larson et al., 1997; Stronge et al., 2005; McNeill et al., 2006). In controlled studies where early embryo development was examined in relationship to maternal progesterone concentrations, greater maternal progesterone concentrations were associated with enhanced embryo development (Maurer and Echternkamp, 1982; Mann and Lamming, 2001; Green et al., 2005). In our previous non-interventional study, 60% of the non-pregnant cows had progesterone concentrations > 2 ng/ml on Day 21 post-breeding, and had lower progesterone concentrations on Day 3.5 post-breeding; we hypothesized that pregnancy recognition had been initiated in many of these cows, yet they failed to maintain pregnancy until the Day 40 pregnancy examination (Larson et al., 1997). A number of studies have examined the utility of post-insemination (or AI) supplementation of progesterone to increase pregnancy rates (for review, see Mann and Lamming, 1999). Results from studies where supplemental progesterone was given have varied widely, with some showing increased pregnancy rates (Robinson et al., 1989; Macmillan and Peterson, 1993), others no effect (Van Cleeff et al., 1991; Villarroel et al., 2004), and still others decreased pregnancy rates (Van Cleeff et al., 1996). Most of the studies where supplemental progesterone increased the pregnancy rate used lactating cattle, while no effect of treatment was observed in most studies using heifers. The present study assessed the efficacy of very modest supplementation of progesterone to establish approximately 1 ng/ml plasma concentration of progesterone around Day 4 post-AI, the time when we detected differences in the onset of the luteal phase (Larson et al., 1997). The objective was to determine if progesterone supplementation soon after AI could increase pregnancy rates in lactating dairy cattle. Performing the present study in the context of a commercial dairy was also a critical factor in the design of the experiment in that we desired to evaluate the efficacy and practicality of the treatment in an industry setting under field conditions. 2. Materials and methods 2.1. Animals Grade Holstein cattle in a commercial dairy (450 milking cows, average rolling herd milk production 9534 kg per cow per year) were used in the study. Cows were milked three times a day and were treated with bST (Posilac, Monsanto, St. Louis, MO) every 2 weeks after reaching 70 days in milk. All lactating cattle presented for breeding between 4 October and 30 October was randomly assigned to either a control (no treatment) or treatment group with the CIDR inserted from Day 3.5 to Day 10 post-AI (Day 0 = day of AI). Random assignment was selected over blocking as the aim of the study was to evaluate the efficacy of routine CIDR treatment of all bred animals managed under commercial conditions. Animals with an abnormal vaginal discharge at AI or those presenting difficulties for the inseminator were not included in the study. Data recorded on each cow included days in milk (DIM) production, lactation number, breeding number for current lactation, and average daily milk production. Progesterone treatment was carried out by insertion of an intravaginal progesterone-releasing device (CIDR-B, starting content of new
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devices 1.9 g P4 , InterAg, Hamilton, New Zealand) that had been previously used in cattle in unrelated experiments for 7 to 9 days for estrous synchronization. Previously used CIDRs ensured that progesterone concentrations would be increased only modestly (approximately 1 ng/ml) to avoid possible disruption of the developing corpus luteum or perturbation of the endocrine status of the animal by achieving luteal-phase concentrations of progesterone too early in the estrous cycle (Van Cleeff et al., 1992). The CIDRs which had been stored at 4 ◦ C since their previous use, were thoroughly sanitized by soaking in warm water and chlorhexidine (Nolvasan Surgical Scrub, Fort Dodge Laboratories, Fort Dodge, IA). For the purposes of statistical analysis, cows that were presented twice for AI during the 27 days were treated as two separate animals and were randomly assigned to a treated or control group on each occasion (n = 7). Data were collected on a total of 177 inseminations, a number that exceeded the a priori (n = 120) determined value for the detection of a 10% increase in the pregnancy rate due to treatment at P < 0.1. Reproductive management on the farm included the use of prostaglandin F2␣ for estrous synchronization. Animals were treated with 25 mg i.m. prostaglandin F2␣ (Lutalyse, Upjohn, Kalamazoo, MI) every 14 days starting at approximately 21 days post-partum but were not inseminated until after 40 days post-partum. All inseminations were performed based on estrous behavior, as monitored by the AFIMILK pedometer system (Germania Dairy Automation, Waunakee, WI). A cow exhibiting estrous activity (>180% of a cow’s normal activity) during two consecutive milkings was considered to be in estrus and was inseminated shortly after the second milking. The timing of insemination was based on the previous 18 months of on-farm experience of inseminating cows at various times after increased pedometer readings. The use of pedometers for estrous detection has been reported to be as accurate as visual observation, and is eminently more practical in a large herd such as this, with >100 animals/pen in a freestall barn (Bilby et al., 2004). Animals that were not detected in estrus for >40 days after this AI were palpated per rectum by the herd veterinarian, who was unaware of assigned treatments, to determine pregnancy status. 2.2. Sample processing and progesterone analysis Pre-milking strip samples were collected from each animal on Day 0 (day of AI), Day 2 or 3 (pre-treatment or control equivalent), Day 4 (to evaluate efficacy of treatment), and Day 22 (to provide an additional check for return to estrus). Samples were processed as described previously and a double-antibody RIA was used to measure progesterone concentrations in skim milk (Larson et al., 1997). Coefficients of variation (CV) for the assay were 12 and 24% within assay, and 13 and 15% between assay for milk with greater and lesser concentrations of progesterone, respectively. The use of skim milk for progesterone analysis as opposed to serum is the likely cause of the somewhat greater than desirable CV’s (Nachreiner et al., 1992). In addition, the greatest variability was seen with the lesser progesterone measurements, yet even with variation of ±25%, a 0.5 ng/ml sample would yield a measured range of 0.375–0.625 ng/ml, well within a range expected in a cow in estrus. 2.3. Statistical analysis After all samples were analyzed for progesterone content, some animals (breedings) were excluded for one or more of the following reasons: (1) >1.5 ng/ml progesterone on day of AI (likely misbred, n = 20, an 11% false positive rate); (2) milk sample sets missing more than 1 sample (n = 17); (3) animals inadvertently injected with prostaglandin after AI (n = 5); (4) animals
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sold before pregnancy determination (n = 5). Data from a total of 130 inseminations were used in the final analysis. Changes in progesterone content of pre- and post-treatment (or control equivalent) samples were calculated for individual animals in order to evaluate the incremental increase in progesterone. This increment and the concentrations of progesterone on individual days were compared by analysis of variance, and differences between means were calculated by Scheffe’s post-hoc test. The outcome of pregnancy determination fit a binary distribution; therefore, a non-parametric test of two proportions (a one-tailed Z-test) was used to determine if pregnancy rate was increased by treatment. After analysis of all cows in the study, the Z-test was applied to the pregnancy data from the group consisting of first and second lactation cows. Logistic regression analysis was carried out using lactation number, days in milk production, number of times bred and CIDR treatment to determine which variables were predictors of pregnancy outcome. Logistic regression analysis was performed on SPSS statistical software (v. 15, Chicago, IL), and Z-tests were performed on Number Cruncher Statistical Systems (Kaysville, UT). 3. Results Treatment increased the mean progesterone concentration by 0.7 ng/ml (P < 0.05) on the day after CIDR placement (Day 4) compared with that of the controls (Table 1). The incremental increase in progesterone from pre- to post-treatment (or equivalent) samples was 1.13 ng/ml in CIDR cows versus 0.43 ng/ml in control cows (P < 0.05). Concentrations of progesterone were not different between pregnant and non-pregnant cows in any milk samples prior to Day 22 samples. On Day 22, cows in both groups that were confirmed as being pregnant had greater concentrations of progesterone (5.45 and 4.93 ng/ml in control and treated cows, respectively) than did the cows that either returned to estrus before the seventh week pregnancy check (1.71 ng/ml) or were non-pregnant when palpated in the seventh week (1.79 ng/ml). Progesterone enhanced pregnancy rates from 35% in controls to 48% in CIDR-treated cows (P = 0.068; Table 2). The random assignment of cows to treatment was successful in creating treatment and control groups that were quite similar in parity, DIM and average daily milk production (Table 3). Logistic regression analysis was performed to verify the results of the Z-test, and to determine whether inclusion of the variables of parity, DIM and average daily milk production improved the predictive capability of the statistical model beyond that of the effect of CIDR treatment alone. The effect of CIDR treatment on pregnancy outcome was identical to the Z-test analysis (P = 0.068), and inclusion of the additional variables did not improve the fit of the model. The similarities between the treated and control groups and the corresponding statistical analysis suggest that the difference in pregnancy rate was due to the progesterone treatment rather than to Table 1 Mean (± S.E.M.) skim-milk concentrations of progesterone (ng/ml) of dairy cows treated with CIDRs starting at Day 3.5 post-insemination Treatment
n
Days 2–3
Day 4
Incrementa
Control CIDR
63 67
1.00 ± 0.05 0.96 ± 0.05
1.43 b ± 0.1 2.09 c ± 0.09
0.43 b ± 0.09 1.13 c ± 0.09
Letters (b, c) indicate differences (P < 0.05) within columns. a Increment from pre- to post-treatment samples were calculated for individual animals to generate means and standard errors.
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Table 2 Effect of CIDR treatment from Day 3.5 to Day 10 post-insemination on pregnancy status of lactating dairy cattle Treatment
All cows Control CIDR
n
Not pregnant
63 67
First and second lactation cows only Control 55 CIDR 61
Pregnant
Estrus-day 21a
Extended estrous cyclesb
31 (49) 24 (36)
10 (16) 11 (16)
22 (35)c 32 (48)c
27 (49) 22 (36)
10 (18) 8 (13)
18 (33)d 31 (51)d
Number of animals per pregnancy status category (%). a Open animals with progesterone <2 ng/ml on Day 22 post-insemination. b Open animals with progesterone ≥2 ng/ml on Day 22 post-insemination. c Treatment effect = + 13%, P = 0.068. d Treatment effect = + 18%, P = 0.03. Table 3 Mean (± S.E.M.) lactation number, days in milk (DIM) and average daily milk production for control and CIDR-treated cows Treatment
n
Lactation number
DIM at AI
Average milk production (kg/d)
Control Not pregnant Pregnant
41 22
1.9 ± 0.2 1.9 ± 0.2
158 ± 20 133 ± 22
31.7 ± 1.1 30.0 ± 1.6
CIDR Not pregnant Pregnant
35 32
1.9 ± 0.1 1.7 ± 0.1
148 ± 19 126 ± 17
32.1 ± 1.2 32.5 ± 1.2
an underlying difference in fertility between the control and treatment group. Considering only first and second lactation cows, the difference in pregnancy rates was even more striking, with controls at 33% pregnant and treated cows at 51% (P = 0.03; Table 2). Our previous survey study (Larson et al., 1997) revealed a population of non-pregnant cows with lesser progesterone early after breeding, yet these cows had greater circulating concentrations of progesterone (mean = 4.5 ng/ml) on Day 21. Cows were classified as having extended estrous cycles, likely due to the presence of an embryo initiating maternal recognition of pregnancy, but failing to maintain pregnancy until the seventh week. The response to treatment in the present study was not due to a decreased incidence of cows with extended estrous cycles (10/63 controls, 11/67 CIDR-treated cows) as had been originally hypothesized from the previous study. 4. Discussion The beneficial effects of augmentation of circulating concentrations of progesterone on the pregnancy rate in lactating dairy cows this study corroborate the results of previous studies (Robinson et al., 1989, Macmillan and Peterson, 1993). The present study was designed to carefully address both the appropriate timing and magnitude of the progesterone rise post-breeding. Utilization of a new CIDR (1.9 g P4 ) through the first week starting immediately after breeding
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caused decreased pregnancy rates in heifers (Van Cleeff et al., 1996). The time period of progesterone supplementation in the study was selected based on previous observations of pregnant cows having greater progesterone around Day 4, and calculations from the previous study of earlier initiation (Day 3.25) of luteal function in pregnant than in non-pregnant cows with extended estrous cycles (Day 3.99) (Butler et al., 1996, Larson et al., 1997, Mann and Lamming, 2001, Gumen et al., 2003, Stronge et al., 2005, McNeill et al., 2006). The experimental use of the previously used CIDR ensured that circulating concentrations of progesterone would not be achieved abruptly in the mid-luteal phase, too early in the estrous cycle (Mann et al., 1998). Previous studies have revealed that CIDRs (starting with 1.9 g P4 ) used for 9 days contain approximately 1 g P4 (Macmillan et al., 1991), and that re-use of the devices increased progesterone concentrations in ovariectomized lactating cows to 1 to 1.5 ng/ml (Van Cleeff et al., 1992), hence the decision in the present study to employ previously used CIDRs. Use of this protocol does not imply advocation of the routine re-use of CIDRs. After the present study was performed, the progesterone content of new CIDR devices made available in the USA was decreased to 1.38 g. These new CIDR devices may prove to be suitable for post-insemination progesterone supplementation with similar success to that described for the present study. Increased pregnancy rates associated with augmentation of progesterone concentrations in the early luteal phase of the estrous cycle are most often found when the lactating cow is the experimental animal. Embryo quality is decreased in lactating cows as compared with non-lactating cows and heifers (Sartori et al., 2002). The rate of metabolism of progesterone is increased in lactating cows compared to heifers (Sangsritavong et al., 2002), and lactating cattle tend to have lesser concentrations of sex steroid hormones than heifers or dry cows (Sartori et al., 2004). The increased productivity associated with bST use likely increases clearance rates of progesterone as is seen in highly productive cattle that are not treated with bST (Sangsritavong et al., 2002), therefore, supplemental progesterone may be particularly beneficial for highly productive cattle regardless of bST use. In the present study, lactating cows were used exclusively, and we also had the advantage of having a large number of cows inseminated within a short interval, minimizing any seasonal effects on reproductive success, within a single herd. In addition, the progesterone regimen in the present study was carefully engineered to mimic the timing of the observed progesterone increase and to establish appropriate circulating concentrations comparable to those in the pregnant cows in a previous survey study (Larson et al., 1997). The positive results from this study may have been due to embryotrophic effects. Maurer and Echternkamp (1982) reported that lesser concentrations of progesterone in the first week after breeding are associated with abnormal embryonic development. A similar pilot study in our laboratory comparing developmental stage of embryos collected from non-superovulated cows to the timing of the onset of the luteal phase indicated that more advanced embryos were collected from animals with earlier onset of the luteal phase (unpublished observations). Green et al. (2005) reported that the production of interferon-tau (IFN-) at Day 16 post-AI was positively correlated with progesterone concentrations on days 4–5. In a previous study, supplementation with progesterone from days 5 to 9 enhanced embryonic IFN- production and trophoblast length (Mann et al., 2006). This would suggest that the maternal progesterone enhances embryonic development and the ability of the embryo to successfully induce maternal recognition of pregnancy. 5. Conclusion The success of the progesterone supplementation regimen in the present study with lactating cattle supports a role for the timely action of progesterone in early embryonic development before
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the maternal recognition of pregnancy. Results of the present study corroborate those from a field trial in New Zealand by (Macmillan and Peterson, 1993), who studied >900 cattle from 11 herds and found that progesterone supplementation only enhanced pregnancy rates (controls = 66.1%, treated = 74.6%; P = 0.02) when administered at Days 4–9 post-insemination. The greater success with the first and second lactation cows is similar to that observed by Villarroel et al. (2004), where PRID administration increased the likelihood of pregnancy only in first and second lactation of repeat breeder cows. The decrease in progesterone content of the CIDR that is currently available in the USA may allow use of new devices with similar results to those of the present study. These results have demonstrated that modest supplementation with progesterone after insemination increased the pregnancy rate in lactating dairy cattle, particularly in first and second lactation cows. The response obtained in the present study is of sufficient magnitude that this method of progesterone supplementation could provide an economic benefit for large, commercial dairy herds, including those where bST is used as a routine management tool. Acknowledgement The authors wish to thank the Aman family of AA Dairy in Candor, New York, for the use of their herd and for the outstanding cooperation of their herd manager and milking crews. References Bilby, T.R., Guzeloglu, A., Kamimura, S., Pancarci, S., Michel, F., Head, H.H., Thatcher, W.W., 2004. Pregnancy and bovine somatotropin in non-lactating dairy cows: I. ovarian, conceptus and insulin-like growth factor system responses. J. Dairy Sci. 87, 3256–3267. Butler, W.R., Calaman, J.J., Beam, S.W., 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74, 858–865. Green, M.P., Hunter, M.G., Mann, G.E., 2005. Relationships between maternal hormone secretion and embryo development on Day 5 of pregnancy in dairy cows. Anim. Reprod. Sci. 88, 179–189. Gumen, A., Guenther, J.N., Wiltbank, M.C., 2003. Follicular size and response to ovsynch versus detection of estrus in anovular and ovular lactating dairy cows. J. Dairy Sci. 86, 3184–3194. Larson, S.F., Butler, W.R., Currie, W.B., 1997. Reduced fertility associated with low progesterone post-breeding and increased milk urea nitrogen in lactating cows. J. Dairy Sci. 80, 1288–1295. Macmillan, K.L., Peterson, A.J., 1993. A new intravaginal progesterone-releasing device for cattle (CIDR-B) for oestrous synchronization, increasing pregnancy rates and the treatment of post-partum anoestrus. Anim. Reprod. Sci. 33, 1–26. Macmillan, K.L., Taufa, V.K., Barnes, D.R., Day, A.M., 1991. Plasma progesterone concentrations in heifers and cows treated with a new intravaginal device. Anim. Reprod. Sci. 26, 25–40. Mann, G.E., Fray, M.D., Lamming, G.E., 2006. Effects of time of progesterone supplementation on embryo development and interferon-tau production in the cow. Vet. J. 171, 500–503. Mann, G.E., Lamming, G.E., 1999. The influence of progesterone during early pregnancy in cattle. Reprod. Dom. Anim. 34, 269–274. Mann, G.E., Lamming, G.E., 2001. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 121, 175–180. Mann, G.E., Lamming, G.E., Payne, J.H., 1998. Role of early luteal phase progesterone in control of the timing of the luteolytic signal in cows. J. Reprod. Fertil. 113, 47–51. Maurer, R.R., Echternkamp, S.E., 1982. Hormonal asynchrony and embryonic development. Theriogenology 17, 11–22. McNeill, R.E., Diskin, M.G., Sreenan, J.M., Morris, D.G., 2006. Associations between milk progesterone concentration on different days and with embryo survival during the early luteal phase in dairy cows. Theriogenology 65, 1435–1441. Nachreiner, R.F., Oschmann, S.J., Edqvist, L.-E., Richards, J.I., 1992. Factors affecting skim milk progesterone assay results. Am. J. Vet. Res. 53, 1085–1089. Robinson, N.A., Leslie, K.E., Walton, J.S., 1989. Effect of treatment with progesterone on pregnancy rate and plasma concentrations of progesterone in holstein cows. J. Dairy Sci. 72, 202–207.
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Sangsritavong, S., Combs, D.K., Sartori, R., Armentano, L.E., Wiltbank, M.C., 2002. High feed intake increases liver blood flow and metabolism of progesterone and estradiol 17B in dairy cattle. J. Dairy Sci. 85, 2831–2842. Sartori, R., Haughian, J.M., Shaver, R.D., Rosa, G.J.M., Wiltbank, M.C., 2004. Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows. J. Dairy Sci. 87, 905–920. Sartori, R., Sartor-Bergfelt, R., Mertens, S.A., Guenther, J.N., Parrish, J.J., Wiltbank, M.C., 2002. Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter. J. Dairy Sci. 85, 2803–2812. Stronge, A.J., Sreenan, J.M., Diskin, M.G., Mee, J.F., Kenny, D.A., Morris, D.G., 2005. Post-insemination milk progesterone concentration and embryo survival in dairy cows. Theriogenology 64, 1212–1224. Van Cleeff, J., Drost, M., Thatcher, W.W., 1991. Effects of post-insemination progesterone supplementation on fertility and subsequent estrous responses of dairy heifers. Theriogenology 36, 795–807. Van Cleeff, J., Lucy, M.C., Wilcox, C.J., Thatcher, W.W., 1992. Plasma and milk progesterone and plasma LH in ovariectomized lactating cows treated with new or used controlled internal drug release devices. Anim. Reprod. Sci. 27, 91–106. Van Cleeff, J., Macmillan, K.L., Drost, M., Lucy, M.C., Thatcher, W.W., 1996. Effects of adminstering progesterone at selected intervals after insemination of synchronized heifers on pregnancy rates and resynchronization of returns to service. Theriogenology 46, 1117–1130. Villarroel, A., Martino, A., BonDurant, R.H., Deletang, F., Sischo, W.M., 2004. Effect of post-insemination supplementation with PRID on pregnancy in repeat-breeder Holstein cows. Theriogenology 61, 1513–1520.
Short communication
Pregnancy rates in lactating dairy cattle following supplementation of progesterone after artificial insemination Sandra F. Larson ∗ , W.R. Butler, W. Bruce Currie Department of Animal Science, Cornell University, Ithaca, NY 14853, USA Received 21 November 2006; accepted 27 February 2007 Available online 3 March 2007
Abstract Poor conception rates in highly productive lactating cattle is especially prevalent in large, intensivelymanaged commercial herds. One of the causative factors is sub-optimal pre-implantation embryonic development which appears to result from inadequate circulating concentrations of progesterone. In the present study, the efficacy of very modest progesterone supplementation, between Days 3.5 and 10 post-AI, on pregnancy rates was determined in a commercial herd where bovine somatotropin (bST) was used as a management tool. All lactating cattle that were deemed to be in estrus and inseminated over a 4-week period were randomly assigned to either a control group (no treatment) or CIDR-1.9 g (previously used for estrous synchronization) treatment from Day 3.5 to Day 10 post-AI. Milk samples were collected four times: on the day of AI, at Day 2 or 3, at Day 4 and at Day 22 post-AI and were analyzed for progesterone content. Data from a total of 130 breedings were used in the final analysis. The CIDR treatment increased circulating concentrations of progesterone in treated animals over those of control animals on Day 4 by 0.7 ng/ml (P < 0.05) and increased pregnancy rate from 35% (22/63) to 48% (32/67) (P = 0.068). The effect of treatment was greater in first and second lactation cows, where pregnancy rates were 33% (18/55) in controls and 51% (31/61) in treated animals (P = 0.03). The results of this study indicate that the timing of onset of the progesterone influence is important for successful pregnancy outcome, particularly in first and second lactation cows. © 2007 Elsevier B.V. All rights reserved. Keywords: Progesterone; Pregnancy; Dairy cattle
∗ Corresponding author. Current address: Department of Biology, Furman University, Greenville, SC 29613, USA. Tel.: +1 864 294 2306; fax: +1 864 294 2058. E-mail address: [email protected] (S.F. Larson).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.02.023
S.F. Larson et al. / Animal Reproduction Science 102 (2007) 172–179
173
1. Introduction One of the factors implicated in low fertility in cattle is low circulating concentrations of progesterone during the pre-implantation phase of embryonic development (reviewed in Mann and Lamming, 1999). Several observational studies of lactating cattle have reported lower postinsemination progesterone in non-pregnant cattle compared to pregnant cattle (Butler et al., 1996; Larson et al., 1997; Stronge et al., 2005; McNeill et al., 2006). In controlled studies where early embryo development was examined in relationship to maternal progesterone concentrations, greater maternal progesterone concentrations were associated with enhanced embryo development (Maurer and Echternkamp, 1982; Mann and Lamming, 2001; Green et al., 2005). In our previous non-interventional study, 60% of the non-pregnant cows had progesterone concentrations > 2 ng/ml on Day 21 post-breeding, and had lower progesterone concentrations on Day 3.5 post-breeding; we hypothesized that pregnancy recognition had been initiated in many of these cows, yet they failed to maintain pregnancy until the Day 40 pregnancy examination (Larson et al., 1997). A number of studies have examined the utility of post-insemination (or AI) supplementation of progesterone to increase pregnancy rates (for review, see Mann and Lamming, 1999). Results from studies where supplemental progesterone was given have varied widely, with some showing increased pregnancy rates (Robinson et al., 1989; Macmillan and Peterson, 1993), others no effect (Van Cleeff et al., 1991; Villarroel et al., 2004), and still others decreased pregnancy rates (Van Cleeff et al., 1996). Most of the studies where supplemental progesterone increased the pregnancy rate used lactating cattle, while no effect of treatment was observed in most studies using heifers. The present study assessed the efficacy of very modest supplementation of progesterone to establish approximately 1 ng/ml plasma concentration of progesterone around Day 4 post-AI, the time when we detected differences in the onset of the luteal phase (Larson et al., 1997). The objective was to determine if progesterone supplementation soon after AI could increase pregnancy rates in lactating dairy cattle. Performing the present study in the context of a commercial dairy was also a critical factor in the design of the experiment in that we desired to evaluate the efficacy and practicality of the treatment in an industry setting under field conditions. 2. Materials and methods 2.1. Animals Grade Holstein cattle in a commercial dairy (450 milking cows, average rolling herd milk production 9534 kg per cow per year) were used in the study. Cows were milked three times a day and were treated with bST (Posilac, Monsanto, St. Louis, MO) every 2 weeks after reaching 70 days in milk. All lactating cattle presented for breeding between 4 October and 30 October was randomly assigned to either a control (no treatment) or treatment group with the CIDR inserted from Day 3.5 to Day 10 post-AI (Day 0 = day of AI). Random assignment was selected over blocking as the aim of the study was to evaluate the efficacy of routine CIDR treatment of all bred animals managed under commercial conditions. Animals with an abnormal vaginal discharge at AI or those presenting difficulties for the inseminator were not included in the study. Data recorded on each cow included days in milk (DIM) production, lactation number, breeding number for current lactation, and average daily milk production. Progesterone treatment was carried out by insertion of an intravaginal progesterone-releasing device (CIDR-B, starting content of new
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devices 1.9 g P4 , InterAg, Hamilton, New Zealand) that had been previously used in cattle in unrelated experiments for 7 to 9 days for estrous synchronization. Previously used CIDRs ensured that progesterone concentrations would be increased only modestly (approximately 1 ng/ml) to avoid possible disruption of the developing corpus luteum or perturbation of the endocrine status of the animal by achieving luteal-phase concentrations of progesterone too early in the estrous cycle (Van Cleeff et al., 1992). The CIDRs which had been stored at 4 ◦ C since their previous use, were thoroughly sanitized by soaking in warm water and chlorhexidine (Nolvasan Surgical Scrub, Fort Dodge Laboratories, Fort Dodge, IA). For the purposes of statistical analysis, cows that were presented twice for AI during the 27 days were treated as two separate animals and were randomly assigned to a treated or control group on each occasion (n = 7). Data were collected on a total of 177 inseminations, a number that exceeded the a priori (n = 120) determined value for the detection of a 10% increase in the pregnancy rate due to treatment at P < 0.1. Reproductive management on the farm included the use of prostaglandin F2␣ for estrous synchronization. Animals were treated with 25 mg i.m. prostaglandin F2␣ (Lutalyse, Upjohn, Kalamazoo, MI) every 14 days starting at approximately 21 days post-partum but were not inseminated until after 40 days post-partum. All inseminations were performed based on estrous behavior, as monitored by the AFIMILK pedometer system (Germania Dairy Automation, Waunakee, WI). A cow exhibiting estrous activity (>180% of a cow’s normal activity) during two consecutive milkings was considered to be in estrus and was inseminated shortly after the second milking. The timing of insemination was based on the previous 18 months of on-farm experience of inseminating cows at various times after increased pedometer readings. The use of pedometers for estrous detection has been reported to be as accurate as visual observation, and is eminently more practical in a large herd such as this, with >100 animals/pen in a freestall barn (Bilby et al., 2004). Animals that were not detected in estrus for >40 days after this AI were palpated per rectum by the herd veterinarian, who was unaware of assigned treatments, to determine pregnancy status. 2.2. Sample processing and progesterone analysis Pre-milking strip samples were collected from each animal on Day 0 (day of AI), Day 2 or 3 (pre-treatment or control equivalent), Day 4 (to evaluate efficacy of treatment), and Day 22 (to provide an additional check for return to estrus). Samples were processed as described previously and a double-antibody RIA was used to measure progesterone concentrations in skim milk (Larson et al., 1997). Coefficients of variation (CV) for the assay were 12 and 24% within assay, and 13 and 15% between assay for milk with greater and lesser concentrations of progesterone, respectively. The use of skim milk for progesterone analysis as opposed to serum is the likely cause of the somewhat greater than desirable CV’s (Nachreiner et al., 1992). In addition, the greatest variability was seen with the lesser progesterone measurements, yet even with variation of ±25%, a 0.5 ng/ml sample would yield a measured range of 0.375–0.625 ng/ml, well within a range expected in a cow in estrus. 2.3. Statistical analysis After all samples were analyzed for progesterone content, some animals (breedings) were excluded for one or more of the following reasons: (1) >1.5 ng/ml progesterone on day of AI (likely misbred, n = 20, an 11% false positive rate); (2) milk sample sets missing more than 1 sample (n = 17); (3) animals inadvertently injected with prostaglandin after AI (n = 5); (4) animals
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sold before pregnancy determination (n = 5). Data from a total of 130 inseminations were used in the final analysis. Changes in progesterone content of pre- and post-treatment (or control equivalent) samples were calculated for individual animals in order to evaluate the incremental increase in progesterone. This increment and the concentrations of progesterone on individual days were compared by analysis of variance, and differences between means were calculated by Scheffe’s post-hoc test. The outcome of pregnancy determination fit a binary distribution; therefore, a non-parametric test of two proportions (a one-tailed Z-test) was used to determine if pregnancy rate was increased by treatment. After analysis of all cows in the study, the Z-test was applied to the pregnancy data from the group consisting of first and second lactation cows. Logistic regression analysis was carried out using lactation number, days in milk production, number of times bred and CIDR treatment to determine which variables were predictors of pregnancy outcome. Logistic regression analysis was performed on SPSS statistical software (v. 15, Chicago, IL), and Z-tests were performed on Number Cruncher Statistical Systems (Kaysville, UT). 3. Results Treatment increased the mean progesterone concentration by 0.7 ng/ml (P < 0.05) on the day after CIDR placement (Day 4) compared with that of the controls (Table 1). The incremental increase in progesterone from pre- to post-treatment (or equivalent) samples was 1.13 ng/ml in CIDR cows versus 0.43 ng/ml in control cows (P < 0.05). Concentrations of progesterone were not different between pregnant and non-pregnant cows in any milk samples prior to Day 22 samples. On Day 22, cows in both groups that were confirmed as being pregnant had greater concentrations of progesterone (5.45 and 4.93 ng/ml in control and treated cows, respectively) than did the cows that either returned to estrus before the seventh week pregnancy check (1.71 ng/ml) or were non-pregnant when palpated in the seventh week (1.79 ng/ml). Progesterone enhanced pregnancy rates from 35% in controls to 48% in CIDR-treated cows (P = 0.068; Table 2). The random assignment of cows to treatment was successful in creating treatment and control groups that were quite similar in parity, DIM and average daily milk production (Table 3). Logistic regression analysis was performed to verify the results of the Z-test, and to determine whether inclusion of the variables of parity, DIM and average daily milk production improved the predictive capability of the statistical model beyond that of the effect of CIDR treatment alone. The effect of CIDR treatment on pregnancy outcome was identical to the Z-test analysis (P = 0.068), and inclusion of the additional variables did not improve the fit of the model. The similarities between the treated and control groups and the corresponding statistical analysis suggest that the difference in pregnancy rate was due to the progesterone treatment rather than to Table 1 Mean (± S.E.M.) skim-milk concentrations of progesterone (ng/ml) of dairy cows treated with CIDRs starting at Day 3.5 post-insemination Treatment
n
Days 2–3
Day 4
Incrementa
Control CIDR
63 67
1.00 ± 0.05 0.96 ± 0.05
1.43 b ± 0.1 2.09 c ± 0.09
0.43 b ± 0.09 1.13 c ± 0.09
Letters (b, c) indicate differences (P < 0.05) within columns. a Increment from pre- to post-treatment samples were calculated for individual animals to generate means and standard errors.
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Table 2 Effect of CIDR treatment from Day 3.5 to Day 10 post-insemination on pregnancy status of lactating dairy cattle Treatment
All cows Control CIDR
n
Not pregnant
63 67
First and second lactation cows only Control 55 CIDR 61
Pregnant
Estrus-day 21a
Extended estrous cyclesb
31 (49) 24 (36)
10 (16) 11 (16)
22 (35)c 32 (48)c
27 (49) 22 (36)
10 (18) 8 (13)
18 (33)d 31 (51)d
Number of animals per pregnancy status category (%). a Open animals with progesterone <2 ng/ml on Day 22 post-insemination. b Open animals with progesterone ≥2 ng/ml on Day 22 post-insemination. c Treatment effect = + 13%, P = 0.068. d Treatment effect = + 18%, P = 0.03. Table 3 Mean (± S.E.M.) lactation number, days in milk (DIM) and average daily milk production for control and CIDR-treated cows Treatment
n
Lactation number
DIM at AI
Average milk production (kg/d)
Control Not pregnant Pregnant
41 22
1.9 ± 0.2 1.9 ± 0.2
158 ± 20 133 ± 22
31.7 ± 1.1 30.0 ± 1.6
CIDR Not pregnant Pregnant
35 32
1.9 ± 0.1 1.7 ± 0.1
148 ± 19 126 ± 17
32.1 ± 1.2 32.5 ± 1.2
an underlying difference in fertility between the control and treatment group. Considering only first and second lactation cows, the difference in pregnancy rates was even more striking, with controls at 33% pregnant and treated cows at 51% (P = 0.03; Table 2). Our previous survey study (Larson et al., 1997) revealed a population of non-pregnant cows with lesser progesterone early after breeding, yet these cows had greater circulating concentrations of progesterone (mean = 4.5 ng/ml) on Day 21. Cows were classified as having extended estrous cycles, likely due to the presence of an embryo initiating maternal recognition of pregnancy, but failing to maintain pregnancy until the seventh week. The response to treatment in the present study was not due to a decreased incidence of cows with extended estrous cycles (10/63 controls, 11/67 CIDR-treated cows) as had been originally hypothesized from the previous study. 4. Discussion The beneficial effects of augmentation of circulating concentrations of progesterone on the pregnancy rate in lactating dairy cows this study corroborate the results of previous studies (Robinson et al., 1989, Macmillan and Peterson, 1993). The present study was designed to carefully address both the appropriate timing and magnitude of the progesterone rise post-breeding. Utilization of a new CIDR (1.9 g P4 ) through the first week starting immediately after breeding
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caused decreased pregnancy rates in heifers (Van Cleeff et al., 1996). The time period of progesterone supplementation in the study was selected based on previous observations of pregnant cows having greater progesterone around Day 4, and calculations from the previous study of earlier initiation (Day 3.25) of luteal function in pregnant than in non-pregnant cows with extended estrous cycles (Day 3.99) (Butler et al., 1996, Larson et al., 1997, Mann and Lamming, 2001, Gumen et al., 2003, Stronge et al., 2005, McNeill et al., 2006). The experimental use of the previously used CIDR ensured that circulating concentrations of progesterone would not be achieved abruptly in the mid-luteal phase, too early in the estrous cycle (Mann et al., 1998). Previous studies have revealed that CIDRs (starting with 1.9 g P4 ) used for 9 days contain approximately 1 g P4 (Macmillan et al., 1991), and that re-use of the devices increased progesterone concentrations in ovariectomized lactating cows to 1 to 1.5 ng/ml (Van Cleeff et al., 1992), hence the decision in the present study to employ previously used CIDRs. Use of this protocol does not imply advocation of the routine re-use of CIDRs. After the present study was performed, the progesterone content of new CIDR devices made available in the USA was decreased to 1.38 g. These new CIDR devices may prove to be suitable for post-insemination progesterone supplementation with similar success to that described for the present study. Increased pregnancy rates associated with augmentation of progesterone concentrations in the early luteal phase of the estrous cycle are most often found when the lactating cow is the experimental animal. Embryo quality is decreased in lactating cows as compared with non-lactating cows and heifers (Sartori et al., 2002). The rate of metabolism of progesterone is increased in lactating cows compared to heifers (Sangsritavong et al., 2002), and lactating cattle tend to have lesser concentrations of sex steroid hormones than heifers or dry cows (Sartori et al., 2004). The increased productivity associated with bST use likely increases clearance rates of progesterone as is seen in highly productive cattle that are not treated with bST (Sangsritavong et al., 2002), therefore, supplemental progesterone may be particularly beneficial for highly productive cattle regardless of bST use. In the present study, lactating cows were used exclusively, and we also had the advantage of having a large number of cows inseminated within a short interval, minimizing any seasonal effects on reproductive success, within a single herd. In addition, the progesterone regimen in the present study was carefully engineered to mimic the timing of the observed progesterone increase and to establish appropriate circulating concentrations comparable to those in the pregnant cows in a previous survey study (Larson et al., 1997). The positive results from this study may have been due to embryotrophic effects. Maurer and Echternkamp (1982) reported that lesser concentrations of progesterone in the first week after breeding are associated with abnormal embryonic development. A similar pilot study in our laboratory comparing developmental stage of embryos collected from non-superovulated cows to the timing of the onset of the luteal phase indicated that more advanced embryos were collected from animals with earlier onset of the luteal phase (unpublished observations). Green et al. (2005) reported that the production of interferon-tau (IFN-) at Day 16 post-AI was positively correlated with progesterone concentrations on days 4–5. In a previous study, supplementation with progesterone from days 5 to 9 enhanced embryonic IFN- production and trophoblast length (Mann et al., 2006). This would suggest that the maternal progesterone enhances embryonic development and the ability of the embryo to successfully induce maternal recognition of pregnancy. 5. Conclusion The success of the progesterone supplementation regimen in the present study with lactating cattle supports a role for the timely action of progesterone in early embryonic development before
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the maternal recognition of pregnancy. Results of the present study corroborate those from a field trial in New Zealand by (Macmillan and Peterson, 1993), who studied >900 cattle from 11 herds and found that progesterone supplementation only enhanced pregnancy rates (controls = 66.1%, treated = 74.6%; P = 0.02) when administered at Days 4–9 post-insemination. The greater success with the first and second lactation cows is similar to that observed by Villarroel et al. (2004), where PRID administration increased the likelihood of pregnancy only in first and second lactation of repeat breeder cows. The decrease in progesterone content of the CIDR that is currently available in the USA may allow use of new devices with similar results to those of the present study. These results have demonstrated that modest supplementation with progesterone after insemination increased the pregnancy rate in lactating dairy cattle, particularly in first and second lactation cows. The response obtained in the present study is of sufficient magnitude that this method of progesterone supplementation could provide an economic benefit for large, commercial dairy herds, including those where bST is used as a routine management tool. Acknowledgement The authors wish to thank the Aman family of AA Dairy in Candor, New York, for the use of their herd and for the outstanding cooperation of their herd manager and milking crews. References Bilby, T.R., Guzeloglu, A., Kamimura, S., Pancarci, S., Michel, F., Head, H.H., Thatcher, W.W., 2004. Pregnancy and bovine somatotropin in non-lactating dairy cows: I. ovarian, conceptus and insulin-like growth factor system responses. J. Dairy Sci. 87, 3256–3267. Butler, W.R., Calaman, J.J., Beam, S.W., 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74, 858–865. Green, M.P., Hunter, M.G., Mann, G.E., 2005. Relationships between maternal hormone secretion and embryo development on Day 5 of pregnancy in dairy cows. Anim. Reprod. Sci. 88, 179–189. Gumen, A., Guenther, J.N., Wiltbank, M.C., 2003. Follicular size and response to ovsynch versus detection of estrus in anovular and ovular lactating dairy cows. J. Dairy Sci. 86, 3184–3194. Larson, S.F., Butler, W.R., Currie, W.B., 1997. Reduced fertility associated with low progesterone post-breeding and increased milk urea nitrogen in lactating cows. J. Dairy Sci. 80, 1288–1295. Macmillan, K.L., Peterson, A.J., 1993. A new intravaginal progesterone-releasing device for cattle (CIDR-B) for oestrous synchronization, increasing pregnancy rates and the treatment of post-partum anoestrus. Anim. Reprod. Sci. 33, 1–26. Macmillan, K.L., Taufa, V.K., Barnes, D.R., Day, A.M., 1991. Plasma progesterone concentrations in heifers and cows treated with a new intravaginal device. Anim. Reprod. Sci. 26, 25–40. Mann, G.E., Fray, M.D., Lamming, G.E., 2006. Effects of time of progesterone supplementation on embryo development and interferon-tau production in the cow. Vet. J. 171, 500–503. Mann, G.E., Lamming, G.E., 1999. The influence of progesterone during early pregnancy in cattle. Reprod. Dom. Anim. 34, 269–274. Mann, G.E., Lamming, G.E., 2001. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 121, 175–180. Mann, G.E., Lamming, G.E., Payne, J.H., 1998. Role of early luteal phase progesterone in control of the timing of the luteolytic signal in cows. J. Reprod. Fertil. 113, 47–51. Maurer, R.R., Echternkamp, S.E., 1982. Hormonal asynchrony and embryonic development. Theriogenology 17, 11–22. McNeill, R.E., Diskin, M.G., Sreenan, J.M., Morris, D.G., 2006. Associations between milk progesterone concentration on different days and with embryo survival during the early luteal phase in dairy cows. Theriogenology 65, 1435–1441. Nachreiner, R.F., Oschmann, S.J., Edqvist, L.-E., Richards, J.I., 1992. Factors affecting skim milk progesterone assay results. Am. J. Vet. Res. 53, 1085–1089. Robinson, N.A., Leslie, K.E., Walton, J.S., 1989. Effect of treatment with progesterone on pregnancy rate and plasma concentrations of progesterone in holstein cows. J. Dairy Sci. 72, 202–207.
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