Effects of Recombinant Bovine Somatotropin Administration at Breeding on the Cow, Conceptus and Subsequent Offspring Performance of Beef Cattle

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1 Effects of Recominnt Bovine Somtotropin Administrtion t Breeding on the Cow, Conceptus nd Susequent Offspring Performnce of Beef Cttle V. R. G. Mercdnte 1, F. M. Cirico 1, D. D. Henry 1, P. L. P. Fontes 1, N. Oosthuizen 1, T. Schulmeister 1, N. DiLorenzo 1, nd G. C. Lm 1. Synpsis The ojective of this study ws to determine if dministrtion of recominnt ovine somtotropin (ST) to eef femles t reeding hs the potentil to increse fertility, conceptus development, nd susequent offspring performnce. Injection of 325 mg of ST round the time of reeding incresed concentrtion of insulin-like growth fctor-1 (IGF-1) nd incresed expression of myxovirus-2 on d 21 of suckled eef cows. However, it filed to lter fetl development nd postntl offspring performnce. Summry To determine the effects of dministrtion of low dose of slow-relese ST (Posilc, Elnco, Greenville, IN) on hormone concentrtion nd conceptus development, totl of 190 suckled eef cows were exposed to the 7-d CO-Synch+CIDR fixed-time AI (TAI) protocol. Cows were locked y dys postprtum, BCS, reed, nd rndomly ssigned to receive one of the following tretments: 1) two injections of 325 mg ST, one t TAI nd second injection 14 d fter TAI (D-ST, n=40); 2) one injection of 325 mg ST t TAI nd plceo (sline) injection 14 d fter TAI (TAI-ST, n=48); 3) plceo injection t TAI nd one injection of 325 mg ST 14 d lter (14D-ST n=49); nd 4) two injections of plceo, one t TAI nd second injection 14 d fter TAI (Ctrl, n=53). Pregnncy ws determined vi trnsrectl ultrsonogrphy 35 d fter TAI nd conceptus development ws ssessed y mesuring crown to rump length (CRL) on d 35 nd crown to nose length (CNL) on d 65 fter TAI. Blood smples were collected on d 0, 7, 14, 21, 35 nd 65 reltive to TAI to determine concentrtions of IGF-1, nd on d 18 nd 21 for isoltion of peripherl lood leukocytes (PBL) nd mrna expression of interferon stimulted genes. Plsm concentrtions of pregnncy-specific protein B (PSPB) were lso ssessed on d 35 nd 65 fter TAI. Individul clf irth weight nd sex were determined t irth. Liver iopsies were performed in suset of clves t 8 ± 3 d of ge, nd expression of trget genes relted to the somtotropic xis were determined. There were no differences (P=0.77) mong tretments on pregnncy to TAI (48.7±0.5%). Administrtion of ST t TAI incresed (P<0.0001) plsm concentrtion of IGF-1 on d 7, 14, nd 21. However, CRL nd CNL (0.48±0.02 in nd 0.67±0.001 in, respectively) did not differ (P=0.23) mong tretments. Concentrtion of PSPB did not differ (P=0.18) mong tretments nd etween dys (P=0.30; 2.69±0.11 ng/ml), nd gesttion length (282±9 d) did not differ (P=0.49) mong tretments. However, mrna expression of myxovirus-2 on d 21 differed (P=0.05) mong tretments. Cows receiving n injection of ST t TAI hd greter fold increse in MX-2 mrna expression compred to cows receiving n injection of ST t TAI nd 14 d lter. Injection of ST did not lter clf liver mrna expression of insulin-like growth fctor-1 (P=0.81), IGF-2 (P=0.29), nd IGFBP-3 (P=0.66). However, injection of ST t TAI tended (P=0.06) to increse clf liver mrna expression of IGFR-1 compred to the clves orn to cows in other tretments. In ddition, clf irth weight ws similr (P=0.52) mong tretments. We conclude tht dministrtion of 325 mg ST during the time of TAI to suckled eef cows enhnced concentrtions of IGF-1, ut filed to improve pregnncy rtes, fetl size, PSPB concentrtions, nd hd no effect on clf irth weight. 1 University of Florid, North Florid Reserch nd Eduction Center, Mrinn, FL

2 Introduction Developmentl progrmming, lso termed fetl progrmming, is the concept tht perturtions during criticl prentl development stges my hve lsting impcts on postntl growth nd dult function (Godfrey, 1998). Growth nd development of the emryo nd fetus re complex iologicl events influenced y genetic, epigenetic, mternl mturity, s well s environmentl nd other fctors. These fctors ffect the size nd functionl cpcity of the plcent, uteroplcentl trnsfer of nutrients nd oxygen from mother to fetus, conceptus nutrient vilility, the fetl endocrine milieu, nd metolic pthwys. The somtotropic xis nd its mjor components growth hormone (GH) nd IGF-1 re n essentil constituent of multiple systems controlling growth nd reproduction (LeRoith et l., 2001). In ddition, the somtotropic xis plys key role on emryonic nd fetl development y cting directly on the oocyte, endometrium, plcent, nd emryo. Erly exposure of ovine emryos to incresed concentrtions of GH nd IGF-1 hs een linked to enhnced prentl development nd incresed postntl growth, with ltertions in the somtotropic xis (Costine et l., 2005; Koch et l., 2010). In ddition, strtegies focusing on supplementtion of GH nd IGF-1, through dministrtion of recominnt ovine somtotropin (ST), during the time of emryonic implnttion nd mternl recognition of pregnncy hve een successful on enhncing conceptus development nd incresing fertility of diry cttle (Rieiro et l., 2013). However, little informtion exists on the effects of ST tretment on eef femles t reeding nd in susequent offspring performnce. Therefore, we hypothesized tht dministrtion of ST to eef femles t reeding hs the potentil to increse fertility, conceptus development nd susequent offspring performnce. Mterils nd Methods A totl of 190 multiprous suckled eef cows composed of Angus, Brngus, nd Brford reeds were enrolled in the experiment. All cows were sujected to the 7-dy CO-Synch + CIDR estrus synchroniztion protocol. In rief, cows received 100-μg injection of GnRH (2 ml Fctrel; Zoetis Animl Helth) t CIDR (1.38 g P4; Zoetis Animl Helth) insertion [d - 10] with 25-mg injection of PGF 2α (5 ml Lutlyse; Zoetis Animl Helth) t CIDR removl [d -3], followed y n injection of 100-μg GnRH nd TAI [d 0] t 66 h fter CIDR removl. Cows were locked y dys postprtum, ody condition score (BCS), reed nd rndomly ssigned to receive one of the following tretments (Figure 1): 1) two injections of plceo (1 ml of 0.9% sline), one t TAI nd second injection 14 d fter TAI (CTRL, n=53); 2) two injections of 325 mg ST (Posilc, Elnco Animl Helth, Greenville, IN, USA), one t TAI nd second injection 14 d fter TAI (2ST, n=40); 3) one injection of 325 mg ST t TAI nd plceo injection 14 d fter TAI (TAIST, n=48); nd 4) plceo injection t TAI nd one injection of 325 mg ST 14 d lter (d14st n=49). Blood smples from ech cow were collected on d 0, 7, 14, 18, 21, 35 nd 65. Concentrtions of totl IGF-1 were determined y commercil ELISA kit (Quntikine ELISA Humn IGF1 Immunossy, R&D Systems, Inc., Minnepolis, MN, USA). Concentrtions of PSPB were nlyzed using commercilly ville quntittive ELISA ssy (BioPRYN, BioTrcking LLC, Moscow, ID). Blood smples were collected on dys 18 nd 21 for isoltion of peripherl lood leukocytes (PBL) nd mrna expression of interferon stimulted genes (ISG). Pregnncy sttus ws determined y trnsrectl ultrsonogrphy on d 35 nd 65 with n Iex portle ultrsound equipped with liner 5 MHz multi-frequency trnsducer (E.I. Medicl Imging, Lovelnd, CO, USA). Emryo crown to rump length (CRL) ws mesured on d 35 nd fetl crown to nose length (CNL) ws mesured on d 65 post-tai. Imges were mesured twice using n imge cpturing softwre (ImgeJ, Ntionl Institute of Helth, Bethesd, Mrylnd, USA) for lter mesurements nd finl CRL nd CNL vlues were clculted s the men etween the two mesurements.

3 Clf ody weight ws determined within 12 hr of irth nd every 30 dys until wening using scle (TRU-TEST, Minerl Wells, TX, USA). Cow-clf pirs were mintined together on psture from irth until wening with d liitum ccess to higrss hy, higrss psture, nd wter. A suset of 40 pregnnt cows of Angus nd Brngus reeds nd 24 clves (12 mles nd 12 femles; not necessrily orn from the suset of 40 cows) eqully distriuted mong tretments were selected for nlysis of mrna expression of trget genes. Liver iopsies were performed in suset of clves t 8±3 d of ge, nd expression of trget genes relted to the somtotropic xis were determined. Extrction of RNA ws conducted ccording to recommendtions of the RNA-extrction kit mnufcturer (PureLink RNA Mini Kit, Invitrogen, Crlsd, CA). A totl of seven genes were investigted, including five trget genes, myxovirus 2 (MX2), 2-5 -oligodenylte synthetse 1 (OAS1), insulin-like growth fctor 1 (IGF1), insulin-like growth fctor 2 (IGF2), insulin-like growth fctor 1 receptor (IGF1R), nd insulin-like growth fctor inding protein 3 (IGFBP3); nd two reference genes, cyclophilin-1 nd riosoml protein S9 (RPS9). Cows were locked y reed nd strtified y dys post-prtum, nd ody condition score (BCS) nd rndomly ssigned to tretments. All dt ws nlyzed s rndomized lock design using the SAS sttisticl pckge (SAS Inst. Inc., Cry, NC) with niml s the experimentl unit. Concentrtions of IGF-1, P4, PSPB, nd the emryo/fetl mesurements CRL nd CNL were nlyzed s repeted mesures using the MIXED procedure. The models included the effects of dy, tretment nd its interctions, nd the rndom effect of niml. Pregnncy rtes were nlyzed using the GLIMMIX procedure. The model included the effects of tretment, dys post-prtum, BCS, reed nd its interctions. Clf ody weight ws nlyzed s repeted mesures using the MIXED procedure. The model included the effects of dy, tretment, clf gender nd its interctions, nd the rndom effect of niml. Reltive mrna expression ws nlyzed within dy using the GLM procedure. The finl model included the effects of sex, tretment nd its interctions. Results Injection of 325 mg of ST to suckled eef cows incresed plsm concentrtion of IGF-1 (Figure 2). Plsm concentrtions of IGF-1 were similr (P=0.92) mong tretments t TAI on d 0 (64.8±2.6 ng/ml). Cows receiving n injection of ST t TAI on d 0 hd greter (P<0.0001) plsm concentrtions of IGF-1 on d 7 nd on d 14. Accordingly, cows receiving n injection of ST t TAI on d 14 hd greter (P<0.0001) plsm concentrtions of IGF-1 on d 21. Concentrtions of progesterone (P4) of pregnnt cows incresed (P<0.0001) fter TAI (0.25, 2.39, 5.37, 4.78 nd 5.09±0.26 ng/ml, for d 0, 7, 14, 21 nd 35, respectively). However, injection of 325 mg of ST did not lter concentrtion of P4 (P=0.49) mong tretments nd no tretment dy interction ws detected (P=0.26). Concentrtions of PSPB did not chnge (P=0.30) etween d 35 (2.76±0.109 ng/ml) nd 65 (2.62±0.110 ng/ml) post TAI, nd did not differ mong tretments (P=055). Pregnncy rtes to TAI were determined y ultrsonogrphy on d 35 post TAI nd ws similr mong tretments (45.3, 45.0, 51.0 nd 54.2 for CTRL, 2ST, TAIST nd d14dst, respectively) nd gesttion length (282±17 d) ws lso similr (P=0.33) mong tretments (Tle 1). Fetl size ws determined y ultrsonogrphy on d 35 nd 65 post TAI (Tle 1) nd ws not ltered y injection of ST. Crown to rump length on d 35 ws similr (0.48±0.02 in; P=0.61) mong tretments. In ddition, crown to nose length on d 65 (0.67±0.001 in) did not differ (P=0.23) mong tretments. Expression of ISG of pregnnt cows ws determined on d 18 nd 21 post TAI. Reltive mrna expression of OAS-1 ws similr mong tretments on d 18 (1.8, 1.0, 1.2 nd 0.9-fold increse for TAIST, d14dst, 2ST nd CTRL, respectively; P=0.22) nd on d 21 (1.6, 1.0, 2.2 nd 0.9-fold increse for TAIST, d14st, 2ST nd CTRL, respectively; P=0.23). In ddition, reltive mrna expression of MX-2 (Figure 3) ws similr mong tretments on d 18 (1.2, 1.3, 0.9 nd 1.5-fold increse for TAIST, d14st, 2ST nd CTRL, respectively;

4 P=0.89). However, mrna expression of MX-2 on d 21 differed (P=0.05) mong tretments. Cows receiving n injection of ST t TAI hd greter fold increse in MX-2 mrna expression compred to cows receiving n injection of ST t TAI nd 14 d lter (2.0 nd 0.8, for TAIST nd 2ST, respectively), while d14st nd CTRL were intermedite (1.2 nd 0.9, respectively). In our study, ST injection hd no effect on clf performnce from irth to wening (Tle 1). Birth weight ws similr (P=0.65) mong tretments (79.4, 72.9, 73.4 nd 75.4±4.47 kg for TAIST, d14st, 2ST nd CTRL, respectively). Furthermore, ody weight did not differ (P>0.10) mong tretments t 30, 60, 90, 120 nd 150±17 d of ge (163.8±5.59, 231.0±8.44, 303.3±9.36, 376.3±8.18, nd 450.4±9.08 kg, respectively). Injection of ST did not lter clf liver mrna expression (Figure 4) of IGF-1 (P=0.81), IGF-2 (P=0.29), nd IGFBP-3 (P=0.66). However, injection of ST t TAI tended (P=0.06) to increse clf liver mrna expression of IGFR-1 compred to the clves orn to cows in other tretments (3.1-fold compred to 1.0, 1.2, nd 1.5-fold for TAIST, 2ST, d14st, nd CTRL, respectively). Conclusion In the present study, injection of 325 mg of ST round the time of reeding incresed concentrtion of IGF-1 nd incresed expression of MX-2 on d 21 of suckled eef cows. However, it filed to lter fetl development nd postntl offspring performnce. Further investigtion is needed on the dose nd timing of dministrtion of ST nd its effects on fetl development nd postntl offspring performnce of eef cttle. Acknowledgements Sincere pprecition is expressed to P. Moriel, A. Ely, S. Johnson, P. Folsom, M. Forn, O. Helms, D. Jones, C. Nowell, nd D. Thoms for their ssistnce with dt collection nd lortory nlysis. The uthors thnk Zoetis Animl Helth (Florhm Prk, NY) for their dontion of PGF 2α (Lutlyse), GnRH (Fertgyl), nd CIDR inserts (CIDR EAZI-Breed). Literture Cited Costine, B. A., et l J. Anim. Sci. 83: Godfrey, K. M Eur. J. Ostet. Gynecol. Reprod. Biol. 78: Koch, J. M.,et l J. Anim. Sci. 88: LeRoith, D., et l Endocr. Rev. 22: Lucy, M. C., et l Domest. Anim. Endocrinol. 12: Rieiro, E. S., et l Biol. Reprod.:1 26.

5 Tle 1. Fertility, gesttion, fetl development nd clf performnce mesurements from suckled eef cows previously treted with ST. Tretments 1 Item CTRL TAIST d14st 2ST SEM P-vlue Pregnncy rte, 24/53 25/49 26/48 18/40 (45) 0.77 no. (%) (45) (51) (54) Gesttion length, d Fetl Size 2, in CRL d CNL d Clf ge, d Body weight, l ± ± ± ± ± ST, injection of 325 mg of ST on d 0 nd 14; TAI-ST, injection of 325 mg of ST on d 0 nd plceo injection on d 14; 14D-ST, plceo injection on d 0 nd injection of 325 mg of ST on dy 14; CTRL, plceo injection on d 0 nd CRL crown-to-rump length; CNL crown-to-nose length.

6 Figure 1. Experiment outline nd schemtic of tretments. All cows were estrous synchronized using 7-Dy CO-Synch + CIDR protocol where cows received n injection of GnRH on d -10 nd CIDR ws inserted, followed y injection of PGF 2α (PGF) nd CIDR removl on d -3, followed y injection of GnRH nd fixed-timed AI (TAI) on d 0. Individul ody condition scores (BCS; 1=thin to 9=oese) were ssigned on d -10. Blood smples (B) were collected on d 0, 7, 14, 18, 21, 35 nd 65. Pregnncy dignosis nd fetl mesurements were performed y ultrsonogrphy (US) on d 35 nd 65. Tretments: CTRL, plceo injection on d 0 nd 14; 2ST, injection of 325 mg of ST on d 0 nd 14; TAIST, injection of 325 mg of ST on d 0 nd plceo injection on d 14; d14st, plceo injection on d 0 nd injection of 325 mg of ST on dy 14.

7 Reltive gene expression Concentrtion of IGF-1, ng/ml CTRL TAIST d14st Dy reltive to TAI 2ST Figure 2. Concentrtions of insulin-like growth fctor 1 (IGF-1) of eef cows on dys reltive to fixed-timed AI (TAI) y tretment. Tretments: CTRL, plceo injection on d 0 nd 14; 2ST, injection of 325 mg of ST on d 0 nd 14; TAIST, injection of 325 mg of ST on d 0 nd plceo injection on d 14; d14st, plceo injection on d 0 nd injection of 325 mg of ST on dy 14., P< CTRL TAIST 1 d14st 2ST Dy reltive to TAI Figure 3. Expression of Myxovirus 2 (MX2) mrna on d 18 nd 21 of gesttion on suckled eef cows treted with ST. Tretments: CTRL, plceo injection on d 0 nd 14; 2ST, injection of 325 mg of ST on d 0 nd 14; TAIST, injection of 325 mg of ST on d 0 nd plceo injection on d 14; d14st, plceo injection on d 0 nd injection of 325 mg of ST on dy 14., P=0.05.

8 Reltive gene expression CTRL TAIST d14st 2ST IGF1 IGF2 IGFBP3 IGFR1 Trget gene Figure 4. Expression of heptic insulin-like growth fctor 1 (IGF1), insulin-like growth fctor 2 (IGF2), insulin-like growth fctor 1 receptor (IGF1R), nd insulin-like inding protein 3 (IGFBP3) mrna of clves orn to suckled eef cows treted with ST. Tretments: CTRL, plceo injection on d 0 nd 14; 2ST, injection of 325 mg of ST on d 0 nd 14; TAIST, injection of 325 mg of ST on d 0 nd plceo injection on d 14; d14st, plceo injection on d 0 nd injection of 325 mg of ST on dy 14., P=0.06.

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