Minimal progesterone concentration required for embryo survival after embryo transfer in lactating Holstein cows

Animal Reproduction Science 136 (2013) 223–230

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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Minimal progesterone concentration required for embryo survival after embryo transfer in lactating Holstein cows A.G. Kenyon a , L.G.D. Mendonc¸a a , G. Lopes Jr. a , J.R. Lima a , J.E.P. Santos b , R.C. Chebel a,c,∗ a b c

Veterinary Medicine Teaching and Research Center, University of California Davis, Tulare 93274, USA Department of Animal Sciences, University of Florida, Gainesville 32911, USA Department of Veterinary Population Medicine, University of Minnesota, Saint Paul 55108, USA

a r t i c l e

i n f o

Article history: Received 10 March 2012 Received in revised form 12 September 2012 Accepted 22 October 2012 Available online 29 October 2012 Keywords: Progesterone Lactating Holstein cow Embryo survival

a b s t r a c t Objectives were to determine progesterone concentration (P4) from days 4 to 28 relative to presumptive estrus necessary for maintenance of pregnancy in lactating Holstein cows. Cows were assigned to the low P4 (LowP4, n = 28) or control (n = 153) treatments. All cows were presynchronized with two injections of PGF2␣ (14 d apart) and an ovulation synchronization protocol (11 d later; GnRH on day −10, PGF2␣ on day −3; and GnRH on day 0 = presumptive estrus). Cows in the Low P4 treatment received 2 injections of prostaglandin F2␣ on days 4 and 5 (day 0 = presumptive estrus) and a new CIDR insert on day 5 that was replaced every 7 d until day 28. Blood was sampled on days −9, −2, 0, 4, 7,14, 21, and 28. Ovaries were examined with ultrasound on days −9, −3, and 7 and cows bearing a corpus luteum ≥20 mm on day 7 received an embryo. On days 0, 4 and 7 P4 did not differ (P ≥ 0.27) but control cows had greater (P < 0.01) P4 on days 14, 21, and 28. Progesterone concentration fold change from day 0 to 7 was not (P = 0.14) affected by treatment, but P4 concentration fold change from day 7 to 14 was (P < 0.01) greater for control cows compared with LowP4 cows. No LowP4 cows became pregnant after embryo transfer, whereas 35.7, 25.5, and 21.4% of control cows were pregnant on day 28, 42, and 63, respectively. Progesterone concentration fold changes from day 0 to 7 (P = 0.03) and from day 7 to 14 (P = 0.05) were associated with pregnancy outcomes on day 63. Among cows that were pregnant on day 63, the minimum P4 concentration fold changes from day 0 to 7 and from day 7 to 14 were 2.71 and 1.48, respectively. Interestingly, cows with P4 concentration <5 ng/mL on day 14 were (P = 0.01) and tended to be (P = 0.07) more likely to lose pregnancy from day 28 to 42 and from day 28 and 63, respectively. Faster rise in P4 concentration during the metestrus and early diestrus are associated with pregnancy establishment following embryo transfer, which suggests that early rise in P4 concentration has an indirect effect on embryo development through modulation of uterine environment and secretion of histotroph. Furthermore, the positive effects of early rise in P4 concentration appear to go beyond the phase of maternal recognition of pregnancy through adhesion and placentation stages. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: 1365 Gortner Ave., Saint Paul, MN 55108, United States. Tel.: +1 612 625 3130; fax: +1 612 625 6421. E-mail address: [email protected] (R.C. Chebel). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.10.014

Several studies have been conducted to determine the association between progesterone (P4) concentrations and embryo development up to the phase of maternal recognition of pregnancy, approximately day 16 of the estrous

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cycle (Thatcher et al., 1984). In general, it is accepted that increasing concentrations of P4 during metestrus and early diestrus enhances the growth of embryos and production of interferon-tau (IFN-␶; Geisert et al., 1992), which is expected to improve the cross-talk between the embryo and the uterus and the likelihood of embryo survival (Mann and Lamming, 2001). It is likely, however, that P4 concentration around the time of maternal recognition of pregnancy and placentation also plays a significant role on pregnancy establishment and retention. In a study in which inseminated cows had blood sampled on week 5 of presumed pregnancy, 50% of cows with P4 concentration ≤2.8 ng/ml aborted before week 9 of gestation and P4 concentration on week 5 that resulted in 95% of pregnancy maintenance was 6.0 ng/ml (Starbuck et al., 2004). Similarly, a non-linear relationship between P4 concentration at 31 d after AI and pregnancy retention from 31 to 66 d has been demonstrated, such that cows with P4 concentration <5ng/ml were more likely to lose pregnancy (Scanavez et al., 2011). Furthermore, when using a P4 concentration cut off ≤4.39 ng/ml on day 31 after insemination, pregnancy loss from 31 to 66 d was predicted with a specificity of 87.1% and a negative predictive value of 93.9%, but sensitivity (46.2%) and positive predictive values (27.3%) were poor (Scanavez et al., 2011). Beef cows treated with two new or two used controlled internal drug-release (CIDR; Pfizer Animal Health, Madison, MI, USA) inserts containing 1.38 g of P4 starting on day 28 of gestation and that had the corpora lutea (CL) enucleated manually were less likely to maintain pregnancy than non-enucleated untreated cows (Rhinehart et al., 2009). As indicated by the authors, however, it was not possible to determine a minimal P4 concentration necessary to maintain pregnancy because the P4 concentration of CIDR-treated enucleated cows did not vary sufficiently (Rhinehart et al., 2009), and likely because of the small number of experimental units. The associations between P4 concentrations during mid to late diestrus and survival of embryos are less understood. Embryo transfer eliminates problems with fertilization and it is a good model to evaluate maintenance of pregnancy in cattle. It was hypothesized that P4 concentrations on days 7 and 14 and average P4 concentration between days 7 and 14 of the estrous cycle are associated with maintenance of pregnancy after ET in lactating dairy cows. Therefore, the objectives of the current experiment were to evaluate the association between P4 concentration at different days of the estrous cycle and pregnancy maintenance after ET to determine the minimal P4 concentration necessary for maintenance of pregnancy in lactating dairy cows. 2. Material and methods

daily for ad libitum consumption to meet or exceed the NRC (NRC, 2001) requirements for dairy cows weighing 680 kg, producing 42 kg of milk/d containing 3.5% fat and consuming 27 kg of dry matter per day. At 32 ± 3 d in milk (DIM) cows had their estrous cycle presynchronized with two injections of prostaglandin (PG) F2␣ (25 mg of dinoprost tromethamine; Lutalyse; Pfizer Animal Health, Madison, NJ, USA) administered 14 d apart with the addition of a CIDR insert containing 1.38 g of P4 from 39 ± 3 to 46 ± 3 DIM. Cows were then enrolled in an ovulation synchronization protocol at 57 ± 3 DIM (day −10; day 0 = presumptive estrus) and received an injection of GnRH (100 ␮g of gonadorelin diacetatetetrahydrate; Cystorelin, Merial Ltd., Iselin, NJ, USA) on the same day, an injection of PGF2␣ on day −3, and a final injection of GnRH on day 0.

2.2. Treatments and embryo transfer Fig. 1 depicts the schedule of activities. On day −10, cows were balanced for parity and body condition score (BCS) and were randomly allocated to control (control, n = 153) or low P4 (LowP4, n = 28) treatments in a 5:1 ratio. This was done because the initial hypothesis was that LowP4 cows would not become pregnant and the study was conducted in a commercial herd. As such, after balancing for parity and BCS cows were assigned to treatments using six pieces of paper being that in five of them control had been written and in one of them LowP4 had been written. Control cows received no further treatment, whereas cows in the LowP4 treatment received two injections of 25 mg of PGF2␣ , on days 4 and 5, and a new CIDR insert every 7 d from day 5 to 28. Cows bearing a CL ≥20 mm in diameter on day 7 received a frozen/thawed embryo on the same day. On Day 7 (day of ET) cows were restrained in self-locking stanchions and regional anesthesia performed using a caudal epidural block with 4 ml of lidocaine hydrochloride 2% (Lidocaine Hydrochloride Injectable-2%; Vedco Inc., St. Joseph, MO, USA). Embryos were thawed for 10 s in air and for 20 s in water bath at 35 ◦ C and were transferred transcervically by a single operator in the uterine horn ipsilateral to the largest CL. All embryos were derived from superstimulated cows from the same dairy. Donors were inseminated twice with frozen-thawed semen from a single sire. Embryos were collected 6.5 d after insemination and only excellent or good quality embryos (IETS, 1998) were frozen using a Dulbecco’s-modified PBS enriched with 1.5 M ethylene glycol, 0.4% BSA, and 0.1 M sucrose (ViGro Freeze Plus; Bioniche Animal Health, Belleville, ON) for later transfer.

2.1. Animals, housing, diets, and synchronization of the estrous cycle 2.3. Ultrasonography and blood samples Lactating Holstein cows (n = 181) from a dairy in the San Joaquin Valley of California were used in the study. The herd had approximately 900 lactating cows with a rolling herd average of 11,236 kg of 3.5% fat-corrected milk. All cows were housed in free-stall barns and milked twice daily. Cows were fed a total mixed ration twice

All cows had their ovaries examined by ultrasonography (5.0 MHz linear transrectal probe, Aloka SSD500, Aloka America, Wallingford, CT) on days −10, −3, and 7. Follicles ≥10 mm in diameter and CL were recorded and measured. Total volume of luteal tissue was calculated

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Fig. 1. Schematic treatment of lactating dairy cows enrolled in the control and LowP4 treatments. CIDR = controlled internal drug release insert containing 1.38 g of progesterone, Pfizer Animal Health, Madison, MI, USA; ET = embryo transfer; GnRH = 100 ␮g of gonadorelin diacetate tetrahydrate; Cystorelin, Merial Ltd., Iselin, NJ, USA; PGF2␣ = 25 mg of dinoprost tromethamine; Lutalyse; Pfizer Animal Health, Madison, NJ, USA; US = ultrasound (5.0 MHz linear transrectal probe, Aloka SSD500, Aloka America, Wallingford, CT) examination of the ovaries; and open circles () = blood sampled for determination of progesterone concentration.

using the formula adapted from Lopez et al. (2005):

total luteal volume =



 4 ×× 3

 −

4 ×× 3





(da /2 + db /2) 2

(ca /2 + cb /2) 2

3 

3 

where da,b and ca,b are orthogonal luteal and cavity dimensions, respectively, for the nth CL. Blood was sampled from all cows on days −10, −3, 0, 7, and 14 for determination of P4 concentrations. Additional blood samples were collected on days 4, 21, and 28 from all LowP4 cows and from a sub-group of control cows (n = 54) and analyzed for P4. Blood was sampled by puncture of coccygeal vessels into evacuated tubes containing K2 EDTA (Becton Dickinson, Franklin Lakes, NJ, USA) and was immediately placed in ice. Chilled samples were then centrifuged at 2000 × g for 15 min and the plasma separated for storage at −20 ◦ C. Progesterone analysis was carried out on thawed plasma samples using commercial RIA kits (Coat-A-Count 17␣-OH Progesterone, Siemens Healthcare Diagnostics, Los Angeles, CA, USA). The sensitivity of the P4 assay was 0.08 ng/ml and the inter- and intra-assay CV were 7.0 and 5.2%, respectively. Among control cows, P4 concentrations on days −3, 0, and 7 were used to determine synchrony of the estrous cycle. As such, cows were considered to have had the estrous cycle precisely synchronized when P4 on day −3 ≥ 1.0 ng/ml, P4 on day 0 < 1.0 ng/ml, and P4 on day 7 ≥ 1.0 ng/ml. Because all control cows with synchronized estrous cycle had P4 ≥ 0.55 ng/ml on day 4, cows in the LowP4 treatment were considered to have had the estrous cycle precisely synchronized when P4 on day −3 ≥ 1.0 ng/ml, P4 on day 0 < 1.0 ng/ml, and P4 on day 4 ≥ 0.55 ng/ml.

Fold change in P4 concentration from day 0 to day 7and from day 7 to day 14 were calculated by dividing the P4 concentration on day 7 by P4 concentration on day 0 and by dividing P4 concentration on day 14 by P4 concentration on day 7, respectively. Cows were classified as having P4 concentration on day 14 < 5.0 ng/ml or as having P4 concentration on day 14 ≥ 5.0 ng/ml to determine if this cut-off, which is similar to that reported by other studies (Scanavez et al., 2011; Starbuck et al., 2004) is associated with incidence of pregnancy loss.

2.4. Pregnancy diagnoses and assessment of reproductive outcomes Pregnancy was diagnosed by ultrasound on day 28, 42, and 63 based on identification of an embryo with a heartbeat. Percentages of cows pregnant on day 28, 42, and 63 were calculated by dividing the number of pregnant cows by the number of cows receiving an embryo.

2.5. Body condition score and milk yield Body condition score (BCS; 1 = emaciated to 5 = obese; Ferguson et al., 1994) was recorded for all cows at the time of the first GnRH injection of the ovulation-synchronization protocol. Yields of milk and milk components were recorded from monthly Dairy Herd Improvement Association tests. Energy corrected milk (ECM) yield was calculated for each test using the formula: ECM (kg) = [(kg milk) × 0.327] + [(kg fat) × 12.95] + [(kg protein) × 7.2] (Orth, 1992). Average ECM value for each cow was then calculated as the average of the first 3 months postpartum.

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2.6. Study design and statistical analysis Design of the experiment was randomized with cows balanced for parity (primiparous and multiparous) and BCS and allocated randomly to one of two treatments in a ratio of 5:1. The number of experimental units (cow) initially planned per treatment (20 cows) was expected to provide sufficient power to determine statistical significance when percentage of cows pregnant after ET between treatments differed in 22 percentage units and pregnancy per ET in the control treatment ranges from 25 to 30% (˛ = 0.05; ˇ = 0.20). For purposes of statistical analyses cows were grouped based on treatment (control or LowP4) or based on treatment and pregnancy outcome on day 63 (control/pregnant, control/non-pregnant, or LowP4). Continuous data were analyzed by ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) and continuous data with repeated measures were analyzed by ANOVA for repeated measures using the MIXED procedure of SAS. Models included treatment or treatment/outcome, parity, ECM yield, and BCS and, when appropriate, day and the interaction between treatment or treatment/outcome and day. Independent variables with P > 0.15 according to the initial model were removed and new models were generated until all independent variables remaining in the model had P ≤ 0.15. Differences in pregnancy outcomes between treatments (control vs. LowP4) were analyzed by Fisher’s exact test using the FREQ procedure of SAS with the ‘fisher’ option for statistical analysis. The associations between P4 concentration at different time points and pregnancy outcomes or pregnancy loss were evaluated by logistic regression using the LOGISTIC procedure of SAS. For such, the model included parity, BCS, ECM yield, and P4 concentration (day 0, day 7, day 14, average between days 7 and 14, P4 concentration fold change from day 0 to 7, and P4 concentration fold change from day 7 to 14). The independent variables referring to P4 concentration (day 0, day 7, day 14, average between days 7 and 14, P4 concentration fold change from day 0 to 7, and P4 concentration fold change from day 7 to 14) were included separately in the model to evaluate their individual effect on the outcome of interest and together in the model to determine which of them were the most important predictor of the outcome of interest. All independent variables were included in the model at first and were removed in a stepwise elimination process if their P > 0.15 according to the Wald statistic criterion. Effects of P4 concentration fold change from day 7 to 14 on probability of pregnancy on day 63 according to different P4 concentration fold change from day 0 to 7 were modeled using logistic regression curves based on the intercept and the coefficient estimates from the analysis applied to the formula P=

1+



1 ea+b1 X1 +a+b2 X2 ...



To evaluate the probability of pregnancy on day 63 based on P4 concentration fold change from day 7 to 14 the lowest, median, and highest P4 concentration fold change from day 0 to 7 were used.

Table 1 Effect of treatment on synchrony of the estrous cycle, luteal volume on day 7, and pregnancy outcomes at different intervals after embryo transfer (ET).

Number of cows Cows receiving an embryo (%) Cows receiving an embryo with synchronized estrous cycle (%) Luteal volume on day 7 (cm3 ) Cows pregnant on day 28 (%) Cows pregnant on day 42 (%) Cows pregnant on day 63 (%) Pregnancy loss from day 28 to 42 (%) Pregnancy loss from day 28 to 63 (%)

Control

LowP4

153 88.2 73.4

28 89.3 68.0

6.4 ± 0.3 36.2 25.5 20.4 29.4 42.4

2.0 ± 0.7 0 0 0 N/A N/A

P-value – 0.73 0.70 <0.01 <0.01 0.02 0.04 – –

Statistical significance was defined as P ≤ 0.05 and statistical tendencies as 0.05 < P < 0.15. 3. Results Days postpartum at enrolment (control = 54.5 ± 0.3 d, LowP4 = 54.8 ± 0.6 d; P = 0.59), daily average ECM yield in the first 90 d postpartum (control = 42.2 ± 0.6 kg/d, LowP4 = 41.2 ± 1.4 kg/d; P = 0.51), and BCS (control = 2.85 ± 0.04, LowP4 = 2.96 ± 0.09; P = 0.21) were not different between treatments. Percentage of cows with a CL ≥ 20 mm on day 7 was not (P = 0.73) different between treatments. Furthermore, among cows that had a CL ≥ 20 mm on day 7, the percentage of them that had the estrous cycle synchronized was not (P = 0.70) different between treatments (Table 1). Only data from cows that had a CL ≥ 20 mm on day 7 and had their estrous cycle synchronized are presented and discussed. Among these cows, the luteal volume on day 7 was greater (P < 0.01) for control cows compared with LowP4 cows (Table 1). 3.1. Progesterone concentration No differences in P4 concentrations on days −10 (control = 3.1 ± 0.2 ng/ml, LowP4 = 3.1 ± 0.5 ng/ml; P = 0.91) and −3 (control = 6.3 ± 0.2 ng/ml, LowP4 = 6.0 ± 0.5 ng/ml; P = 0.65) were observed between treatments. Concentration of P4 from day 0 to 14 was (P < 0.01) greater for control cows compared with LowP4 cows (2.7 ± 0.1 vs. 1.5 ± 0.2 ng/ml). Concentration of P4 from day 0 to 14 was (P < 0.01) affected by the interaction between treatment and day. Such an interaction was observed because on day 0 (P = 0.64), day 4 (P = 0.78), and day 7 (P = 0.27) there were no differences in P4 concentrations between treatments (Fig. 2A). On day 14, however, control cows had (P < 0.01) greater P4 concentration than LowP4 cows (Fig. 2A). There was no (P = 0.14) difference between treatments on P4 concentration fold change from day 0 to 7 (control = 12.5 ± 1.6 vs. LowP4 = 6.4 ± 3.8). Progesterone concentration fold change from day 7 to 14 was (P < 0.01) different between treatments (control = 2.6 ± 0.1 vs. LowP4 = 1.1 ± 0.2). When cows were grouped based on treatment and pregnancy outcome on day 63 to evaluate P4 concentration from day 0 to 14, pregnant cows (3.0 ± 0.2 ng/ml) and

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Progesterone, ng/ml

6

LowP4

A

Control

5 4 3 2 1 0

Progesterone, ng/ml

7

0

B

4 7 Days relative to presumptive estrus LowP4

Control/Pregnant

14

Control/Not pregnant

6 5 4 3 2 1 0

0

4 7 Days relative to presumptive estrus

14

Fig. 2. (A) Progesterone concentrations according to treatment. Effect of treatment – P < 0.01, day – P < 0.01, and the interaction between treatment and day – P < 0.01. (B) Progesterone concentrations according to treatment and pregnancy outcome 56 d after ET. Effect of treatment – P < 0.01, day–P< 0.01, and the interaction between treatment and day – P < 0.01.

non-pregnant cows (2.7 ± 0.1 ng/ml) had (P < 0.01) greater P4 concentrations than LowP4 cows (1.5 ± 0.2 ng/ml), but P4 concentration from day 0 to 14 did not (P = 0.21) differ between pregnant and non-pregnant cows. The interaction between treatment/pregnancy outcome on day 63 and day affected (P < 0.01) P4 concentration from day 0 to 14 (Fig. 2B). Pregnant cows had (P ≤ 0.03) greater P4 concentration on day 14 compared with non-pregnant and LowP4 cows but no differences were observed on day 0 (P ≥ 0.64), 4(P ≥ 0.22), and 7(P ≥ 0.64). Pregnant cows (6.4 ± 1.7 ng/ml) tended to (P = 0.08) and had (P = 0.02) greater P4 concentration on day 21 than nonpregnant (3.2 ± 0.6 ng/ml) and LowP4 (1.9 ± 0.7 ng/ml) cows, respectively, but there was no (P = 0.20) difference in P4 concentration on day 21 between non-pregnant and LowP4 cows. On day 28, P4 concentrations of pregnant (6.3 ± 2.2 ng/ml) and non-pregnant cows (5.6 ± 0.7 ng/ml) tended to be (P = 0.06) and were (P < 0.01), respectively, greater than P4 concentration of LowP4 cows (1.6 ± 0.9 ng/ml), but P4 concentration on day 28 of pregnant and non-pregnant cows did not (P = 0.78) differ.

3.2. Pregnancy and pregnancy loss As expected, treatment affected (P ≤ 0.04) percentage of cows pregnant at different intervals after ET because no cows from the LowP4 treatment were diagnosed pregnant, whereas in the control treatment 36.2, 25.5, and 20.4% of cows were diagnosed pregnant on day 28, 42, and 63, respectively (Table 1).

227

3.2.1. Analysis of the association between P4 concentration at different time points and pregnancy outcomes Percentage of cows pregnant on day 28 tended to (P = 0.10) be affected by P4 concentration on day 0. Progesterone concentration on day 7 was not (P = 0.57) associated with percentage of cows pregnant on day 28. On the other hand, P4 concentration on day 14 (P < 0.01), average P4 concentration on day 7and 14 (P = 0.01), P4 concentration fold change from day 0 to 7 (P = 0.03), and P4 concentration fold change between day 7 and 14 (P = 0.02) were positively associated with percentage of cows pregnant on day 28. Percentage of cows pregnant on day 42 was (P = 0.03) positively affected by P4 concentration on day 0. Progesterone concentration on day 7 was not (P = 0.16) associated with percentage of cows pregnant on day 42. On the other hand, P4 concentration on day 14 (P < 0.01), average P4 concentration on day 7 and 14 (P < 0.01), P4 concentration fold change from day 0 to 7 (P < 0.01), and P4 concentration fold change between day 7 and 14 (P = 0.05) were positively associated with percentage of cows pregnant on day 42. Percentage of cows that lost pregnancy from day 28 to 42 tended (P = 0.09) to be affected by P4 concentration on day 0. On the other hand, P4 concentration on day 7 (P = 0.37), P4 concentration on day 14 (P = 0.13), average P4 concentration from day 7 to 14 (P = 0.14), P4 concentration fold change from day 0 to 7 (P = 0.13), and P4 concentration fold change from day 7 to 14 (P = 0.98) were not associated with pregnancy loss from day 28 to 42. Percentage of cows pregnant on day 63 was not affected by P4 concentration on day 0 (P = 0.19) or P4 concentration on day 7 (P = 0.31). On the other hand, P4 concentration on day 14 (P = 0.03), average P4 concentration on day 7 and 14 (P = 0.04), and P4 concentration fold change from day 0 to 7 (P = 0.04) were positively associated with percentage of cows pregnant on day 63. Percentage of cows pregnant on day 63 tended (P = 0.06) to be positively affected by P4 concentration fold change between day 7 and 14. Percentage of cows that lost pregnancy from day 28 to 63 was not affected by P4 concentration on day 0 (P = 0.80), P4 concentration on day 7 (P = 0.40), P4 concentration on day 14 (P = 0.45), average P4 concentration from day 7 to 14 (P = 0.37), P4 concentration fold change from day 0 to 7 (P = 0.83), or P4 concentration fold change from day 7 to 14 (P = 0.95).

3.2.2. Analysis of the association between P4 concentration and pregnancy outcomes Percentage of cows pregnant on day 28 was positively affected by P4 concentration fold change from day 0 to 7 (P = 0.02) and by P4 concentration fold change from day 7 to 14 (P = 0.01). On the other hand, P4 concentration on day 0 (P = 0.55), on day 7 (P = 0.21), on day 14 (P = 0.91), and average P4 concentration from day 7 to 14 (P = 0.59) were not associated with pregnancy outcome on day 28. Pregnancy outcome on day 42 was positively affected by P4 concentration fold change from day 0 to 7 (P < 0.01) and by average P4 concentration from day 7 to 14 (P = 0.02). Progesterone concentration on day 0 (P = 0.99), on day 7 (P = 0.44), and on day 14 (P = 0.36), and P4 concentration

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1.0

Probability of pregnancy

according to P4 concentration on day 14 as <5 ng/ml and ≥5 ng/ml, however, cows with P4 concentration <5 ng/ml were (P = 0.01) and tended (P = 0.07) to be more likely to lose pregnancy from day 28 to 42 (53.9 vs. 14.3%) and from day 28 to 63 (61.5 vs. 30%), respectively, than cows with P4 concentration ≥5 ng/ml.

P4 fold-change from Day 0 to 7 = 1.2

0.9

P4 fold-change from Day 0 to 7 = 6.1 P4 fold-change from Day 0 to 7 = 96.4

0.8 0.7 0.6 0.5 0.4 0.3

4. Discussion

0.2 0.1 0.0 0

1

2

3

4

5

6

7

P4 fold-change from Day 7 to 14 Fig. 3. Probability of pregnancy on day 63 according to progesterone fold-change from day 0 to 7 and progesterone fold-change from day 7 to 14. According to the logistic regression, effect of: P4 concentration foldchange from day 0 to 7 – P = 0.03; and, P4 concentration fold-change from day 7 to 14 – P = 0.05.

fold change from day 7 to 14 (P = 0.17) were not associated with percentage of cows pregnant on day 42. Percentage of cows that lost pregnancy from day 28 to 42 was associated with P4 concentration on day 0 (P = 0.05) and tended (P = 0.09) to be associated with average P4 concentration from day 7 to 14. Progesterone concentration on day 7 (P = 0.52) and on day 14 (P = 0.69) was not associated with percentage of cows that lost pregnancy from day 28 to 42. Similarly, P4 concentration fold change from day 0 to 7 (P = 0.70) and P4 concentration fold change from day 7 to 14 (P = 0.70) were not associated with pregnancy loss from day 28 to 42. Pregnancy outcome on day 63 was positively affected by P4 concentration fold change from day 0 to 7 (P = 0.03) and P4 concentration fold change from day 7 to 14 (P = 0.05). On the other hand, P4 concentration on day 0 (P = 0.68), on day 7 (P = 0.97), and on day 14 (P = 0.14), and average P4 concentration from day 7 to 14 (P = 0.20) were not associated with percentage of cows pregnant on day 63. Fig. 3 depicts the probability of pregnancy on day 63 according to the P4 concentration fold change from day 7 to 14 according to the minimal (1.2), median (6.1), and maximum (96.4) P4 concentration fold change from day 0 to 7 observed in the current experiment. Ultimately, no cows with P4 concentration fold change from day 0 to 7 < 2.70 and no cows with P4 concentration fold change from day 7 to 14 < 1.48 were pregnant on day 63. Among cows in the control treatment, the average P4 fold change from day 0 to 7 (19.1 ± 3.5 vs. 10.0 ± 1.6) was greater (P = 0.02) for cows diagnosed pregnant on day 63 compared with cows that were not pregnant on day 63. Furthermore, P4 fold change from day 7 to 14 (2.8 ± 0.3 vs. 2.2 ± 0.1) tended to be (P = 0.06) greater for cows diagnosed pregnant on day 63 compared with cows that were not pregnant on day 63. Interestingly, percentage of cows that lost pregnancy from day 28 to 63 was not affected by P4 concentration on day 0 (P = 0.90), P4 concentration on day 7 (P = 0.40), P4 concentration on day 14 (P = 0.77), average P4 concentration from day 7 to 14 (P = 0.34), P4 concentration fold change from day 0 to 7 (P = 0.70), or P4 concentration fold change from day 7 to 14 (P = 0.28). When cows were classified

Treatment of cows with two PGF2␣ on days 4 and 5 followed by P4 supplementation via new CIDR inserts every 7 d starting on day 5 resulted in P4 concentrations of approximately 2 ng/ml from days 7 to 28 and resulted in no LowP4 cows maintaining pregnancy after ET. Considering that treatment of high-producing Holstein cows that do not have a CL with a new intravaginal P4 insert results in P4 concentrations of approximately 0.8 ng/ml (Cerri et al., 2009) and that treatment of lactating Holstein cows with PGF2␣ on day 5 of the estrous cycle does not result in complete luteolysis in 50% of the cows (Santos et al., 2010), it appears that our strategy to reduce P4 secretion by the CL without causing complete luteolysis was effective, as demonstrated by the P4 concentration of approximately 2 ng/ml up to day 21 in LowP4 cows. Nonetheless, it is important to note that by inserting new CIDR inserts in the vagina of the cows every 7 days we may have introduced iatrogenic contaminants in the vagina and caused vaginitis in LowP4 cows, which would not had been caused in control cows. In the current experiment, the vaginal exudate of cows receiving CIDR was recorded and did not exceed 2 (1 = no exudate, 4 = brown/fetid exudate). In several experiments that we have conducted with CIDR inserts (Chebel et al., 2006, 2010; Lima et al., 2009) we have not been able to establish a correlation between vaginal exudate after CIDR treatment and decline in pregnancy per AI. Cows in the LowP4 treatment did not establish pregnancy after embryo transfer. When analyzing the association among different P4 parameters (concentration, average concentration from day 7 to 14, and fold change) and pregnancy outcomes, fold change from day 0 to 7 was associated with pregnancy outcome on days 28, 42, and 63. On the other hand, P4 fold change from day 7 to 14 was associated with pregnancy outcome on days 28 and 63, whereas average P4 concentration from day 7 to 14 was only associated with pregnancy outcome on day 42. Progesterone fold change from day 0 to 7 and from day 7 to 14 was reduced by approximately 50% in LowP4 compared with control cows. Therefore, we determine that the hampered rise in P4 from day 0 to 14 is likely to have precluded the establishment of pregnancies in LowP4 cows. Among cows that were diagnosed pregnant on day 63, the minimum P4 concentration fold change from day 0 to 7 and from days 7 to14 were 2.71 and 1.48, respectively. It is important to note that in the current experiment only 21.4% of cows (19 cows) were pregnant on day 63 and, even though we controlled for possible confounding effects (i.e. synchrony of the estrous cycle, BCS, milk yield, parity), this is a relatively small sample size. Despite the biological importance of the findings of the current experiment, to determine more precisely the minimal P4 concentration fold change during

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metestrus and early diestrus for pregnancy establishment after embryo transfer would require a larger sample size. We are unaware of other studies that have used an ET model to evaluate the minimum P4 concentrations necessary for establishment and maintenance of pregnancies up to 63 d of gestation in high producing dairy cows. By utilizing ET, fertilization and the effect of recipient uterine environment on embryo development from ovulation to day 7 of development were eliminated. Also, only using cows with precisely synchronized estrous cycle eliminated the confounding effect of lack of synchrony of the estrous cycle on P4 concentration on day 7 and 14. In cows that were inseminated and later slaughtered (day 16) those cows with greater P4 concentration during metestrus and early diestrus had larger embryos and greater concentrations of IFN-␶ in the uterus than cows with slower rise in P4 concentration early after AI (Mann and Lamming, 2001; Mann et al., 2006). Several researchers have demonstrated the effect of P4 concentration during metestrus and early diestrus on embryo development of cows that were inseminated, but the positive effects of P4 concentration fold change from day 0 to 7 on pregnancy establishment in the current experiment demonstrate that the importance of early increase in P4 concentration during metestrus for pregnancy establishment goes beyond the early embryo. The positive effects of P4 concentration on embryo quality, elongation, and IFN-␶ production (Mann and Lamming, 2001) appear to be indirect through its effects on endometrial luminal epithelium and glandular epithelium secretions (histotroph; Lonergan, 2011). A series of in vitro experiments demonstrated that, even though mRNA for P4 receptor is present in all stages of embryo development, culture of embryos in the presence of P4 but in the absence of oviduct epithelial cells had no effect on embryo development or blastocyst cell number (Clemente et al., 2009). Early rise in P4 concentration results in greater uterine capacity for production and secretion of histotroph as demonstrated by the increased mRNA expression for lipoprotein lipase and connective tissue growth factor in cows with earlier rise in P4 concentration (Forde et al., 2010). Furthermore, increasing concentrations of P4 and IFN-␶ result in up regulation of genes related to glucose and fructose transport (Forde et al., 2011). These findings indicate that early rise in P4 concentration increases availability of nutrients necessary for embryo growth and development. Progesterone concentration fold change from day 7 to 14 also was associated with establishment of pregnancy in the current experiment. Furthermore, P4 concentration on day 14 ≥ 5 ng/ml resulted in fewer pregnancies lost between days 28 and 42 and between days 28 and 63. Even though a significant down-regulation in P4 receptors in the endometrial luminal and superficial glandular epithelia occurs before maternal recognition of pregnancy, P4 receptors are still present in endometrial stroma and myometrium throughout pregnancy (Bazer et al., 2009; Forde et al., 2011). This results in P4 regulation of stroma expression and secretion of progestamedins (i.e. fibroblast growth factors 7 and 10, and hepatocyte growth factor) that have a paracrine effect on endometrial luminal and glandular epithelia and trophectoderm (Bazer et al., 2009). Thus,

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P4 concentration beyond maternal recognition of pregnancy is likely to indirectly regulate histotroph secretion, endometrial structure, adhesion, placentation, and consequently survival of the embryo and/or fetus. Furthermore, priming of the endometrium with P4 and down-regulation of P4 receptors in the endometrial luminal and superficial glandular epithelia seems to be necessary for the optimum response to placental lactogen produced by binucleatetrophectoderm cells responsible for glandular epithelium differentiation and secretary proteins production and the growth hormone induced glandular epithelium hyperplasia from day 20 to 50 and glandular epithelium hypertrophy beyond day 50 (Bazer et al., 2009). It is important to note that these findings are mostly related to ovine and it is still not clear if they accurately translate to the bovine species. Nonetheless, 19.6% of cows with P4 concentration on day 31 < 5 ng/ml lost pregnancy from 31 to 66 d after AI, whereas only 6.6% of cows with P4 concentration on day 31 ≥ 5 ng/ml lost pregnancy during the same period (Scanavez et al., 2011). Similarly, Starbuck et al. (2004) demonstrated, based on a prediction equation, that only 5% of cows would have pregnancy loss if P4 concentration on week 5 of gestation were approximately 6 ng/ml. We conclude from the current experiment that the early rise in P4 concentration from day 0 to 14 is associated with establishment of pregnancy after ET and that P4 concentration on day 14 tends to be associated with maintenance of pregnancy from day 28 to 63. Ultimately, pregnancy may be established in cows with P4 concentration fold change from day 0 to 7 ≥ 2.71 and P4 concentration fold change from days 7 to 14 ≥ 1.48, respectively; however, pregnancy is less likely to be maintained if P4 concentration on day 14 < 5.0 ng/ml. Reproductive management of lactating dairy cows receiving embryos should strive to maximize synchrony of the estrous cycle and fast rise in P4 concentration from day 0 to 14.

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