BIOLOGY OF REPRODUCTION (2014) 90(1):10, 1–12 Published online before print 27 November 2013. DOI 10.1095/biolreprod.113.114694

Low Doses of Bovine Somatotropin Enhance Conceptus Development and Fertility in Lactating Dairy Cows1 Eduardo S. Ribeiro,3 Ralph G.S. Bruno,4 Alexandre M. Farias,4 Juan A. Herna´ndez-Rivera,4 Gabriel C. Gomes,3 Ricardo Surjus,3 Luis F.V. Becker,3 Alyssa Birt,5 Troy L. Ott,5 Josh R. Branen,6 R. Garth Sasser,6 Duane H. Keisler,7 William W. Thatcher,3 Todd R. Bilby,4 and Jose´ E.P. Santos2,3 3

Department of Animal Sciences, University of Florida, Gainesville, Florida Texas A&M AgriLife Research and Extension, Stephenville, Texas 5 Department of Animal Sciences, Penn State University, University Park, Pennsylvania 6 BioTracking, LLC, Moscow, Idaho 7 Department of Animal Sciences, University of Missouri, Columbia, Missouri 4

Objectives were to evaluate the effects of administering either one or two low doses of slow-release recombinant bovine somatotropin (bST) on hormone concentrations, conceptus development, and fertility in dairy cows. Cows from two farms were detected in estrus on or after 50 days postpartum (n ¼ 1483), inseminated, and enrolled in the study (Day 0). Within farm, cows were blocked by parity and assigned randomly to receive a single placebo injection at insemination (control), a single injection with 325 mg of bST at insemination (S-bST), or two injections with 325 mg of bST administered on Days 0 and 14 (T-bST). From a subset of cows, blood was collected twice weekly from Day 0 to 42 for determination of hormone concentrations and on Day 19 for isolation of leucocytes and analysis of transcript abundance of selected interferon-stimulated genes. Pregnancy was diagnosed on Days 31 and 66, and ultrasonographic morphometry of the conceptus was performed on Days 34 and 48 in a subset of cows. Cows that received T-bST had increased plasma concentrations of GH and IGF1 for 4 wk, increased mRNA expression of ISG15 and RTP4 in leukocytes, earlier rise in the pregnancy-specific protein B in plasma of pregnant cows, increased conceptus size, and enhanced fertility. Cows that received S-bST had increased concentrations of GH and IGF1 for only 2 wk and it was insufficient to alter conceptus development and fertility. In conclusion, supplementation with low doses of bST during the pre- and peri-implantation periods enhanced conceptus development, reduced embryonic losses, and improved fertility in dairy cows. bST, conceptus, dairy cow, fertility

INTRODUCTION Although reproductive management in dairy farms has evolved in the last decade, reproductive efficiency in dairy 1 Supported by Elanco Animal Health, Greenfield, IN (Study GN4US100015). This research was presented at the 2013 Annual Meeting of the American Dairy Science Association, July 8–12, 2013, Indianapolis, Indiana. 2 Correspondence: Jose´ E.P. Santos, Department of Animal Sciences, 2250 Shealy Drive, University of Florida, Gainesville, FL 32611-0910. E-mail: [email protected]

Received: 6 October 2013. First decision: 31 October 2013. Accepted: 18 November 2013. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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cattle is still not optimal [1]. For instance, the average 21-daycycle pregnancy rate, a common metric used to evaluate reproduction, in the United States is only 16% [2], and increments beyond 16% are usually economically attractive [3]. After the advent of timed artificial insemination (AI) programs, low pregnancy per AI (P/AI) remained the major contributor to suboptimal reproduction in lactating dairy cows. Although fertilization is estimated to be above 80% in cows not exposed to heat stress, P/AI on Day 28 after insemination is usually around 40%, indicating that failures in normal embryo development during pre- and peri-implantation stages are substantial [4]. Suboptimal uterine conditions and less competent embryos originating from low-quality oocytes are likely major causes for such failures [5]. Development of strategies that rescue less competent embryos either by direct effects on conceptus development or indirectly through the oviductal and uterine environment have the potential to improve reproductive efficiency in dairy cows. Growth hormone or somatotropin is secreted by the anterior pituitary gland into the blood circulation of animals. Binding of GH to receptors in target tissues induces cell proliferation/ differentiation and controls cell metabolism [6]. Intracellular signaling after activation of GH receptors induces a wide range of cellular responses, including synthesis and secretion of IGF1 [6]. The bovine uterus and embryo/conceptus have receptors for GH and IGF1 that allow endocrine, paracrine, and autocrine actions of both hormones during establishment of pregnancy [7–11]. In fact, in vitro and in vivo studies have demonstrated positive effects with these two hormones in stimulating uterine function and conceptus development during the pre- and periimplantation stages in many species [12–17]. Thus, the GHIGF system constitutes an important target for the development of strategies to enhance conceptus development, minimize embryonic loss, and improve fertility in lactating dairy cows. Slow-release formulations of recombinant bovine somatotropin (bST) have been used extensively in dairy cows as a management tool to enhance lactation performance by antagonizing actions of insulin and altering nutrient partitioning to favor milk synthesis [18]. Additionally, studies have shown that long-term treatment with 500 mg of bST at 14-day intervals improves the fertility of dairy cows subjected to synchronized ovulation [19–21], although some have suggested that the benefits are dose dependent [22, 23]. In some cases, high doses of bST might have negated [22] or even had a detrimental effect on establishment and maintenance of pregnancy in cattle [23]. In fact, research with pregnant mice showed that exposure in utero to an excess of IGF1 increased embryo mortality [24]. Thus, it is possible that benefits to

ABSTRACT

RIBEIRO ET AL. distiller’s grains, whole cottonseed, minerals, and vitamins. Diets were formulated to meet the nutrient requirements for lactating Jersey and crossbred cows weighing 500 and 550 kg, respectively, producing 35 kg of 4.5% fatcorrected milk with a daily intake of 22 kg of dry matter. On both farms, all cows received two injections of PGF2a (500 lg of cloprostenol; Estrumate; Merck Animal Health, Summit, NJ), with the first injection administered at 38 6 3 days postpartum and the second at 52 6 3 days postpartum. Cows were observed for signs of estrus once daily, after the morning milking, based on removal of tail chalk that was re-applied daily. Cows not detected in estrus within 14 days of the second PGF2a received a third treatment at 66 6 3 days postpartum. Cows detected in estrus were inseminated the same morning. On farm A, all cows were inseminated with sexed semen originating from 10 Jersey sires, whereas on farm B, all cows were inseminated with conventional semen originating from 11 sires, including Jersey, Holstein, Swedish Red, and Angus sires. After first insemination, cows were continually evaluated for signs of estrus and reinseminated if diagnosed in estrus. On farm A, interval to second postpartum AI and pregnancy at second AI were evaluated. Calving data, including gestation length, calf sex, calving assistance, twin birth, and stillbirth were collected at both farms. Calving assistance and stillbirths were grouped as calving problems.

fertility in dairy cows could occur at doses of less than the standard 500 mg administered at 14-day intervals to stimulate milk yield. The gestation period between early implantation and early pregnancy diagnosis is not well studied in cattle despite being critical for maintenance of gestation in ruminants [25]. The lack of detailed information is probably due to the inability to examine conceptuses at this period without slaughtering cows. Measuring peripheral responses to pregnancy might be a noninvasive alternative for comparative studies with large numbers of animals. Recent studies demonstrated that interferon-tau (IFN-s) produced by trophectoderm cells reaches the maternal circulation [26] and induces expression of interferon-stimulated genes (ISGs) in peripheral tissues (e.g., leukocytes; [27–29]), which parallels the total amount of IFN-s in utero [30]. In addition, pregnancy-associated glycoproteins (PAG) secreted by binucleated cells in early placentation, such as pregnancy-specific protein B (PSPB; [31]), are abundantly expressed and have been used as markers for pregnancy in cattle [32, 33]. Thus, expression of ISGs in leucocytes and PSPB concentration in plasma could be used as noninvasive indicators to study bovine pregnancy at the pre- and periimplantation stages. The hypotheses of the current study were as follows: Administration of a low dose of bST at insemination would enhance conceptus development at the pre-implantation stages and improve the fertility of lactating dairy cows. Administration of a second dose 14 days later would extend the benefits to the peri-implantation period and further improve fertility. Enhanced conceptus development would be evidenced indirectly by increased transcript abundance for ISGs in leucocytes and PSPB concentrations in plasma, and directly by ultrasonography morphometry. Therefore, the objectives were to evaluate the effects of administering a low dose of slow-release bST either as a single or as two sequential treatments on plasma hormone concentrations, conceptus development, and fertility in lactating dairy cows inseminated after detection of estrus.

Treatments

Blood Sampling and Analyses of Plasma Blood was collected from a subset of 117 cows (60 cows on farm A and 57 on farm B), comprising 20 primiparous and 19 multiparous cows per treatment from common experimental blocks, by puncture of the coccygeal blood vessels using evacuated tubes containing K2 ethylenediaminetetraacetic acid (EDTA; Vacutainer; Becton Dickinson, Franklin Lakes, NJ). Blood was sampled on Days 0, 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, and 42. Samples were placed immediately in ice and later centrifuged at 2000 3 g for 15 min for plasma separation. Three aliquots of each plasma sample were frozen at 258C for later analyses. All plasma samples were analyzed for concentration of progesterone; samples from Day 0 to Day 31 were analyzed for concentrations of GH, total IGF1, and insulin; samples from Day 17 to Day 42 were analyzed for concentrations of PSPB. An additional 184 cows had blood sampled on Day 31, and the plasma was analyzed for concentrations of PSPB. Therefore, on Day 31, a total of 301 cows (104 controls, 97 S-bST, and 100 T-bST) had plasma analyzed for concentrations of PSPB. Concentrations of GH were analyzed by radioimmunoassay (RIA) according to the method of Lalman et al. [36] in three assays, with triplicates of each sample. Concentrations of total IGF1 were determined by a commercial ELISA kit (Quantikine ELISA Human IGF1 Immunoassay; R&D Systems, Inc., Minneapolis, MN) designed for human IGF1 but with 100% crossreactivity with bovine IGF1. Concentrations of insulin were determined by a commercial bovine ELISA kit (Mercodia Bovine Insulin ELISA; Mercodia AB, Uppsala, Sweden). Concentrations of progesterone were determined by RIA using a commercial kit (Coat-a-Count; Siemens Healthcare Diagnostics, Los Angeles, CA) and performed in two assays. Concentrations of PSPB were analyzed using a commercially available quantitative ELISA assay (BioPRYN; BioTracking LLC, Moscow, ID) according to the method used in Sasser et al. [31]. At least two control samples of known hormone concentrations were included multiple times in all assays. The intra- and interassay coefficients of variation were 4.5% and 6.1% for GH, 3.9% and 9.1% for IGF1, 3.3% and 4.1% for insulin, 4.5% and 5.5% for progesterone, and 7.0% and 5.9% for PSPB, respectively.

MATERIALS AND METHODS All procedures involving cows were approved by the animal care and use committees of the University of Florida and the Texas A&M University System.

Eligibility for Enrollment Only cows observed in estrus during the period from 50 to 100 days postpartum were eligible for enrollment in the study. On the day of enrollment, cows had to meet the following requirements: no previous AI, no cesarean surgery in the current lactation, a body condition score (BCS) .2.25, a lameness score ,4, no more than two cases of mastitis in the current lactation, and at least three functional mammary glands. Cows were scored for body condition on a scale of 1 to 5 in increments of 0.25 units (1 ¼ emaciated, 5 ¼ obese; [34]), and for lameness on a scale of 1 to 5 scale in increments of 1 unit (1 ¼ normal, 5 ¼ severely lame; [35]). The BCS were reevaluated 31 6 3 days after enrollment for evaluation of BCS changes.

Cows, Housing, and Management The study was conducted in two commercial dairy farms located in Hartley County, TX. Farm A milked approximately 2400 Jersey cows thrice daily in a 72-stall rotary parlor. Farm B milked approximately 4300 Holstein/Jersey/ Swedish Red crossbred cows thrice daily in an 80-stall rotary parlor. The rolling herd averages for milk yield during the study period were approximately 8000 kg per cow on farm A and 9000 kg per cow on farm B. On both farms, primiparous and multiparous cows were housed separately in free-stall barns throughout the lactation. All cows received a total mixed ration fed twice daily, with amounts adjusted daily to allow 5% of feed refusals. On both farms, lactating cow diets consisted primarily of corn silage, alfalfa hay, steam-flaked corn, solvent-extracted soybean meal, solvent-extracted canola meal, corn

Isolation of Leukocytes, RNA Extraction, and Quantitative RT-PCR Blood was sampled on Day 19 from a subgroup of 164 cows in farm A for isolation of peripheral blood leukocytes (PBL) according to the method in Gifford et al. [28]. Briefly, after centrifugation and harvest of plasma, buffy

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In total, 1483 cows were enrolled in the study, 733 Jersey cows in farm A and 750 Holstein/Jersey/Swedish Red crossbreed cows in farm B. Daily, within each farm, cows detected in estrus in the period from 50 to 100 days postpartum and receiving their first AI in the current lactation were blocked by parity (primiparous or multiparous) and assigned randomly to receive a single placebo injection at AI (control; mixture of aluminum monostearate and food grade oil), a single treatment injection with bST at AI (S-bST; 325 mg of sterile sometribove zinc suspension; Elanco Animal Health, Greenfield, IN), or two sequential treatments with bST (T-bST), the first concurrent with AI and the second 14 days later. All treatments were administered subcutaneously in the ischial fossa area. The day of AI was considered Day 0.

LOW-DOSE bST AND FERTILITY IN DAIRY COWS TABLE 1. Primer reference and sequences for genes investigated by quantitative real-time PCR. Target gene

Gene name

NCBI sequence

ISG15

Interferon stimulated gene 15

NM_174366

RTP4

Receptor transporter protein 4

BC105539

MX1

Myxovirus 1

AF047692

MX2

Myxovirus 2

NM_173941

ACTB

b-actin

AY141970

RPL19

Ribosomal protein L19

NM_001040516

Primer

Primer sequence

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5 0 -GGTATGAGCTGAAGCAGTT-3 0 5 0 -ACCTCCCTGCTGTCAAGGT-3 0 5 0 -CACATGTACCTGGAGAACCAGA-3 0 5 0 -AGGTAGCTCTGAAACCTTCCTG-3 0 5 0 -GTACGAGCCGAGTTCTCCAA-3 0 5 0 -ATGTCCACAGCAGGCTCTTC-3 0 5 0 -CTTCAGAGACGCCTCAGTCG-3 0 5 0 -TGAAGCAGCCAGGAATAGTG-3 0 5 0 -CTGGACTTCGAGCAGGAGAT-3 0 5 0 -GGATGTCGACGTCACACTTC-3 0 5 0 -ATCGATCGCCACATGTATCA-3 0 5 0 -GCGTGCTTCCTTGGTCTTAG-3 0

Ultrasonographic Morphometry of the Amniotic Vesicle and Embryo/Fetus A subset of 151 cows diagnosed as pregnant on Day 31 was reexamined by transrectal ultrasonography using a real-time B-mode ultrasound scanner (Aloka SSD-500V; Hitachi Aloka Medical, Ltd., Wallingford, CT) equipped with a 7.5-MHz linear-array transducer on Days 34 and 48 for amniotic vesicle and embryo/fetus morphometry according to Bertolini et al. [40], but with some modifications. Briefly, on each gestational day, several images of the same conceptus were recorded on an audio video interleaved for 1–2 min in a laptop (HP Pavilion dv6 Notebook PC; Hewlett-Packard, Palo Alto, CA) by capturing the ultrasonographic images using a USB device with an audio-video input cable (EZ Grabber; MyGica, Shenzhen, China). Video fragments were analyzed carefully in a frame-by-frame fashion using an open-source image processing software, ImageJ (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/index.html; [41]). The frame containing the ideal positioning and orientation of the conceptus was identified and utilized to measure the amniotic vesicle longitudinal and transversal diameter by drawing two cross-sectional lines of 908 angle, and the embryo/fetus crown-rump length and abdominal diameter was also measured. As discussed by Bertolini et al. [40], the detailed process of comparing snapshots and selecting the best orientation for the specific trait evaluated in a frame-by-frame fashion improves the certainty of measurements taken from the conceptus. All ultrasonographic examinations were performed by the same person using the same ultrasound machine. Images were then evaluated by the same person, who was blinded to treatments.

Experimental Design and Statistical Analyses

Diagnoses of Pregnancy, Pregnancy Loss, and Reinsemination

The experiment followed a randomized complete block design. Within farm, pre-assigned enrollment lists of cows were prepared for primiparous and multiparous cows in blocks of three. Randomization was based on sequence of daily identification of eligible cows as they were observed in estrus and inseminated. Therefore, within farm and parity, cows were assigned randomly within each block to one of the three treatments. All responses were analyzed using the GLIMMIX procedure of SAS version 9.3 (SAS/STAT; SAS Institute Inc., Cary, NC). Binary responses were analyzed by logistic regression fitting a binary data distribution. The models included the effects of treatment (control vs. S-bST vs. T-bST), farm (A vs. B), parity (primiparous vs. multiparous), interactions between treatment and farm and treatment and parity, BCS at artificial insemination, sire nested within farm, and technician nested within farm. Continuous data were analyzed with models

Pregnancy was diagnosed in all cows, including those reinseminated, on Day 31 6 3 via transrectal ultrasonography of the uterus and its contents using a portable ultrasound (Easy Scan; BCF Technology, Rochester, MN) equipped with a 7.5-MHz linear-array transducer. A pregnant cow was characterized by visualization of an embryo. Cows diagnosed as pregnant were reexamined 35 days later for detection of viable pregnancy on Day 66 6 3. Throughout the manuscript, Days 31 and 66 refer to the respective study days 63. Pregnancy per AI was calculated as the number of pregnant cows on Day 31 or Day 66 divided by the number of cows enrolled in the study and evaluated for pregnancy. Pregnancy loss between Days 31 and 66 was calculated as the

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number of pregnant cows on Day 31 minus the number of pregnant cows on Day 66, divided by the number of pregnant cows on Day 31. Pregnancy loss after Day 66 was calculated as the number of pregnant cows on Day 66 minus the number of cows calving, divided by the number of pregnant cows on Day 66. The overall pregnancy loss was calculated as the number of pregnant cows on Day 31 minus the number of cows calving, divided by the number of pregnant cows on Day 31. On farm A, cows reinseminated after the first AI were examined for pregnancy by transrectal palpation 48 6 3 days after reinsemination. The proportion of nonpregnant cows that were reinseminated was calculated as the number of cows reinseminated until the day of pregnancy diagnosis of first AI (study Day 31), divided by the number of nonpregnant cows at first pregnancy diagnosis. Pregnancy per AI for reinsemination was calculated as the number of cows pregnant 48 6 3 days after reinsemination, divided by the number of cows reinseminated.

coat fractions were collected by pipetting and transfer to 15-ml conical tubes. Red cell lysis buffer (150 mM NH4Cl, 10 mM NaHCO3, and 1 mM EDTA; pH 7) was added to the buffy coat for a total volume of 15 ml. Tubes were inverted several times and incubated at room temperature for 5 min. Samples were then centrifuged at 300 3 g for 10 min at 48C, and the supernatant was discarded. The PBL pellet was mixed with 5 ml of red cell lysis buffer, incubated at room temperature for 5 min, and centrifuged at 300 3 g for 10 min at 48C, and the supernatant was discarded. The PBL pellet was washed with ice-cold PBS and centrifuged at 300 3 g for 10 min at 48C, and the supernatant was discarded. The PBL pellet was resuspended with 0.8 ml of Trizol (Molecular Research Center, Inc., Cincinnati, OH), transferred to 1.5-ml microtubes, and stored at 808C. The time interval from blood collection and PBL sample storage at 808C was no longer than 6 h. Extraction of RNA was conducted according to the manufacturer’s recommendations for the RNA-extraction kit (PureLink RNA Mini Kit; Invitrogen, Carlsbad, CA). The concentration of RNA was calculated by measuring absorbance at 260 nm, and 1 lg of total cellular mRNA was treated with DNase (RQ1 RNase-Free DNase; Promega, Madison, WI) and used to synthesize complementary DNA using the DyNAmo cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). Complementary DNA was then used for quantitative real-time RT-PCR (ABI 7500 Sequence Detector; Applied Biosystems Inc., Foster City, CA). Six genes were investigated (Table 1): four target genes—interferon-stimulated gene 15 (ISG15), receptor transporter protein 4 (RTP4), myxovirus 1 (MX1), and myxovirus 2 (MX2)—and two reference genes—beta-actin (ACTB) and ribosomal protein L19 (RPL19). Each reaction mixture consisted of 3 ll of a 1:5 dilution of the cDNA, gene-specific forward and reverse primers, SYBR Green (Applied Biosystems Inc.), and nuclease-free water in a total reaction volume of 20 ll. Reactions were run in duplicate and comprised 40 cycles of a three-step amplification protocol (30 sec at 958C followed by 45 sec at the optimized annealing temperature [578–608C] and 1 min at 728C). Primer efficiency ranged from 90% to 105%. Melting curve analysis was also performed to ensure amplification of a single product. Data are presented using the comparative method developed by Livak and Schmittgen [37], using nonpregnant cows from the control group as the reference for comparisons. Relative expression values were obtained by raising the PCR amplification efficiency (E ¼ 2) to the power of the delta-delta threshold cycle (DDCT) obtained from the DCT least square mean differences of pairwise comparisons among treatment and reference groups [38]. The DCT values subjected to statistical analysis were generated by normalization of CT values from target genes with the geometric mean of CT values from reference genes according to the method of Vandesompele et al. [39]. Confidence intervals for graphical representations of relative expression were generated from the lower and upper confidence limits obtained for DCT least square mean differences as described by Yuan et al. [38].

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fitting a Gaussian or a logarithmic distribution, and residuals were tested for normality. The models included all predictors of binary responses except sire and technician. For analyses of mRNA expression of ISGs and concentrations of PSPB in plasma, the models included the effects of treatment, farm, parity, interactions between treatment and farm and treatment and parity, BCS at artificial insemination, sire nested within farm, pregnancy status (pregnant vs. nonpregnant, or nonpregnant on Day 31 vs. pregnant on Day 31 but not on Day 66 vs. pregnant on Days 31 and 66), and presence of twins (singleton vs. twin). Pregnancy maintenance between Days 31 and 66 (lost vs. maintained), presence of twins, and sire were also included in models analyzing conceptus morphometry. For repeated measures, models included the effects of day of measurement, interaction between treatment and day, and the random effect of cow nested within treatment. Measurements on study Day 0 were used as covariates. The covariance structure that resulted in the smallest Bayesian information criterion was selected from the mixed models. Treatment differences with P  0.05 were considered as significant, and 0.05 , P  0.10 were considered as a tendency.

Compared with controls, cows receiving bST had greater (P , 0.05) concentrations of GH from Day 3 to Day 17. Concentrations of GH did not differ between S-bST and TbST in the first 14 days. Nevertheless, an injection of bST in TbST cows on Day 14 resulted in a second increase in GH, with a sustained elevation in concentrations until Day 28. Concentrations of GH were greater (P , 0.05) for T-bST than S-bST from Day 17 to Day 24, after which they no longer differed. On Day 28, only a tendency (P ¼ 0.06) for difference was observed between T-bST and control cows. For the entire period, concentrations of GH averaged 3.49 6 0.54, 6.38 6 0.57, and 7.65 6 0.55 ng/ml for controls, S-bST, and T-bST, respectively. Similar to GH, plasma concentrations of IGF1 did not differ among treatments on Day 0 and averaged 59.6 ng/ml. Concentrations of IGF1 remained unaltered in controls for the first 31 days of the study (Fig. 1B). Concentrations of IGF1 increased (P , 0.01) in cows receiving bST in the first 7 days after treatment, reached a peak on Day 7, and then declined. Concentrations of IGF1 were greater (P , 0.01) in bST-treated cows compared with those of controls in the first 14 days of the study. A second injection of bST on Day 14 resulted in a second increase in concentrations of IGF1 in T-bST cows. This second increase resulted in greater (P , 0.01) concentrations of IGF1 for T-bST than those in control and S-bST from Day 17 to Day 31. For the entire period, concentrations of IGF1

RESULTS Systemic Effects in Plasma Hormone Concentrations and Body Condition Plasma concentrations of GH did not differ among treatments on Day 0 and averaged 5.4 6 0.5 ng/ml (Fig. 1A). Concentrations of GH remained unaltered in control cows from Day 3 to Day 31. However, in cows treated with bST, concentrations of GH increased (P , 0.01) in the first 7 days after injection, reached a peak on Day 7, and then declined. 4

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FIG. 1. Plasma concentrations (ng/ml) of GH (A), IGF1 (B), insulin (C), and progesterone (D) according to treatment. Control, single placebo injection at AI; S-bST, single treatment with 325 mg of bST at AI; T-bST, two sequential treatments with 325 mg of bST, the first at AI and the second treatment 14 days later. Within a day, pairwise differences (P , 0.05) are represented as follows:  control versus S-bST; zcontrol versus T-bST; *S-bST versus T-bST.

LOW-DOSE bST AND FERTILITY IN DAIRY COWS

bST ¼ 3.07 6 0.01), although differences were small. This difference in BCS was caused by a greater (P ¼ 0.02) gain of body condition between Days 0 and 31 in control and S-bST than in T-bST cows (control ¼ 0.11 vs. S-bST ¼ 0.11 vs. T-bST ¼ 0.07).

increased (P , 0.001) with use of bST and with the additional treatment on Day 14, and averaged 64.1 6 3.1, 77.4 6 3.3, and 98.5 6 3.2 ng/ml for controls, S-bST, and T-bST, respectively. Concentrations of insulin in plasma were influenced (P ¼ 0.01) by day in the study (Fig. 1C). Treatment with bST did not have an overall effect on plasma insulin, although T-bST tended (P , 0.10) to have greater concentrations of insulin than did control cows on Days 21 and 28, and it was greater (P , 0.05) than S-bST on Day 28. Overall, concentrations of insulin averaged 0.59 6 0.04, 0.60 6 0.05, and 0.67 6 0.04 ng/ml for controls, S-bST, and T-bST, respectively. Similarly, concentrations of progesterone in plasma were influenced (P , 0.01) by day in the study, but treatment did not have an overall effect (Fig. 1D). Within day, the only difference (P ¼ 0.04) detected was between control and S-bST on Day 7 (Fig. 1D). Among pregnant cows, however, there was an interaction (P ¼ 0.02) between treatment and day. Progesterone concentrations on Day 3 in pregnant cows were greater (P ¼ 0.04) for T-bST compared with those in control and tended (P ¼ 0.09) to be greater for S-bST compared with control (control ¼ 0.52 6 0.08 vs. S-bST ¼ 0.73 6 0.10 vs. T-bST ¼ 0.76 6 0.08). As expected, the BCS did not differ among treatments on Day 0 and averaged 3.03 6 0.01. However, on Day 31, control and S-bST cows had greater (P ¼ 0.02) BCS than those on TbST (control ¼ 3.11 6 0.01 vs. S-bST ¼ 3.12 6 0.01 vs. T-

Conceptus Development and Ultrasonography Morphometry Cows diagnosed as pregnant on Day 31 had greater (P , 0.01) mRNA expression of ISG15, RTP4, MX1, and MX2 on Day 19 than did nonpregnant cows, on average an increase of 2.8-, 1.5-, 1.7-, and 2.2-fold, respectively (Fig. 2). Messenger RNA abundance of ISG15 was also affected (P ¼ 0.04) by treatment. Pregnant cows from T-bST had a 2.0- and 1.9-fold increase in mRNA expression of ISG15 compared with that from pregnant cows from control and S-bST, respectively (Fig. 2A). Transcript abundance of RTP4 tended (P ¼ 0.07) to be affected by treatment, and there was an interaction (P ¼ 0.01) between treatment and pregnancy status on Day 30 (Fig. 2B). Among pregnant cows, T-bST treatment had a 1.7- and 1.9fold increase in mRNA expression of RTP4 compared with that of control and S-bST, respectively. On the other hand, no statistical differences were observed for mRNA abundance among treatments in nonpregnant cows, which explains the interaction observed between treatment and pregnancy status 5

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FIG. 2. Relative mRNA expression of interferon-stimulated genes ISG15 (A), RTP4 (B) MX1 (C), and MX2 (D) according to bST treatments and pregnancy status on Day 31. Nonpregnant cows from the control group were used as reference for comparison. Control, single placebo injection at AI; S-bST, single treatment with 325 mg of bST at AI; T-bST, two sequential treatments with 325 mg of bST, the first at AI and the second treatment 14 days later. Different letters (a, b, c) represent different (P , 0.05) least square means. Error bars represent the 95% confidence intervals for group means.

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0.01) among treatment, day after AI, and farm was also detected. For pregnant cows on farm A, control cows had lower concentrations of PSPB than did S-bST on Days 17 and 21, and lower than did T-bST on Day 21 (Fig. 3B), but these differences were not observed on farm B (Fig. 3C). When data on Day 31 were analyzed including the additional cows (n ¼ 301), the concentration of PSPB was less (P ¼ 0.04) for T-bST compared with that of control (1.62 vs. 1.87 ng/ml). This difference was mainly determined by PSPB concentrations of nonpregnant cows, which tended (P ¼ 0.08) to be greater for control compared with T-bST cows (Fig. 3D). No differences were detected among pregnant cows. Longitudinal diameter of the amniotic vesicle, crown-rump length, and abdominal diameter of embryos/fetuses, but not transversal diameter of the amniotic vesicle, were affected by treatment (Table 2). Cows from T-bST tended (P , 0.07) to have a larger amniotic vesicle and embryo crown-rump length on Day 34 than did cows from S-bST or control treatments. On Day 48, cows from T-bST had a larger (P , 0.05) amniotic vesicle, fetus crown-rump length, and abdominal diameter than did those from S-bST or control groups. These differences in the size of the amniotic vesicle and embryo/fetus were more evident when analyses included only data from farm A, which had more uniform genotypes with only Jersey cows inseminated with Jersey sires (data not shown). No differences in

(Fig. 2B). Within treatment groups, only cows from T-bST had a distinction in RTP4 transcript abundance between pregnant and nonpregnant cows, and it increased 2.4-fold with pregnancy (Fig. 2B). Relative mRNA expressions of MX1 and MX2 were not affected by treatment (Fig. 2, C and D). Parity had an effect (P ¼ 0.02) on mRNA expression of ISG15 but not on mRNA expression of RTP4, MX1, and MX2. Within pregnant cows, primiparous cows had a 1.9-fold increase in mRNA expression of ISG15 compared with that of multiparous cows. Treatment did not influence the concentrations of PSPB in plasma, and no interaction between treatment and day of the study or treatment and pregnancy status on Day 31 was observed. Nonetheless, a distinction in plasma concentrations of PSPB between pregnant and nonpregnant cows occurred earlier for those treated with bST compared with those of control (Fig. 3A). There was no distinction in plasma concentrations of PSPB between pregnant and nonpregnant cows on Day 17. On Day 21, however, cows receiving bST had greater (P , 0.04) concentrations of PSPB when pregnant than nonpregnant. On the same day, no difference was observed between pregnant and nonpregnant cows in the control group (Fig. 3A). On Day 24, however, plasma concentrations of PSPB were distinct (P , 0.01) between pregnant and nonpregnant cows in all treatments. An interaction (P , 6

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FIG. 3. Plasma concentrations of PSPB on Day 21 according to treatments and pregnancy status on Day 31 (A); from Day 17 to Day 42 in pregnant cows on farm A (B) and on farm B (C); and on Day 31 according to treatment and pregnancy status on the same day (D). Control, single placebo injection at AI; S-bST, single treatment with 325 mg of bST at AI; T-bST, two sequential treatments with 325 mg of bST, the first at AI and the second treatment 14 days later. Different letters in bar graphics represent different least square means (a, b, c: P , 0.05; A, B: P , 0.10). In B, within a day, pairwise differences (P , 0.05) are represented as follows:  control versus S-bST; zcontrol versus T-bST. Error bars represent 95% confidence intervals (A) or SEM (B, C, D).

LOW-DOSE bST AND FERTILITY IN DAIRY COWS TABLE 2. Effect of bST treatments on ultrasonographic morphometry of amniotic vesicle and embryo/fetus on Gestation Days 34 and 48. Treatment* Parameter Pregnancies evaluated Total Twins Conceptuses Live on Gestation Day 66 Lost by Gestation Day 66 Amniotic vesicle diameter Longitudinal (mm)  Day 34§ Day 48§ Transversal (mm) Day 34§ Day 48§ Embryo/fetus Crown-rump length (mm)z Day 34§ Day 48§ Abdominal diameter (mm)z Day 34§ Day 48§

Control

S-bST

T-bST

54 2 56 49 7

46 3 49 41 8

51 2 53 46 7

12.5 6 0.55B 29.3 6 0.80b

12.5 6 0.59B 28.5 6 0.83b

13.9 6 0.63A 31.8 6 0.84a

8.6 6 0.38 18.7 6 0.72

8.6 6 0.41 19.6 6 0.75

8.6 6 0.44 19.9 6 0.74

10.6 6 0.52B 23.6 6 0.76b

10.5 6 0.56B 22.5 6 0.79b

12.0 6 0.60A 26.1 6 0.80a

4.8 6 0.23 9.2 6 0.33b

4.9 6 0.25 8.9 6 0.34b

5.3 6 0.27 10.4 6 0.34a

P – – – – – ,0.01 0.63

,0.01 ,0.01

observed between control and S-bST cows. Similarly, a greater (P , 0.04) proportion of cows calved from first AI in the TbST group compared with control or S-bST. Pregnancy losses from Day 31 to Day 66 and from Day 31 to calving were fewer (P , 0.03) for T-bST compared with S-bST, and both treatments did not differ from controls (Table 3). There were no differences in gestation length, proportion of female calves, and twin births among treatments. Nonetheless, incidence of calving problems (calving assistance þ stillbirths) was greater (P ¼ 0.04) for control compared to S-bST, and tended (P ¼ 0.09) to be greater for control compared with TbST (Table 3). On farm A, treatment had no effects on the proportion of nonpregnant cows reinseminated before Day 31, on the interval between first and second AI, and on P/AI in the second insemination (Table 4). No interaction between treatment and farm was observed for the fertility responses evaluated.

embryo/fetus morphometry were observed between control and S-bST in any of the analyses performed. Cows that experienced pregnancy loss between Days 31 and 66 had intermediate mRNA expression of ISG15 on Day 19 compared with those never diagnosed pregnant, which had the least expression, and those that remained pregnant by Day 66, which had the greatest expression (Fig. 4A). On average, there was a 1.7-fold reduction in mRNA expression of ISG15 in cows that lost pregnancy compared with those that maintained by Day 66. For mRNA expression of RTP4, MX1, and MX2, no differences were observed between cows that lost or maintained pregnancy by Day 66, and except for RTP4, these groups had greater mRNA expression than cows never diagnosed pregnant (Fig. 4A). The mean concentrations of PSPB were also intermediate for cows that experienced pregnancy loss between Days 31 and 66 (Fig. 4B). Cows that lost pregnancies had reduced (P , 0.05) concentration of PSPB between Days 24 and 42 than cows that maintained their gestation. The mean concentration of PSPB in the same period was always greater for cows that lost their pregnancy compared with those not diagnosed nonpregnant on Day 31 (Fig. 4B). The mean concentrations from Day 17 to Day 42 were 0.05, 1.52, and 2.21 ng/ml for cows never diagnosed pregnant, those that lost the pregnancy between Days 31 and 66, and those that remained pregnant by Day 66, respectively. Cows that lost pregnancy by Day 66 had smaller amniotic vesicle and embryo/fetus crown-rump lengths on Days 34 and 48 compared with those viable on Day 66 (Fig. 4C).

DISCUSSION This study comprehensively evaluated the effects of supplementing low doses of bST at and after AI on the fertility of lactating dairy cows. Treatment with two sequential doses of 325 mg of bST administered on the day of AI and again 14 days later increased plasma concentrations of GH and IGF1 for a period of 3–4 wk, enhanced conceptus development and, more importantly, improved overall fertility by 27%. Effects on conceptus development were evidenced by increased transcript abundance of ISG15 and RTP4 in PBL at the onset of implantation, by an early rise in PSPB in the plasma of pregnant cows, and by enhanced embryo/fetus and amniotic vesicle sizes at the peri-implantation stages. On the other hand, a single treatment with bST at AI increased concentrations of GH and IGF1 for only 2 wk, but was insufficient to improve conceptus development, survival, and the other measures of fertility.

Fertility Responses The P/AI on Day 31 tended (P ¼ 0.08) to be influenced by treatment, and it was greater (P ¼ 0.03) for T-bST compared with control, but both groups did not differ from S-bST (Table 3). On Day 66, cows in T-bST had greater (P , 0.02) P/AI than those in control or S-bST, but no differences were 7

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* Control, single placebo injection at AI; S-bST, single treatment with 325 mg of bST at AI; T-bST, two sequential treatments with 325 mg of bST, the first at AI and the second treatment 14 days later.   Tendency for interaction between treatment and day of gestation (P , 0.10). z Interaction between treatment and day of gestation (P , 0.05). § Data are presented as least square means 6 SEM. a,b Least square means on the same row with different superscripts differ (P , 0.05). A,B Least square means on the same row with different superscripts differ (P , 0.07).

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Downloaded from www.biolreprod.org. FIG. 4. Relative mRNA expression of interferon-stimulated genes (A) and plasma concentrations of PSPB (B) in cows diagnosed as nonpregnant on Day 31, pregnant on Day 31 but not on Day 66, and pregnant on Days 31 and 66. Amniotic vesicle and embryo/fetus crown-rump sizes on Days 34 and 48 in pregnancies that were lost or maintained by Day 66 (C). Different letters in bar graphics represent different least square means (a, b, c: P , 0.05; A, B, C: P , 0.10). RIn A, nonpregnant cows on Day 31 are the reference group for comparison. In B, within a day, pairwise differences (P , 0.05) are represented as follows: pregnant on Days 31 and 66 versus nonpregnant on Day 31; fl pregnant on Days 31 and 66 versus pregnant on Day 31 but not on Day 66; }pregnant on Day 31 but not on Day 66 versus nonpregnant on Day 31.

implantation stages, finally enhancing fertility. These positive responses likely resulted from systemic increases in GH and IGF1 concentrations, combined with their local actions on the uterus and conceptus [17, 42]. The rise in plasma IGF1 concentrations paralleled the increase in GH following injections. The liver has a large abundance of GH receptors,

Although one cannot discount that bST and IGF1 might have enhanced fertilization [15], given the timing of treatment administration and the observed responses, the two low doses of bST likely improved the uterine environment and its receptivity for pregnancy establishment, and stimulated conceptus development and survival during pre- and peri8

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LOW-DOSE bST AND FERTILITY IN DAIRY COWS TABLE 3. Effect of bST treatments on fertility responses, pregnancy maintenance, and calving features in lactating dairy cows. Treatment*  Parameter

S-bST

T-bST

P

500 35.6b 29.9b 27.3b 26.6b

493 37.5ab 29.2b 25.0b 25.2b

490 43.1a 38.0a 34.0a 34.0a

– 0.08 0.01 0.01 0.01

12.6ab 4.8 19.7ab 276.5 70.5 4.8 9.6a,A

19.1a 7.3 26.8a 275.9 79.6 6.1 1.1b,B

8.6b 4.0 15.7b 276.8 68.5 6.1 3.8ab,B

0.04 0.58 0.08 0.41 0.29 0.94 0.05

245 32.6ab 24.9b 24.5ab,B

244 29.0b 23.1b 20.4b,B

244 40.8a 35.2a 33.7a,A

– 0.06 0.02 0.01

255 39.4B 35.9 30.0

249 45.1AB 35.5 29.5

246 46.7A 42.1 35.4

– 0.26 0.29 0.34

* Control, single placebo injection at AI; S-bST, single treatment with 325 mg of bST at AI; T-bST, two sequential treatments with 325 mg of bST, the first at AI and the second treatment 14 days later.   Adjusted proportions for categorical data and least square means for continuous data. a,b Proportions on the same row with different superscripts differ (P , 0.05). A,B Proportions on the same row with different superscripts differ (P , 0.10).

growth. Exposure in vitro of zygotes to GH and/or IGF1 enhanced early embryo development, depicting the direct effects of both hormones on the early embryo [15]. The IGF system is composed of ligands, cell membrane receptors, and binding proteins that control ligand bioavailability, transport, and degradation [6]. All these components are expressed in cells of the oviduct, endometrium, and embryo/conceptus and are important for conceptus development, uterine receptivity, and pregnancy establishment [10–12, 17, 43]. Receptors for IGF1 are considered ‘‘dependence’’ receptors, and in addition to survival signals by ligand binding, the absence of ligand binding induces proapoptotic cell signaling [44]. Dairy cows receive the first postpartum AI at early stages of lactation, when IGF1 concentration is low [45], which could potentially limit embryo development. Supplementation with T-bST restored IGF1 concentrations to those

which results in production of most of the circulating IGF1 that can then act on other tissues, including the uterus [6, 42]. The bovine endometrium and conceptus express receptors for GH [7–9]; therefore, paracrine and autocrine responses on those tissues are expected to occur when intense crosstalk between the conceptus and endometrium takes place [13, 32, 43]. In fact, expression of GH receptors in the endometrium increased with pregnancy establishment and progression [7]. Interestingly, Rhoads et al. [9] observed a small variation in the transcript expression of GH receptor in the uterus of lactating dairy cows that was correlated (r ¼ 0.76) with IGF1 transcript expression in the same tissue, potentially indicating a high sensitivity of the uterus to GH stimulus. Therefore, in addition to increasing the systemic supply of IGF1 to the uterus, it is anticipated that bST treatment increased uterine IGF1, which could have aided in enhancing conceptus nourishment and

TABLE 4. Effect of bST treatments on reinsemination and fertility of second AI in cows nonpregnant from first AI (farm A only). Treatment* Parameter Number of cows evaluated Reinseminated (%) § Day of reinseminationz} Pregnant Day 48 after 2nd AI (% )§

Control

S-bST

T-bST

P

166 77.1 23.4 6 0.3 50.7

167 74.1 23.7 6 0.3 54.1

152 74.8 23.5 6 0.3 53.8

– 0.89 0.64 0.89

* Control, single placebo injection at AI; S-bST, single treatment with 325 mg of bST at AI; T-bST, two sequential treatments with 325 mg of bST, the first at AI and the second treatment 14 days later. Proportion of nonpregnant cows on Day 31 6 3 that were reinseminated before the pregnancy diagnosis. z Day of reinsemination relative to first AI. § Adjusted proportion. } Data are presented as least square means 6 SEM.  

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Both farms Number of cows enrolled Pregnant Day 31 (%) Pregnant Day 66 (%) Calving (%) Live calf (%) Pregnancy loss Between Days 31 and 66 (%) After Day 66 (%) Total (%) Gestation length (days) Female calf (%) Twins (%) Calving problems (%) Farm A (sexed semen) Number of cows enrolled Pregnant Day 31 (%) Pregnant Day 66 (%) Calving (%) Farm B (conventional semen) Number of cows enrolled Pregnant Day 31 (%) Pregnant Day 66 (%) Calving (%)

Control

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immunomodulatory features [50]. Administration of IFN-s to lambs caused drastic changes in PBL recirculation and redistribution [50]. These changes might occur in early pregnancy in cattle and could affect the population, type, and activity of immune cells in the endometrium, which might be important for tolerance of conceptus alloantigens and tissue remodeling as described in humans [51]. Comparative analyses of endometrial transcriptome in cows and women showed changes during early pregnancy involving the immune system and highlighted the possible role of immune cells during conceptus implantation in cattle [52]. In the present study, the group of cows with increased modification in transcript abundance in PBL had greater fertility and reduced pregnancy losses, which suggests that changes in the immune system in response to pregnancy might, in fact, be important to pregnancy establishment and survival in dairy cows. PSPB is one of the modern pregnancy-associated glycoproteins secreted by binucleated cells of the synepitheliochorial placenta [53]. The specific functions of PSPB are not totally understood but may play roles for conceptus development and pregnancy establishment, and they have been used as markers for conceptus viability and pregnancy in cattle [32, 33]. In fact, cows that lost the pregnancy after 31 days of gestation had smaller concentrations of PSPB in plasma, as early as 24 days after AI, than did cows that maintained the pregnancy. Interestingly, cows that lost the pregnancy tended to have less mRNA expression of ISG15 in PBL on Day 19 after AI compared with those that maintained pregnancy, indicating differences in the biology of those pregnancies early in the preimplantation period. A number of studies have demonstrated the beneficial effects of bST on the fertility of dairy cows either by increasing P/AI or by reducing pregnancy loss [19–21, 54, 55]. In most cases, cows were either subjected to an estrous synchronization protocol when bST was used [19–21], or bST was administered when the cow was detected in estrus [54, 55]. Although one cannot overlook the potential effects of GH/IGF1 on follicle development, oocyte quality, and fertilization [15, 56], it is thought that the beneficial effects of bST supplementation occur primarily after ovulation, during the pre- and periimplantation periods [20]. In fact, benefits of bST in the current study were only observed when cows received a second treatment 14 days after AI, reinforcing the importance of sustained increases in GH and IGF1 during early embryo development. The dose of bST that was lower than the typical 500 mg at a 14-day interval used to stimulate milk production was chosen as a conservative approach based on evidence that 500 mg might be the upper limit to stimulate reproduction of dairy cows [22]. When lactating cows received two treatments of 500 mg of bST at 11-day intervals, conceptus size and P/AI on Day 17 of gestation increased [17]; however, when nonlactating cows received the same treatment regimen, bST decreased P/AI [23]. The authors suggested that bST may have excessively increased plasma IGF1 and insulin in nonlactating cows, which could have caused asynchrony between the conceptus and uterus that was detrimental to pregnancy [23]. Rivera et al. [22] failed to demonstrate any positive effects of bST on the fertility of dairy cows when 500 mg was administered at 10-day intervals. In fact, not only did large doses of bST fail to improve P/AI, but they suppressed estrous behavior [22]. Administration of bST to beef cattle or dairy heifers, animals that typically have greater concentrations of IGF1 than lactating dairy cows, usually failed to improve fertility [46, 55]. In laboratory species, excessive IGF1 in utero negatively affected embryo survival [24], and high concentrations of IGF1

observed in nonlactating cows [46], which typically have increased fertility. Growth hormone can directly influence embryo development and histotroph production by endometrial cells. For instance, during early embryo development, GH stimulated proliferation of trophectoderm and had antiapoptotic activity in both the trophectoderm and the inner cell mass [14, 16]. Kolle et al. [8] demonstrated that GH receptors are present starting at the 2-cell stage in bovine embryos, and they are important for metabolism of glycogen and transport of lipids in blastocysts. Additionally, the glandular epithelium of the ovine uterus responded to intrauterine administration of GH by increasing mRNA expression of histotroph proteins when IFN-s was present in the uterus [13]. Starting at pre- and peri-implantation transition, GH-like peptides referred to as placental lactogens are synthesized by the bovine placenta, coordinate endometrial responses that are important for conceptus development, and further substantiate the importance of somatogenic hormones during pregnancy [47, 48]. Induction of cell proliferation, antiapoptotic effects, and improved histotroph secretion by GH and IGF1 are all factors that potentiate embryo development and are consistent with the increased embryo survival and larger amniotic vesicle and embryo sizes observed in T-bST cows. Conceptus development and survival depends on an intimate crosstalk between trophectoderm and endometrial cells for establishment of a servomechanism of conceptus nourishment and pregnancy signaling [13]. IFN-s secreted by trophectoderm cells is of central importance in those mechanisms in ruminants, which involve the stimulation of ISG expression in endometrial cells and blockage of the luteolytic cascade. Concentration of IFN-s in uterine secretions is mainly determined by the size of the conceptus [17], which reflects the number of trophectoderm cells. Therefore, cell proliferation and consequent elongation of the conceptus becomes extremely important to prevent the demise of the corpus luteum and to maintain the pregnancy [13]. The actions of IFN-s are not restricted to the uterine lumen, and small quantities reach the systemic circulation, inducing expression of ISGs in peripheral cells such as the leukocytes [27–29]. Responses to IFN-s by PBL seem to be related to the amount of IFN-s in utero [30] and were used in the present study as an indicator of conceptus development around the onset of implantation, on Day 19 after AI. Corroborating our hypothesis, cows treated with two doses of bST had increased expression of ISG15 and RTP4, suggesting that the conceptuses at the preimplantation period were already larger and secreted more IFN-s in utero. In addition, among pregnant cows, those treated with bST had an earlier rise of PSPB in plasma than did control cows, detected at 21 days after insemination, which supports the idea of advanced development of the conceptus and earlier signaling of pregnancy. These responses early in gestation are likely linked with the increased size of the conceptus and support the enhanced P/AI observed later in gestation. Nonetheless, the advanced conceptus development was not followed by changes in gestation length or altered incidence of calving complications that could indicate delivery of larger calves. It is not clear why expression of MX1 and MX2 did not follow the same pattern in response to treatments. Perhaps different ISGs responded differently to increased amounts of IFN-s, or direct effects of GH and IGF1 on PBL [49] could influence their responses to IFN-s. It remains unclear the importance of peripheral immune responses to IFN-s to pregnancy establishment and maintenance in cattle. IFN-s is a type I IFN that has several

LOW-DOSE bST AND FERTILITY IN DAIRY COWS

increased apoptosis in the inner cell mass of embryos [57]. The negative effects of high IGF1 are thought to be mediated by reduced IGF-receptors in embryonic cells, which impairs glucose uptake and triggers apoptosis [57]. Embryo responses to GH in vitro are usually dose-dependent [16], which emphasizes the importance of the dose of bST and subsequent concentrations of GH and IGF1 to stimulate fertility. Nonetheless, the critical timing of administration and dose regimen of bST supplementation to enhance fertility in lactating dairy cows is still not clearly established. Others have demonstrated that a single treatment with 500 mg of bST at insemination increased P/AI in lactating dairy cows inseminated at detected estrus [54, 55]. In addition, concentrations of both GH and IGF1 in the blood of cattle usually follow the dose of bST administered, and as the dose increases, so do the concentrations of GH and IGF1 in the serum of dairy cows [46]. Although S-bST did not improve P/ AI, cows receiving S-bST had greater pregnancy loss between gestational Days 31 and 66 than did T-bST. It is possible that cows receiving S-bST had a delay in pregnancy loss, which was observed after Day 31 of gestation. On the other hand, cows not receiving bST might have had more pregnancy losses occurring before Day 31. This might explain the increased concentrations of PSPB in the plasma of nonpregnant control cows on Day 31 compared with those of nonpregnant bSTtreated cows and the delayed distinction of PSPB concentration in the plasma of pregnant and nonpregnant cows in the control group. Perhaps a single dose of bST partially stimulated the development of less-competent embryos, but it was not sufficient to extend beyond the preimplantation period. In other studies, use of 500 mg of bST at 10- to 14-day intervals reduced expression or detection of estrus [21, 22]. Thus, it was thought that bST treatment could reduce the rate of reinsemination of cows that failed to become pregnant to the first AI. However, treatment with 325 mg of bST either as SbST or T-bST did not affect the proportion of nonpregnant cows reinseminated by Day 31 or the interval between AI. Therefore, it is possible that the negative effects of bST on estrous expression are observed at higher doses, but not when 325 mg is used. In summary, treatment of lactating dairy cows with 325 mg of bST increased concentrations of GH and IGF1 in plasma in a dose- and time-dependent fashion but had minor effects on plasma concentrations of insulin and progesterone. A single treatment with 325 mg of bST at AI was not sufficient to alter conceptus development and fertility. On the other hand, two sequential treatments of 325 mg of bST administered on Days 0 and 14 relative to AI increased mRNA expression of ISG15 and RTP4 in leukocytes, induced an earlier rise in PSPB in plasma of pregnant cows, increased amniotic vesicle and embryo/fetus sizes, and improved P/AI and calving of healthy calves compared with cows receiving either no bST or a single treatment at insemination. Additionally, cows receiving two sequential treatments had reduced pregnancy loss compared with those receiving a single bST treatment at AI. Rate and timing of reinsemination and fertility of reinseminated cows that failed to become pregnant to the first AI were not affected by bST treatments. In conclusion, treatment with two sequential doses of 325 mg of bST on Days 0 and 14 relative to AI increased fertility of lactating dairy cows likely because of sustained increases in GH and IGF1 during the pre- and periimplantation periods, resulting in improvements in conceptus development and a reduction in embryonic mortality. Supplementation with a low dose of bST at AI and 14 days later might be used strategically to improve the fertility of lactating dairy cows.

ACKNOWLEDGMENT The authors thank the owners and staff of Full Circle Jerseys (Dalhart, TX) and Frisia West Dairy (Hartley, TX) for use of cows and facilities for this study. Our appreciation is extended to Dr. Howard Green, Dr. Thomas Bailey, and Dr. Christopher Ashworth (Greenfield, IN) for assistance with the study.

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