Metabolic and endocrine profiles and hepatic gene expression in periparturient, grazing primiparous beef cows with different body reserves

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Metabolic and endocrine profiles and hepatic gene expression in periparturient, grazing primiparous beef cows with different body reserves A.L. Astessiano, R. Pérez-Clariget, G. Quintans, P. Soca, A. Meikle, B.A. Crooker, M. Carriquiry

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Received date: 12 April 2014 Revised date: 3 October 2014 Accepted date: 6 October 2014 Cite this article as: A.L. Astessiano, R. Pérez-Clariget, G. Quintans, P. Soca, A. Meikle, B.A. Crooker, M. Carriquiry, Metabolic and endocrine profiles and hepatic gene expression in periparturient, grazing primiparous beef cows with different body reserves, Livestock Science, http://dx.doi.org/10.1016/j. livsci.2014.10.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Metabolic and endocrine profiles and hepatic gene expression in periparturient, grazing primiparous beef cows with different body reserves

A.L. Astessiano

1a

, R. Pérez-Clariget1, G. Quintans2, P. Soca1, A. Meikle3, B.A. Crooker4, M.

Carriquiry1

1

Facultad de Agronomía, Universidad de la República, Montevideo, Av. E. Garzón 780,

Uruguay. 2

Instituto Nacional de Investigación Agropecuaria Treinta y Tres, Ruta Nº 8 km 281, Treinta y

Tres, Uruguay. 3

Facultad de Veterinaria, Universidad de la República, Montevideo, Lasplaces 1550

4

Department of Animal Sciences, University of Minnesota, 1364 Eckles Ave, St. Paul 55108-

6118

Corresponding author: Ana Laura Astessiano. E-mail: [email protected] Tel: (+598)23559636; Fax: (+5982)3543460.

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Abstract The objective was to determine effects of prepartum BCS on metabolic/endocrine profiles and hepatic gene expression and their associations with cow and calf performance in grazing suckled-primiparous beef cows from -49 to 49 days postpartum (DPP). Twenty crossbred cows selected according to expected calving date, were classified at -35 DPP into thin (BCS<4.5) or moderate (BCS•4.5) BCS groups and blocked by calving date. Blood samples were obtained weekly for metabolite and hormone analyses and liver biopsies were collected at −11, 7, 31, and 49 DPP. Cow BW and BCS were greater in moderate than thin cows throughout the period. Estimated energy intake was greater in moderate than thin cows Moderate BCS cows produced more milk than thin cows at 35 DPP and calves from moderate BCS cows had greater BW and average daily gain than calves from thin cows. Serum leptin tended to be greater while adiponectin was less in moderate than thin BCS cows. Overall serum insulin was less in moderate than thin cows while serum IGF-I during the prepartum was greater in moderate than thin BCS cows. Growth hormone receptor (GHR) mRNA was 2-fold greater at -11 DPP while GHR1A and IGF1 mRNA were 2.5-fold less at 49 DPP in moderate than thin BCS cows. The IGFBP2 mRNA decreased in moderate but increased in thin BCS cows from -11 to 49 DPP. These results were associated with changes in body reserves during prepartum and may indicate that prepartum differences in BCS lost can affect nutrient partitioning towards the mammary gland, and subsequent milk production and calf weight. Key words: cattle; pastures; mRNA; metabolic profile, body condition

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Introduction The adaptability of ruminants to periods of nutritional restriction depends on the capacity of their endocrine and metabolic mechanisms to maintain homeostasis (Chilliard et al., 1998). Rangeland cows that calve in winter or early spring experience moderate to severe nutritional restrictions during the last months of gestation due to the reduced quantity and quality of pastures which decreases energy intake and increases the energy costs of grazing (Soca et al., 2013). This determines the onset of a prepartum negative energy balance and loss of BCS (Quintans et al., 2010) to help meet the greater energy demands of the developing fetus and mammary gland (Bell et al., 1995). Greater pre and postpartum body reserves BCS increased milk yield and calf performance in grazing conditions (Quintans et al., 2010) and increased the overall profits from cow-calf operations. In contrast, in primiparous beef cows grazing grasslands, a small difference in BCS at calving (3.5 vs. 4.0, scale 1 - 8) increased probability of cyclicity and early pregnancy (0.2 to 0.7 of probability) when cows maintained BCS during the postpartum but it did not affect calf weaning weight (Soca et al., 2013). Adaptive mechanisms during periods of dietary restriction and/or negative energy balance include increased blood concentrations of GH, which in turn stimulates a state of insulin resistance in the peripheral tissues that increases fat mobilization (increased NEFA) and redirects nutrients – especially glucose - to the fetus and/or milk production (Bell et al., 1995, Bauman, 2000). This increased GH is accompanied by reduced insulin concentrations, which are believed to be part of the consequence of uncoupling of the GH-IGF axis, and the reduced IGF-I concentrations (Lucy et al., 2008). Reduced IGF-I concentrations in dairy cows during early lactation are associated with reduced expression of the liver-specific GH 3

receptor-1A (GHR1A), IGF1 and IGFBP3 mRNA expression (Kobayashi et al., 1999, Loor et al., 2005, Carriquiry et al., 2009). However, in beef cows, hepatic expression of GHR1A and IGF1 mRNA did not change after calving in ad libitum-fed (Jiang et al., 2005) or grazing (Schneider et al., 2010) cows, indicating that this mechanism could be related to the genetic potential for milk production. Leptin and adiponectin, two of the best-characterized adipokines, are signals from adipose tissue that regulate metabolic, endocrine and behavioral adaptations to changes in energy balance and alter energy intake and expenditure, carbohydrate and lipid metabolism, and insulin sensitivity/resistance (Chilliard et al., 2005; Guerre-Millo, 2008; Galic et al., 2010). However, leptin concentrations increased while adiponectin concentrations decreased with increased adiposity (Chilliard et al., 2005; Guerre-Millo, 2008; Galic et al., 2010). While circulating leptin concentrations decreased from pre to postpartum in dairy cows (Meikle et al., 2004; Thorn et al., 2008), leptin concentrations they have been reported to be less (Gentry et al., 2008) or unchanged (Strauch et al., 2002) in pregnant vs. lactating beef cows. Thorn et al. (2008) reported greater hepatic mRNA expression of both short and long isoforms of leptin receptor in early postpartum than in the prepartum period in dairy cows, which was associated with reduced plasma insulin and leptin and with increased plasma growth hormone. Few reports of bovine plasma adiponectin profiles are available but adiponectin concentrations increased during the first month postpartum in dairy cows (Ohtani et al., 2012). Beef cows with different BCS have distinctive productive and reproductive performances (Lake et al., 2006, Soca et al., 2012). Lake et al. (2006) reported elevated GH and reduced IGF-I concentrations in serum from postpartum beef cows managed nutritionally 4

during gestation to achieve a low rather than a high BCS (4 vs. 6 on a 1 to 9 unit scale) at calving. However, it appears that there are no reports of simultaneous insulin, IGF-I, leptin and adiponectin profiles and/or changes in hepatic gene expression during the peripartum of beef cows calving with moderate vs. thin BCS. Improved better understanding of the mechanisms that regulate nutrient partitioning could help improve nutritional management of the beef cow. We hypothesized that the differential energy reserves at calving in terms of BCS (moderate vs. thin BCS) in primiparous grazing beef cows will be reflected in a distinct nutritional partitioning (in terms of metabolic and endocrine profiles and hepatic gene expression) which may explain differences in their productive responses (milk production and calf performance). The objectives of this study were to determine 1) relationships among metabolic and endocrine profiles and hepatic expression of genes associated with the somatotropic axis and the full-length leptin receptor (LEPR-b) and 2) associations among these components with changes in BCS, milk production, and calf performance in grazing, suckled, primiparous beef cows during the peripartum period.

Materials and methods The experiment was carried out at Palo a Pique Experimental Unit of the Instituto Nacional de Investigación Agropecuaria (Treinta y Tres, Uruguay; 33º S, 56º W) from May to November 2007. Animal procedures were approved by the Animal Experimentation Committee of Universidad de la Republica (UdelaR, Montevideo, Uruguay).

Animals and Experimental Design 5

Twenty primiparous crossbred Aberdeen Angus x Hereford cows (449 ± 6.5 kg of BW and 30 ± 1.8 month of age) were selected from a group (n = 64; managed together as a contemporary group since the breeding period in November 2006 and with a similar BCS in May 2007) according to expected calving date (all expected calvings were within a 28 ± 3 days period) for the study. Cows were classified at -35 days postpartum (DPP) according to BCS (scale from 1.0 (very thin) to 8.0 units (very obese); Soca et al., 2012) into thin (BCS < 4.5; 4.2 ± 0.05) or moderate (BCS • 4.5; 4.8 ± 0.05) BCS groups and blocked by calving date. A BCS of 4.5 has been reported as the critical BCS at calving because subsequent reproductive performance of primiparous cows is reduced if their BCS at calving is < 4.5 (Soca et al., 2012). Differences in BCS were not due to dietary treatments, because all 20 cows were managed together in the same pasture. All cows and calves were from the same herd (similar genetic background) and in a good sanitary status from the beginning of the study. During the study (pre and postpartum periods), cows grazed together in the herd of 64 cows a native pasture paddock (60 ha) with good access to water. Available forage mass was determined by cutting squares (0.3 x 0.3 m, n = 60; every 28 ± 3 days from -49 to 49 ± 9 DPP). Forage provided 453 ± 34 kg dry matter (DM)/ha with 132 ± 19 g of crude protein (CP) and 244 ± 6 g of acid detergent fiber (ADF) per kg DM during the prepartum (-49 to 0 ± 9 DPP) and 552 ± 86 kg DM with 144 ± 7 g of CP and 251 ± 1 g of ADF per kg DM during the postpartum (0 to 49 ± 9 DPP) periods. Due to low forage mass available during the early postpartum period (from 17 to 28 ± 9 DPP), all cows were offered an additional 4.7 kg DM/cow/day of pasture hay (137 g/kg DM of CP and 300 g/kg DM of ADF) during 12 days.

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Calves were managed with their dams and did not receive any supplementation during the study.

Data and Sample Collection Body weight and BCS were measured every 14 days from -49 to 49 ± 9 DPP and at calving. Cow BCS was determined by the same observer throughout the study who did not know to which BCS group cows belonged. Blood samples were collected weekly from -49 to 49 ± 9 DPP via jugular venipuncture using Vacutest® tubes (8 mL, Vacutest Kima, Arzergrande, Italy) that contained clot activator gel. Samples were centrifuged (2000 X g for 15 min at 4°C) within 2 h after collection and serum was stored at -20° C until assayed. Milk production was determined from a subset of 12 cows (n = 6 per BCS group) at 14 and 35 ± 4 DPP according to Quintans et al. (2010). Briefly, cows were separated from calves and milked in the morning (to empty the udder) and in the afternoon using a portable milking machine previous intramuscular injection of 20 IU of oxytocin (Hipofamina, Laboratorio Dispert SA, Uruguay), to facilitate milk letdown. Milk weight was recorded in the afternoon collection and adjusted to a 24 h period. Samples from the afternoon milking were preserved with potassium dichromate upon collection and analyzed for fat, protein, and lactose by infrared analyses (Bently 2001; Bently, USA; Dairy Laboratory; INIA-La Estanzuela, Colonia, Uruguay). Liver biopsies (approximately 0.5 g of tissue) were collected at -11 ± 4, and 7, 31, and 49 ± 2.5 DPP from the same subset of cows in which milk production was determined. Liver biopsies were obtained using a 14-gauge biopsy needle (Tru-Core®-II Automatic Biopsy 7

Instrument; Angiotech, Lausanne, Suitzerland) as described by Carriquiry et al. (2009). Liver samples were placed in a screw-cap DNAse free microcentrifuge tube DNAse / RNAse free, immediately frozen in liquid nitrogen and stored at −80°C until total RNA was extracted. Calf weight was determined at birth and at 40 ± 9 days of age. Serum analyses Metabolites, insulin, IGF-I, and leptin concentrations were measured every 14 days in all cows on the study while adiponectin concentration were determined at -49, -21, 7, 21, 35 and 49 DPP. Non-esterified fatty acids, cholesterol, and urea concentrations were determined spectrophotometrically using commercial kits (Wako NEFA-HR(2) from Wako Pure Chemical Industries, Ltd. Osaka, Japan, Cholesterol , and Urea/BUN - Color from BioSystems S.A., Barcelona, Spain; respectively) according to Astessiano et al. (2012). The intra and interassay CV values for low, medium and high controls were not greater than 12.3% Insulin concentrations were quantified by solid-phase RIA (Coat and Count, Diagnostic Products Co, Los Angeles, CA, USA). The sensitivity of the assay was 1.2 μIU/mL; intra and inter-assay CV for control 1 (2.8 μIU /mL) and control 2 (16 μIU /mL) were not greater than 7 and 6.3%, respectively. Concentrations of IGF-I were quantified by RIA (Weber et al., 2007). The sensitivity of the assay was 5.9 ng/mL; intra and inter-assay CV were 8.0 and 3.5% and 10.6 and 6.1%, for low (81.4 ng/mL) and high (223.1 ng/mL) controls, respectively. Leptin concentrations were determined by a liquid-phase RIA using a commercial Multi-Species Leptin kit (RIA kit, Millipore, USA) previously validated for bovines (Pinotti and Rosi, 2006). The RIA had a sensitivity of 2.9 ng/mL. All samples were determined in the same assay and the intra-assay CV for control 1 (4.2 ng/mL) and control 2 (18.8 ng/mL) were 8 and 10.3%, 8

respectively. In the absence of purified bovine adiponectin, concentrations of adiponectin were measured with a human RIA kit (HADP-61 HK, Millipore, USA) using undiluted serum samples. The sensitivity of the assay was 4.3 ng/ml. All samples were determined in the same assay and the intra-assay CV for control 1 (12.2 ng/mL) and control 2 (95.4 ng/mL) were 3.6 and 4.2%, respectively. Quantitative real time PCR Isolation of total RNA from hepatic tissue and synthesis of cDNA by reverse transcription was performed according with Carriquiry et al. (2009) (Supplementary Material S1). Primers (Supplementary Table S1) were obtained from literature (Carriquiry et al., 2009; Astessiano et al., 2012; Lemor et al., 2009) or designed (Primer Express Software; Applied Biosystems, Foster City, CA, USA) to specifically amplify cDNA for GHR, GHR1A, IGF1, IGFBP2, IGFBP3, insulin receptor (INSR), LEPR-b as target genes of interest and for ß-actin (ACTB) and hypoxanthine phosphoribosyltransferase (HPRT) as endogenous controls. Before use, primer product size (1% agarose gel separation) and sequence (Macrogen Inc., Seoul, Korea) were determined to ensure that the primers produced the desired amplification products (data not shown). Both ACTB and HPRT have been used before as endogenous control genes in tissues from ruminants (Loor et al., 2005; Carriquiry et al., 2009; Astessiano et al., 2012) and their expression was stable between BCS groups and across time points in the samples of this study. Real time PCR reactions were performed in a final volume of 20 μL using SYBR®Green mastermix (Quantimix EASY SYG kit, Biotools B&M Labs, Madrid, Spain), according with Astessiano et al. (2012) (Supplementary Material S1). Dissociation curves were generated after the last cycle. The absolute mRNA expression was determined based 9

on a standard curve constructed from a cloned plasmid cDNA containing the amplified fragment for each target and control gene (n = 6 dilutions, from 106 to 101). Intra and interassay CV values were 1.9 and 4.2 %, respectively. Expression of each target gene was normalized to the mean expression of ACTB and HPRT controls genes.

Energy calculations Estimates of energy requirements were calculated according to NRC (NRC, 2000) and average individual net energy (NEm) intakes for the pre and postpartum periods were estimated from animals energy requirements (Smit et al., 2005). Milk energy output was calculated as NEL (MJ) = milk yield × ((0.3883 × fat %) + (0.2353 × true protein %) + (0.1651 × lactose %)), using composition data derived from analysis of the samples collected.

Statistical analyses Data were analyzed in a randomized block design (cows of different BCS were blocked by calving date) using the SAS Systems program (SAS 9.0V; SAS Institute Inc., Cary, NC, USA). Univariate analyses were performed on all variables to identify outliers and inconsistencies and to verify normality of residuals. Cow BW, BCS, milk production and composition, serum metabolite and hormone concentrations, and hepatic mRNA expression were analyzed as repeated measures using the MIXED procedure with DPP as the repeated effect and first-order autoregressive (for evenly spaced data) or spatial power law (for unevenly spaced data) as the covariance structure. The Kenward-Rogers procedure was used to adjust the denominator degrees of freedom. The model included BCS group, DPP, and their interaction as fixed effects, and block and cow as random effects. Calf BW and 10

average daily gain were analyzed using the same model with calf sex included also as a fixed effect. Tukey–Kramer tests were conducted to analyze differences between cow groups and DPP. In addition, BCS response was fitted to appropriate order-polynomial curves. Correlation analysis (the CORR procedure) was used to describe relationships between variables. Results are expressed as LSMeans ± SE. Results

Cow and calf performances Moderate cows had greater (P < 0.001) BW (413 vs. 394 ± 2.5 kg) and BCS (4 vs. 3.6 ± 0.03 units) than thin cows throughout the 98-day period and there was an effect of DPP (P < 0.001) on both variables (Figure 1A and B). Evolution of BCS fitted a quadratic model (P < 0.001) for both BCS groups (Figure 1B). Intercept values indicate that BCS was 0.5 greater (P < 0.001) in moderate than thin cows (3.9 vs. 3.4 ± 0.05) at calving. Linear coefficients did not differ (-0.012 ± 0.001; P = 0.334) but the quadratic coefficient tended (0.00014 vs. 0.00021 ± 0.00002; P = 0.095) to be less for moderate than thin cows which is consistent with a similar rate of peripartum reduction in BCS but a slower postpartum recovery of BCS in the moderate BCS cows. Cow BCS at nadir was greater (P < 0.001) in moderate than thin cows (3.6 vs. 3.3 ± 0.04) but the moment of nadir did not differ between groups (28 vs. 42 ± 5 DPP for thin and moderate cows, respectively). Calf BW at birth (34.2 vs. 29.0 ± 1.0 kg) and at 40 days of age (64.4 vs. 55.4 ± 3.2 kg), and average daily gain (ADG, 0.81 vs. 0.65 ± 0.05 kg/day) from birth to 40 days of age were greater (P < 0.05) for calves from moderate than thin cows. Insert Fig1

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Milk production and energy requirements and intake Average milk production did not differ between BCS groups, but was affected by DPP (P = 0.020) and an interaction (P = 0.037) of BCS group and DPP (Figure 2). While no differences in milk production were observed among groups on day 14 (Table 1), thin cows decreased their production on day 35 and had lower milk production than moderate cows on this day. Milk composition did not differ between BCS groups and averaged 2.70 ± 0.30, 2.96 ± 0.11, and 4.95 ± 0.6% for fat, protein, and lactose, respectively. Milk protein and fat percentages tended to decrease (P < 0.080) while lactose percentage increased (P < 0.001) from 14 to 35 DPP in both groups. Consistent with the impact on milk yield, there was a trend for an interaction (P = 0.080) of BCS group and DPP as milk energy output decreased (P < 0.05) only in thin cows (16.0 to 14.0 ± 1.3 MJ/d and 16.7 to 10.2 MJ/d from 14 to 35 DPP in moderate and thin cows, respectively). Insert Table 1 Total energy requirements for maintenance and gestation were greater (P < 0.05) in moderate than thin cows from -49 DPP to calving (49.1 and 42.0 ± 1.3 MJ/d of NEm, respectively) and did not increase dramatically from late gestation to early lactation (42.6 to. 48.5 ± 1.3 MJ/d of NEm for both groups). Average estimated energy intake increased (P < 0.05) from pre to postpartum (32.1 ± 0.75 to 49.0 ± 0.0.83 MJ/d of NEm) and was greater (P < 0.001) for the moderate than thin cows during the prepartum and postpartum period (34.5 vs. 29.6 ± 0.75 and 50.5 vs. 47.6 ± 0.83 MJ/d of NEm, respectively). Both cow BCS groups mobilized energy from tissues during the prepartum period (-2.79 and -2.69 ± 0.24 MJ/d of NEm), but during the postpartum period moderate cows maintained (-0.12 ± 0.23 MJ/d of NEm) while thin cows recovered (-0.35 ± 0.17 MJ/d of NEm) tissue energy.

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Metabolites and hormones Serum NEFA concentration (Figure 2A) did not differ between BCS groups but was affected by DPP (P < 0.001). Concentrations of NEFA were elevated during the prepartum period, peaked at -7 DPP, decreased until 21 DPP, and were maintained thereafter. However, the peak in NEFA at -7 DPP was only evident in moderate BCS cows. Serum cholesterol (Figure 2B) did not differ between BCS groups, but was affected by DPP (P < 0.001) as concentrations remained low from -49 to 21 DPP and increased thereafter. Although the interaction between BCS group and DPP was not significant, serum cholesterol was greater (P < 0.05) in moderate than thin BCS cows at 35 and 49 DPP. Serum urea concentration (Figure 2C) did not differ between BCS groups, but was affected by DPP (P < 0.001) and by the interaction (P = 0.033) between BCS group and DPP. Concentrations of urea in moderate BCS cows were increased at -7 DPP and decreased at 7 DPP whereas serum urea remained constant in thin cows. Insert Fig 2 Although there was no effect of DPP on insulin concentrations (Figure 3A), serum insulin was less (P=0.024) in moderate than thin cows (1.28 vs. 1.88 ± 0.38 μUI/mL), due to increased (P < 0.05) serum insulin after 7 DPP in thin cows. Concentrations of IGF-I (Figure 3B) were only affected by DPP (P=0.020) as serum IGF-I did not change from -49 to -7 DPP, decreased at 7 DPP, increased at 21 DPP, and remained stable thereafter. However, when only the prepartum period was considered, serum IGF-I was greater (P = 0.026) in moderate than thin cows (53.5 and 44.8 ± 2.5 ng/mL, respectively). Leptin concentrations (Figure 3C) tended (P = 0.108) to be greater in moderate than thin cows (4.8 vs. 4.3 ± 0.3 ng/mL, respectively) and did not vary during the period evaluated. Concentrations of adiponectin (Figure 3D) were less (P = 0.004) in moderate than thin cows (152 vs. 106 ± 18 ng/mL, 13

respectively) and were affected by DPP (P = 0.013). Serum adiponectin increased from -49 to -21 DPP, decreased from -21 to 21 DPP, and remained stable through 49 DPP. Insert Fig 3

Hepatic mRNA expression Expression of GHR mRNA tended to be greater (P = 0.075) for moderate than thin cows (1.4 vs. 1.1 ± 0.1) but was not affected by DPP or their interaction (Table 2). Expression of GHR1A was not affected by BCS group or DPP but there was an interaction between BCS group and DPP (P = 0.054) as GHR1A mRNA increased (P < 0.05) at 49 DPP only in thin cows (Table 2). Hepatic IGF1 mRNA expression was more reduced (P = 0.041) in moderate than thin cows, but was not affected by DPP or the interaction of BCS and DPP. The difference in IGF1 mRNA between moderate and thin cows was only evident at 49 DPP (Table 2). Expression of IGFBP2 mRNA was affected by the interaction of BCS group and DPP (P = 0.045) as abundance of this transcript decreased from -11 to 49 DPP in moderate BCS cows, while it tended (P = 0.082) to increase in thin cows during the same period (Table 2). Expression of IGFBP3 mRNA and LEPR-b were not affected by BCS group, DPP or their interaction (Table 2). Hepatic INSR mRNA increased (P= 0.008) from pre to postpartum in both, moderate and thin, BCS groups (Table 2). Independent of BCS groups or DPP (n = 48 samples: 12 cows per 4 time points), serum IGF-I tended to be correlated with hepatic GHR1A (r = 0.33, P = 0.057) and IGF1 (r = 0.31, P < 0.074) mRNA, while hepatic expression of IGF1 mRNA was correlated with GHR1A (r = 0.56, P < 0.001) and IGFBP3 (r = 0.52, P < 0.001) mRNA. Expression of GHR and GHR1A mRNA were also correlated (r = 0.47, P = 0.003). Serum insulin concentrations were 14

positively correlated with expression of IGF1 mRNA (r = 0.45, P = 0.010) but they were negatively correlated with IGFBP2 mRNA (r = -0.37, P = 0.036). Concentrations of adiponectin were correlated (r = 0.74, P < 0.001) with hepatic INSR and IGF1 mRNA and expression of INSR was correlated (r > 0.50, P = < 0.015) with IGF1 and LEPR-b mRNA. Insert Table 2 Discussion Loss of BCS during the prepartum period can vary among cows and the magnitude of this loss affects subsequent cow performance (Lake et al., 2005, Soca et al., 2012). Our moderate and thin cows were managed together under the same grazing conditions but achieved -35 DPP with different BCS. Actual reasons for this differential BCS between moderate and thin cows are unknown but it could be due to a combination of individual animal variation in intake (i.e. better grazing ability, greater social hierarchy) or efficiency (reduced demand for energy maintenance) when quantity and quality of forage in native pasture is limiting (winter). In agreement with the estimated energy intakes, cows in both groups lost more BCS during the pre than the postpartum interval (1.2 vs. 0.2 units and 1.1 vs. 0.1 units for moderate and thin cows, respectively). The larger prepartum losses reflected the reduced availability of forage in native pastures during winter which prevented cows from consuming enough feed to meet the energy demands of the last trimester of gestation. Moderate BCS cows, had a greater energy intake and BCS during both the pre and postpartum intervals. Similarly, cows fed during gestation to achieve greater differences in BCS than those in this study, calved with greater BCS and maintained greater BCS postpartum (Lake et al., 2006). In addition, the small difference in BCS at calving (0.5 units) and/or other differences in 15

metabolism between the groups appear to have been sufficient to elicit differences in calf birth weight. This difference in calf birth weight was probably the result of the greater supply of nutrients to the growing fetus (Bell, 1995). No differences in milk production were found at 14 DPP which is in accordance with reports that milk production in early lactation is not influenced by BCS at calving (Lake et al., 2005) or energy intake during the prepartum period (Radunz et al., 2012). However, moderate BCS cows produced more milk on day 35 which was associated with a more rapid growth of their calves during the first 40 days of age in agreement with previous reports (Beal et al., 1990). Concentrations of NEFA were elevated during the prepartum period in both BCS groups reflecting mobilization of lipid stores (Chilliard et al., 1998; Bauman, 2000). A peak in NEFA and urea concentrations was observed only in moderate BCS cows at -7 DPP and could reflect greater mobilization of reserves from adipose tissue (Meikle et al., 2004) and muscle (Chimonyo et al., 2002). This greater mobilization could be caused by greater energy requirements for conceptus growth (Bell, 1995) and maintenance of maternal non-uterine tissues (Houghton et al., 1990). Indeed total energy requirements for maintenance and gestation (estimated by NRC 2000) were 11% greater in moderate than thin cows during this period, which were supplied by an increase in both energy intake and energy tissue mobilization. The increase in cholesterol concentrations on 35 DPP is consistent with NEFA decrease, as it reflects a better energy status (Chimonyo et al., 2002). Insulin concentrations in both groups were low around calving as has been reported earlier in dairy and beef cows (Meikle et al., 2004; Quintans et al., 2010). Reduced insulin concentrations reflect the low energy intake caused by lack of available forage. Reduced insulin concentrations favor gluconeogenesis and lipolysis while increased insulin 16

concentrations are associated with reduced gluconeogenesis and greater lipogenesis (Bauman, 2000). The insulin increase in thin cows after 7 DPP is an interesting finding and was associated with lower milk production and calf ADG and a faster recovery of BCS (reflected in a greater quadratic coefficient of the BCS regression) and greater estimated body energy deposition. Although serum insulin was stable around calving in both groups, thin cows tended to present reduced leptin concentrations and greater adiponectin concentrations than cows with moderate BCS. Leptin, adiponectin and other compounds from adipose depots are involved in regulation of energy homeostasis (Galic et al., 2009; Liu et al., 2010) and reduced leptin and greater adiponectin are associated with increased feed intake, reduced energy expenditure, and favor insulin synthesis and secretion and insulin sensibility (Chilliard et al., 2005; GuerreMillo et al., 2008; Thorn et al., 2008). Greater leptin and reduced adiponectin concentrations during the postpartum in moderate than thin cows would facilitate postpartum insulin resistance, which would increase the supply of glucose to the mammary gland and help in the metabolic support of lactation (Ohtani et al., 2012) and likely contribute to their greater milk yield. Serum IGF-I during the prepartum and hepatic expression of GHR mRNA was greater at -11 DPP in moderate than thin cows, probably associated with their better metabolic status due the greater energy intake. Similarly, greater prepartum IGF-I concentrations have been reported in high than in low BCS grazing dairy cows when cows were classified according with their BCS at calving (Meikle et al., 2004) or when cows were fed during gestation to achieve greater BCS at -30 days (Adrien et al., 2012). The greater total GHR mRNA expression in moderate BCS cows at -11 DPP was not only due to a greater expression of 17

GHR1A mRNA, as ratio of GHR to GHR1A mRNA was similar (0.41 vs. 0.35) between BCS groups, which would suggest that other transcripts of the GHR gene were involved (Wang et al., 2003). Indeed this ratio was relatively stable (039 ± 0.02) throughout the study and only differed (0.74) at 49 DPP for the thin cows. Serum IGF-I decreased on 7 DPP after calving and increased thereafter, as previously reported in dairy cows (Meikle et al., 2004; Lucy et al., 2008). Concentrations of IGF-I have been showed to increase (Spicer et al., 2002; Lake et al., 2006) or not change (Spicer et al., 2002; Schneider et al., 2010) in beef cows during the postpartum period. However, these reports did not include prepartum sampling. The decrease in circulating IGF-I at calving is consistent with an uncoupled somatotropic axis, which mediates nutrient partitioning for milk production (Bauman, 2000; Lucy et al., 2008). However, in contrast with the pattern of GHIGF axis gene expression in dairy cows (Kobayashi et al., 1999; Lucy et al., 2008), no pre to postpartum (-11 vs. 7 DPP) changes in hepatic GHR1A and IGF1 mRNA expression were detected in our beef cows. Similar results have been reported in ad libitum fed and grazing periparturient beef cows (Jiang et al., 2005; Schneider et al., 2010). Expression of hepatic GHR1A and IGF1 in our moderate BCS cows was in fact stable throughout the study period, while in thin cows GHR1A mRNA increased by 49 DPP and expression of IGF1 mRNA trended upward and was greater than in moderate cows at 49 DPP. These expression profiles in thin cows did not correspond with the serum IGF-I profile and this indicates potential post-transcriptional regulation of IGF1mRNA. Alterations in relative amounts of the IGFBPs (Fenwick et al., 2008; Gross et al., 2011) could also be affecting IGF-I availability and ability of serum IGF-I to elicit tissue responses. In contrast to the postpartum decrease in hepatic IGFBP3 mRNA expression in dairy cows (Loor et al., 18

2005; Carriquiry et al., 2009; Gross et al., 2011), no changes were observed in the expression of this transcript in the present study while expression of IGFBP2 mRNA decreased in moderate cows but tended to increase in thin cows from pre to postpartum. The decreased IGFBP2 mRNA in moderate BCS cows towards 49 DPP would agree with their greater milk production and indicate a better nutritional status (ie. BCS; Rajaram et al., 1997) as reflected in the negative correlation between circulating insulin concentrations and IGFBP2 mRNA determined in this and other (Fenwick et al., 2008) studies. Greater hepatic IGFBP2 mRNA has been associated with reduced insulin resistance in mice (Hedbacker et al., 2010). Hepatic INSR mRNA increased from the pre to postpartum period. Previous reports have associated increased hepatic expression of INSR mRNA in early postpartum dairy cows with negative energy balance (Gross et al., 2011) and suggested reduced insulin concentrations cause an up regulation of hepatic INSR mRNA in order to maintain insulin function in the liver, while maximizing nutrient supply to the mammary gland. Hepatic INSR mRNA expression tended to increase earlier in thin cows, which had greater serum insulin concentrations, compared to moderate BCS cows. The positive correlations between serum adiponectin and hepatic expression of INSR and IGF1 in our cows are also consistent with the reduced milk yield by thin BCS cow (Ohtani et al., 2012) Hepatic expression of LEPR-b was very low and did not change with DPP in our study. Thorn et al. (2008) reported that increased hepatic LEPR-b mRNA was associated with decreased serum leptin and insulin concentrations in the transition dairy cow. Given that in the present study peripartum leptin and insulin concentrations were not affected by day postpartum or by the interaction of day and BCS , changes in LEPR-b expression might not be expected in our cows. 19

In summary, grazing primiparous beef cows that calved in late winter with a reduced BCS (3.4 vs. 3.9, scale 1-8) produced less milk and delivered lighter calves that grew less rapidly through the first 40 d of life. This difference in body reserves was reflected in metabolic/endocrine profiles and liver expression of somatotropic axis genes that are consistent with a greater partitioning of nutrients towards milk production and calf growth.

Acknowledgments The authors thank Palo a Pique Experimental Unit staff for excellent animal care and courteous assistance throughout the study. This work was supported by research project ANII-FCE2007-614.

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Hedbacker, K., Birsoy, K., Wysocki, R.W., Asilmaz, E., Ahima, R.S., Farooqi, I.S., Friedman, J.M., 2010. Antidiabetic effects of IGFBP2, a leptin-regulated gene. Cell Metab. 11, 1122 Galic ,S., Oakhill, J.S., Steinberg, G.R., 2010. Adipose tissue as an endocrine organ. Mol. Cell. Endocrinol. 25, 316,129-39. Gentry, L.R., Thompson, D.L., Gentry, G.T., 2002. The relationship between body condition, leptin, and reproductive and hormonal characteristics of mares during the seasonal anovulatory period. J. Anim. Sci. 80, 2695–2703. Gross, J., van Dorland, H.A., Bruckmaier, R.M., Schwarz, F.J., 2011. Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation. J.Dairy Sci. 94, 1820–1830. Guerre-Millo, M., 2008. Adiponectin: an update. Diabetes & Metabolism 34, 12-18. Houghton, P.L., Lemenager, R.P., Horstman, A., Hendrix, K.S., Moss, G.E., 1990: Effects of body composition, pre- and postpartum energy level and early weaning on reproductive performance of beef cows and preweaning calf gain. J. Anim. Sci. 68, 1438-1446. Jiang, H., Lucy, M.C., Crooker, B.A., Beal, W.E., 2005. Expression of growth hormone receptor 1A mRNA is decreased in dairy cows but not in beef cows at parturition. J.Dairy Sci. 88,137-7. Kobayashi, Y., Boyd, C.K., Bracken, C.J., Lamberson, W.R., Keisler, D.H., Lucy, M.C., 1999. Reduced growth hormone receptor (GHR) messenger ribonucleic acid in liver of periparturient cattle is caused by a specific down-regulation of GHR1A that is associated with decreased insulin-like growth factor I. Endocrinology 140, 3947–54. 22

Lake, S.L., Scholljegerdes, E.J., Atkinson, R.L., Nayigihugu, V., Paisley, S.I., Rule, D.C., Moss, G.E., Robinson, T.J., Hess, B.W., 2005. Body condition score at parturition and postpartum supplemental fat effects on cow and calf performance. J. Anim. Sci. 83, 2908–2917 Lake, S.L., Scholljegerdes, E.J., Hallford, D.M., Moss, G.E., Rule, D.C., Hess, B.W., 2006. Effects of body condition score at parturition and postpartum supplemental fat on metabolite and hormone concentrations of beef cows and their suckling calves. J. Anim. Sci. 84, 1038-1047. Lemor, A., Hosseini, A., Sauerwein,,H., Mielenz. M., 2009. Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows. Dom. Anim. Endocrin. 37, 37–44. Liu, G.W., Zhang, Z.G., Wang, J.G., Wang, Z., Xu, C., Zhu, X.L., 2010. Insulin receptor gene expression in normal and diseased bovine liver. J. Comp. Pathol.143, 258-61. Loor, J., Dann, H.M., Everts, R.E., Oliveira, R., Green, C.A., Janovick Guretzky, N.A., Rodriguez-Zas, S.L., Lewin, H.A., Drackley, J.K., 2005. Temporal gene expression profiling of liver from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiology Genomics 23, 217–226. Lucy, M.C., 2008. Functional differences in the growth hormone and insulin-like growth factor axis in cattle and pigs: implications for post-partum nutrition and reproduction. Reprod Domest Anim. 43, 31-9.

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Meikle, A., Kulcsar, M., Chilliard, Y., Febel, H., Delavaud, C., Cavestany, D., Chilibroste, P., 2004. Effects of parity and body condition at parturition on endocrine and reproductive parameters of the cow. Reproduction 127, 727–737. NRC., 2000. Nutrient Requirements of Beef Cattle: Seventh Revised Edition: Update 2000. Washington, D.C: National Academy Press, 234 pp. Ohtani, Y., Takahashi, T., Sato, K., Ardiyanti, A., Song, S.H., Sato, R., Onda, K., Wada, Y., Obara, Y., Suzuki, K., Hagino, A., Roh, S.G., Katoh, K., 2012. Changes in circulating adiponectin and metabolic hormone concentrations during periparturient and lactation periods in Holstein dairy cows. J. Anim. Sci. 83, 788-95 Pinotti, L., Rosi, F., 2006. Leptin in bovine colostrums and milk. Horm Metab Res.38(2): 89-93 Quintans, G., Banchero, G., Carriquiry, M., López, C., Baldi, F., 2010. Effect of body condition and suckling restriction with and without presence of the calf on cow and calf performance. Anim. Prod. Sci.50, 931–938. Radunz, A.E., Fluharty, F.L., Day, M.L., Zerby, H.N., Loerch, S.C., 2010. Prepartum dietary energy source fed to beef cows: I. Effects on pre- and postpartum cow performance. J. Anim. Sci. 88, 2717-2728. Rajaram, S., Baylink, D.J., Mohan, S., 1997. Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr. Rev. 18: 801–831. Schneider, A., Machado, P., Feifer, L.F., Teixeira, Hax L, Paludo GR, Burkert Del Pino FA, Laurino Dionello NJ and Nunes Corrêa M. 2010. Insulin like growth factor and growth hormone receptor in postpartum lactating beef cows. Pesquisa Agropecuária Brasileira 45, 925-931.

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Smit, H.J., Taweel, H.Z., Tas, B.M., Tamminga, S., Elgersma, A., 2005. Comparison of techniques for estimating herbage intake of grazing dairy cows. J.Dairy. Sci. 88, 18271836. Soca, P., Carriquiry, M., Keisler, D.H., Claramunt, M., Do Carmo, M., Olivera-Muzante, J., Rodríguez, M., Meikle, A., 2013. Reproductive and productive response to suckling restriction and dietary flushing in primiparous grazing beef cows. Anim. Prod. Sci.53, 283-291. Spicer, L.J., Chase, C.C., Rutter, L.M., 2002. Relationship between serum insulin-like growth factor-I and genotype during the postpartum interval in beef cows. J. Anim. Sci. 80, 716-722. Strauch, T.A., Neuendorff, D.A., Brown, C.G., Wade, M.L., 2003. Effects of lasalocid on leptin concentrations and reproductive performance of postpartum Brahman cows. J. Anim. Sci. 81, 1363-70. Thorn, S.R., Ehrhardt, R.A., Butler, W.R., Quirk, S.M., Boisclair, Y.R., 2008. Insulin regulates hepatic leptin receptor expression in early lactating dairy cows. Am. J. Physiol. Regul. Integr. Comp. Physio.l 295, 1455–1462. Wang, Y., Eleswarapu, S., Beal, W.E., Swecker, W.S., Akers, R.M., Jiang, H., 2003. Reduced serum insulin-like growth factor (IGF) I is associat ed with reduced liver IGF-I mRNA and liver growth hormone receptor mRNA in food-deprived cattle. J. Nutr. 133, 25552560

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Figure captions Figure 1 Evolution of body weight (A) and body conditions score (B; quadratic regression) during the peripartum period (from -49 to 49 days relative to parturition) in primiparous beef cows classified according to BCS at -35 days prior to parturition (10 moderate Ŷ vs. 10 thin Ƒ). Day of calving is indicated by the dashed vertical line. Differences between BCS groups according to Tukey–Kramer test (P ” 0.05) are indicated with asterisk (*).

Figure 2 Concentrations of NEFA (A), cholesterol (B) and urea (C) during the peripartum period (from -49 to 49 days relative to parturition) in primiparous beef cows classified according to BCS from -35 days to parturition (10 moderate Ŷ vs. 10 thin Ƒ). Calving is indicated with dashed lines. Differences between cow groups according to Tukey–Kramer test (P ” 0.05) are indicated with asterisks (*).

Figure 3 Concentrations of insulin (A), IGF-I (B), leptin (C), and adiponectin (D) during the peripartum period (from -49 to 49 days relative to parturition) in primiparous beef cows classified according to BCS from -35 days to parturition (10 moderate Ŷ vs. 10 thin Ƒ). Calving is indicated with dashed lines. Differences between cow groups according to Tukey–Kramer test (P ” 0.05) are indicated with asterisks (*).

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Table 1 Milk production and composition of primiparous beef cows classified according to BCS from -35 days to parturition (10 moderate vs. 10 thin) at 14 and 35 days of lactation.

Days Milk (kg/d) Fat (%) Protein(%) Lactose (%) Energy Output in Milk (MJ/d)

Treatments Moderate 14

P-value se

35

Thin 14

35

6.02 3.10 3.02 4.80

5.87 2.28 2.93 5.15

6.26 3.09 3.00 4.70

4.39 2.30 2.92 5.17

16.00

BCS

DPP

BCSxDPP

0.64 0.36 0.11 0.06

0.480 0.978 0.914 0.596

0.020 0.060 0.077 <.0001

0.037 0.968 0.933 0.152

13.97 16.73 10.22 1.54

0.455

0.006

0.080

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Table 2. Hepatic expression of genes related to the GH-IGF-I axis, LEPR-b and, INSR mRNA (amount of mRNA expressed relative to the mean expression of endogenous control genes, ACTB and HPRT) during peripartum period in primiparous beef cows classified according to BCS at -35 days relative to parturition.

Treatments Days

Moderate 7 31

-11

49

P-value se

Thin 7 31

-11

49

BCS

DPP BCSxDPP

Gene2 GHR

1.60a 1.01b 1.47ab 1.33ab 0.77b 0.97b 1.21ab

1.48a 0.25

0.075 0.19

0.247

0.18

0.363 0.203

0.054

0.20a 0.04

0.041 0.865

0.334

IGFBP2 84.9a 75.9ab 59.7cd 53.5cd 45.7d 49.3cd 59.9cd 70.1abc 15.2

0.134 0.947

0.045

IGFBP3 1.05

0.362 0.169

0.910

0.726 0.008 0.787 0.543

0.738 0.756

GHR1A 0.65 IGF1

b

0.37

b

0.54

b

0.44

b

0.27

b

0.51

b

0.52

b

0.11ab 0.11ab 0.10ab 0.08b 0.12ab 0.13ab 0.17a

b

1.16

1.04

1.27

1.04

b

b

a

b

1.23 ab

1.15 ab

INSR 5.82 5.74 6.28 8.41 5.76 7.29 7.24 LEPR-b 0.002 0.003 0.003 0.003 0.002 0.002 0.003

1.10

a

1.46

0.17

a

8.37 1.35 0.003 0.001

1

BCS = body condition score, DPP = days pospartum

2

GHR = growth hormone receptor, GHR1A= growth hormone receptor 1A, IGF-1 = insulin-like

growth factor-I, IGFBP-2 = IGF-binding protein-2; IGFBP-3 = IGF-binding protein-3, LEPR-b = full-length leptin receptor, INSR = insulin receptor, ACTB= ß-actin (endogenous control gene), HPRT = hypoxanthine phosphoribosyltransferase (endogenous control gene). Letters denote least squares means differ (P ” 0.05), according to Tukey–Kramer test for

a,b

the interaction between cow groups and DPP.

28

Montevideo, April 10, 2014

LIVESTOCK SCIENCE An International Journal Editor-in-Chief: J.E. Hermansen,, Dear Sir,

Find enclosed please, the manuscript under the title “Metabolic and endocrine profiles and hepatic gene expression in periparturient, grazing primiparous beef cows with different body reserves” by A. L Astessiano and co-authors, which we would like to be considered to be published at Animal.

The objectives of this study were to determine 1) relationships among metabolic and endocrine profiles and hepatic expression of genes associated with the somatotropic axis and the full-length leptin receptor (LEPR-b) and 2) associations among these components with changes in BCS, milk production, and calf performance in grazing, suckled, primiparous beef cows during the peripartum period.

All co-authors have seen and agreed with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication. We believe that our findings could be of interest to the readers of this Journal and we hope that the editorial board and the reviewers will agree on the interest of this study.

This data has not been published and/or sent to other journal before. The authors have no

29

conflict of interest. Animal procedures were approved by the Animal Experimentation Committee of Universidad de la Republica (UdelaR, Montevideo, Uruguay).

Sincerely yours,

A. L Astessiano

30

• Grazing primiparous beef cows with moderate BCS at calving had greater estimated energy intake during pre and postpartum • Had greater prepartum IGF-I but less postpartum insulin concentrations • Tended to present greater leptin and had less adiponectin concentrations pre and postpartum • Although serum IGF-I decreased around calving, no pre to postpartum changes in hepatic GHR1A and IGF1 were detected • IGFBP2 mRNA changed according with BCS at calving

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Figure

Figure

Figure