Influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow

Postpartum dairy cows enter a period of negative energy balance (NEB) associated with low circulating IGF1, during which the uterus must undergo extensive repair following calving. This study investigated the effects of NEB on expression of IGF family members and related genes in the involuting uterus. Cows were allocated to two treatments using differential feeding and milking regimes to produce mild NEB or severe NEB (SNEB). Uterine endometrial samples collected 2 weeks post partum were analysed by quantitative PCR. The expression of IGF-binding protein 4 (IGFBP4) mRNA increased in the endometrium of SNEB cows, with trends towards increased IGFBP1 and reduced IGFBP6 expression. There were no significant differences between treatments in mRNA expression of IGF1, IGF2 or of any hormone receptor studied, but significant correlations across all cows in the expression levels of groups of receptors suggested common regulatory mechanisms: type 1 IGF receptor (IGF1R), IGF2R and insulin receptor (INSR); GHR with ESR1; and ESR2 with NR3C1. The expression of IGF1R and INSR also positively correlated with the circulating urea concentration. Matrix metalloproteinases (MMPs) are important in tissue remodelling and can affect IGF signalling via interaction with IGFBPs. The expression levels of MMP1, MMP3, MMP9 and MMP13 mRNAs all showed major upregulation in the endometrium of cows in SNEB and all except MMP9 were highly correlated with expression of IGFBP4. Alpha(2)-HS-glycoprotein (AHSG) and PDK4, two genes implicated in insulin resistance, were also highly expressed in SNEB. These results suggest that cows in SNEB experience alterations to the IGF and insulin signalling pathways in the postpartum endometrium. This may affect the rate of tissue repair with a possible negative impact on subsequent fertility.     

Nitric oxide in blastocyst implantation in the rhesus monkey

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N(G)-nitro-l-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.     

Immunoneutralization of vascular endothelial growth factor inhibits pregnancy establishment in the rhesus monkey (Macaca mulatta)

Maternal endometrial vascular endothelial growth factor (VEGF) is considered important in blastocyst implantation. However, there is no direct evidence to support this conjecture in the primate. In the present study, we have examined this hypothesis by testing whether immunoneutralization of VEGF during the peri-implantation stage of gestation affects embryo implantation in the rhesus monkey. Adult female animals (n = 36) during mated ovulatory cycles were randomly assigned to one of the experimental groups treated subcutaneously with either isotype-matched mouse immunoglobulin (group 1: control, n = 8) or monoclonal mouse antibody against VEGF-A (anti-VEGF Mab; group 2: 10 mg on day 5 after ovulation, n = 8; group 3: 20 mg on day 5 after ovulation, n = 8; group 4: 10 mg on day 10 after ovulation, n = 4; group 5: 10 mg on days 5 and 10 after ovulation, n = 8). Anti-VEGF Mab-treated animals in groups 2-4 did not show any marked inhibition in pregnancy establishment. On pooled analysis, however, anti-VEGF Mab administration in groups 2-5 (n = 28) resulted in a significant (P < 0.04) decline in the number of viable term pregnancy when compared with control animals. The observed difference was explained by the fact that 10 mg anti-VEGF Mab given to each animal on days 5 and 10 after ovulation in group 5 (n = 8) inhibited pregnancy establishment significantly (P < 0.02) when compared with control group 1. There was no significant change in serum concentrations of estradiol-17beta, progesterone, and free VEGF among groups. Furthermore, animals treated with anti-VEGF Mab (n = 8) as in group 5 revealed marked decrease in immunoreactive VEGF, fms-like tyrosine kinase-1, and kinase-insert domain region in trophoblast cells associated with shallow uterine invasion on day 13 of gestation when compared with samples from control group animals (n = 8). Thus, VEGF action is required for successful blastocyst implantation in the rhesus monkey.     

The role of nitric oxide on matrix metalloproteinase 2 (MMP2) and MMP9 in placenta and fetus from diabetic rats

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling that accompanies the rapid growth, differentiation, and structural changes of the placenta and several fetal organs. In the present study, we investigated whether the diabetic maternal environment may alter the regulatory homeostasis exerted by nitric oxide (NO) on MMPs activity in the feto-placental unit from rats at midgestation. We found that NADPH-diaphorase activity, which reflects the distribution and activity of NO synthases (NOS), was increased in both placenta and fetuses from diabetic rats when compared with controls. In addition, while a NO donor enhanced MMP2 and MMP9 activities, a NOS inhibitor reduced these activities in the maternal side of the placenta from control rats. This regulatory effect of NO was only observed on MMP9 in the diabetic group. On the other hand, the NO donor did not modify MMP2 and MMP9 activities, while the NOS inhibitor reduced MMP9 activity in the fetal side of both control and diabetic placentas. In the fetuses, MMP2 was enhanced by the NO donor and reduced by the NO inhibitor in both fetuses from control and diabetic rats. Overall, this study demonstrates that NO is able to modulate the activation of MMPs in the feto-placental unit, and provides supportive evidence that increased NOS activity leads to NO overproduction in the feto-placental unit from diabetic rats, an alteration closely related to the observed MMPs dysregulation that may have profound implications in the formation and function of the placenta and the fetal organs.     

EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways

Epidermal growth factor (EGF) is present in the maternal-fetal environment and has an important role in placental development. Matrix metalloproteinase-9 (MMP-9) expression/activation is a pre-requisite in extravillous trophoblast invasion. Whereas EGF up-regulates MMP-9 activity in a variety of cell types, there is no direct evidence for the stimulation of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion by EGF in extravillous trophoblasts. In addition, the signalling pathways involved in this regulation are not clear. In the present study, we have examined the possible involvement of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in the regulation of the MMP-9/TIMP-1 system by EGF in vitro. We used a well-established invasive extravillous trophoblast cell line (HTR8/Svneo) and measured gene and protein expression by semi-quantitative RT-PCR and western analysis respectively. MMP activity was determined by zymography. We showed for the first time that EGF activated both PI3K/Akt and MAPK/extracellular-signal regulated kinase (ERK) signalling in HTR8/SVneo, and increased both MMP-9 and TIMP-1 mRNAs and protein concentrations. Interfering with either signalling pathway via PI3K inhibitor LY294002 or MEK inhibitor U0126 in EGF-stimulated HTR8/SVneo cells blocked the induction of MMP-9 and TIMP-1. LY294002 inhibited Akt phosphorylation, but had no effect on ERK phosphorylation; U0126 suppressed ERK phosphorylation without interfering with the phosphorylation of Akt. In addition, expression of constitutively active Akt (Myr-Akt1, Myr-Akt2, Myr-Akt3) was not sufficient to induce proMMP-9 and TIMP-1 secretion. Our results suggest that the activation of both PI3K and MAPK pathways in extravillous trophoblasts is necessary for the up-regulation of MMP-9 and TIMP-1 expression by EGF.     

Dynamic expression of matrix metalloproteinases (MMP-2, -9 and -14) and the tissue inhibitors of MMPs (TIMP-1, -2 and -3) at the implantation site during tubal pregnancy

Matrix metalloproteinases (MMPs) are responsible for extracellular matrix (ECM) degradation, and their functions are regulated by tissue inhibitors of MMPs (TIMPs). The evidence for the roles of MMPs and TIMPs in implantation and placentation has remained insufficient in humans, especially during the early stages. Tubal pregnancy has some similarities to normal intrauterine pregnancy and therefore may provide a unique model for implantation studies. In the present study, the expression of MMP-2, -9 and -14, and TIMP-1, -2 and -3 at the feto-maternal interface during tubal pregnancy was examined by immunohistochemistry and in situ hybridization. We found that MMP-9 and TIMP-1, -2 and -3 are produced by all types of extravillous cytotrophoblast (EVCT) cells, while MMP-2 and -14 mainly exist in distal column cytotrophoblast (CCT) cells and invasive EVCT cells. Meanwhile, the intensity of MMP-14 and TIMP-1 and -2 increased along the invasive pathway toward maternal interstitium. In addition, MMP-2, -9 and -14 and TIMP-1, -2 and -3 were all detected in the villous CT (VCT) cells. Furthermore, both the mRNA level and immunoreactivity of MMP-9, TIMP-1 and -3 increased, while those of TIMP-2 decreased concurrent with the progression of pregnancy during weeks 3-9. The unique expression pattern of various MMPs and TIMPs at the feto-maternal interface suggests that they may have roles in regulating the controlled invasion of trophoblasts during implantation and placentation. Meanwhile, the study provides a better understanding of the mechanisms involved in cellular events during human pregnancy, especially at the initiation stage of implantation.     

Seasonal effects on the response of ovarian follicles to IGF1 in mares

The response of follicles to IGF1 was compared between the transition into the ovulatory season (transitional period) and the ovulatory season (ovulatory period) in eight mares using a cross-over experimental design within periods. Granulosa cells were collected from follicles 15-24 or 25-34 mm and expression of IGF1R, IGF2R, FSHR, LHCGR and PAPPA was determined by qPCR. In addition, 10 mg IGF1 or vehicle were injected into the largest follicle (transitional period) or the second largest follicle (ovulatory period) of a follicular wave before the beginning of diameter deviation between the two largest follicles (mean diameters at injection 19.2 and 20.0 mm during transitional and ovulatory periods respectively). Follicular fluid was collected 24 h after injection for determination of free IGF1, IGFBP, inhibin A and oestradiol levels. Granulosa cells from follicles 25-34 mm, but not follicles 15-24 mm, expressed higher levels of IGF1R (P=0.01), FSHR (P<0.007) and LHCGR (P=0.09) during the ovulatory period than during the transitional period, whereas IGF2R expression was higher in transitional than ovulatory follicles (P=0.06). Follicular IGFBP2 levels were not different (P>0.1) between periods and treatments, whereas IGFBP5 levels were higher (P<0.05) during the ovulatory period. Finally, IGF1 injection before the beginning of deviation induced an approximately twofold increase (P=0.01) in follicular inhibin A levels during each period and did not affect oestradiol (P>0.1). These results suggest that, as during ovulatory waves, equine follicles during transitional waves are responsive to IGF1 before the beginning of deviation and that, therefore, inadequate IGF1 responsiveness before deviation may not underlie the deficient development of dominant follicles during transition.     

Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles

Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.     

Expression of integrins and extracellular matrix proteins at the maternal-fetal interface during tubal implantation

Investigation of the expression pattern of integrins and their extracellular matrix (ECM) ligands in trophoblasts at the maternal-fetal interface during tubal pregnancy may aid better understanding of the adhesion and invasion of acceptable maternal endometrium by trophoblast cells at the very early stage of human gestation. In this study, spatial and temporal alterations of integrins and ECM ligands were examined in specimens of tubal pregnancies during weeks 3-9 of gestation. In situ hybridization and immunohistochemistry revealed that relatively high levels of integrin alpha(1), beta(1), alpha(5) subunits and heterdimer alpha(5)beta(1) as well as ECM ligands, were displayed in trophoblast cells as early as weeks 3-4 of gestation. Expression peaked during weeks 5-7 and then, with the exception of integrin alpha(1), which remained high, declined slightly up to weeks 8-9 of gestation. Immunoreactive fibronectin, laminin and type IV collagen were detected in column cytotrophoblastic cells (CTB) and some invasive extravillous cytotrophoblast (EVCT) cells and the alterations were coincident with those of the corresponding integrin receptors in EVCT cells. Laminin was strongly stained in EVCT cells that had invaded maternal blood vessels and deep into the interstitium. Maternal epithelial, endothelial and stromal cells also expressed these integrins and ECM ligands. The results indicate their involvement in mediating the adhesion of trophoblasts to the epithelium of the maternal Fallopian tube. The upregulated expression of these molecules in column CTB and invasive EVCT cells may also facilitate the invasion of trophoblasts into the maternal interstitium. Moreover, trophoblasts possessed the potential for self-controlled adhesion and invasion and appear to reach peak invasive capability in the second month of tubal implantation.     

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus)

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.     

Production of capsular material by equine trophoblast transplanted into immunodeficient mice

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.     

Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinases during mouse embryonic development

Mouse embryo implantation is a highly invasive and controlled process that involves remodeling and degradation of the extracellular matrix of the uterus. Matrix metalloproteinases (MMPs) are the main proteinases facilitating this process. Extracellular matrix metalloproteinase inducer (EMMPRIN) can stimulate the production of MMPs and is required for successful implantation in the mouse. The aims of the present study were to examine the expression profiles of mRNA and proteins for EMMPRIN and MMPs in the developing mouse embryo in vitro, and to study whether EMMPRIN protein induces the production of MMPs by mouse blastocysts. EMMPRIN mRNA, detected by RT-PCR, was present at all stages of embryo development from the one-cell to the blastocyst outgrowth. EMMPRIN protein, observed by confocal microscopy, was present on the cell surface at the same stages of development as was the mRNA. Of seven MMPs studied, murine collagenase-like A (Mcol-A), murine collagenase-like B (Mcol-B) and gelatinase A (MMP-2) mRNAs were detected only in blastocyst outgrowths by RT-PCR. Gelatinase B (MMP-9) mRNA was detected both in expanded blastocysts and blastocyst outgrowths. MMP-2 and -9 proteins were detected in the cytoplasm of outgrowing trophoblast cells. Collagenase-2 (MMP-8), collagenase-3 (MMP-13), or stromelysin-1 (MMP-3) mRNAs were not present at any stage of pre- or peri-implantation mouse embryo development. Quantitative RT-PCR analyses showed that recombinant EMMPRIN protein did not stimulate MMP-2 or -9 expression by mouse blastocyst outgrowths. These data suggest that EMMPRIN may regulate physiological functions other than MMP production by mouse embryos during implantation.     

Comparison of mRNA for IGFs and their binding proteins in the oviduct during the peri-oestrous period between dairy heifers and lactating cows

The oviduct provides the environment to support gamete maturation, fertilisation and early embryo development. As there is a high incidence of early embryonic death in lactating dairy cows, this study compared expression of IGF family members in the oviduct between lactating Holstein-Friesian dairy cows (n=16, 81±2.4 days in milk) and nulliparous heifers (n=16, age 1.6±0.07 years) at three stages of the oestrous cycle: A) newly selected dominant follicle in the luteal phase, B) follicular phase before the LH surge and C) pre-ovulatory phase after the LH surge. Expression of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGFBP3 and IGFBP6 mRNA was determined in the ampulla of the oviduct. Oviduct side (ipsilateral or contralateral) with respect to the dominant follicle did not affect gene expression. Expression of IGF1 and all three IGFBPs increased significantly between the luteal and the pre-ovulatory phases, with no further significant alteration post-LH surge. Concentrations of circulating IGF1 were higher in heifers than in cows, as was the mRNA expression of IGF1, IGFBP3 and IGFBP6. The pre-LH surge rise in IGFBP2 mRNA was only observed in heifers. IGF2 expression was not influenced by either age or stage of cycle. These three IGFBPs are generally considered to inhibit IGF action. These results indicate tight regulation of IGF bioavailability in the oviductal environment around oestrus, with pronounced differences between cows and heifers, which are likely to influence early embryonic development. Further studies are required to assess the implications for embryo survival.     

Mouse pregnancy-specific glycoproteins: tissue-specific expression and evidence of association with maternal vasculature

The pregnancy-specific glycoproteins (Psg) are secreted hormones encoded by multiple genes in rodents and primates, and are thought to act as immune modulators. The only Psg receptor identified is CD9, through which Psg17 induces cytokine production from macrophages cultured in vitro. We examined temporal and spatial aspects of Psg and CD9 expression during mouse pregnancy to determine whether their expression patterns support a role in immune modulation. Using in situ hybridisation, immunohistochemistry and RT-PCR we found Psg expression in trophoblast giant cells and in the spongiotrophoblast. Psg22 is the predominant Psg family member expressed in giant cells. Detectable Psg is associated predominantly with endothelial cells lining vascular channels in the decidua, rather than with maternal immune cell markers. CD9 expression exhibited partial overlap with Psg, but without exclusive co-localisation. CD9 was observed in decidual cells surrounding early implantation sites, and in the endometrium. However, embryo transfer of wild-type embryos to CD9-deficient females indicates that maternal CD9 is not essential for successful pregnancy.     

Influence of ad libitum milk replacer feeding and butyrate supplementation on the systemic and hepatic insulin-like growth factor I and its binding proteins in Holstein calves

Ad libitum milk feeding and butyrate (B) supplementation have the potential to stimulate postnatal growth and development in calves. The somatotropic axis is the main endocrine regulator of postnatal growth and may be affected by both ad libitum milk replacer (MR) feeding and B supplementation in calves. We hypothesized that ad libitum MR feeding and B supplementation stimulate systemic and hepatic insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP) in preweaning calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (6 L/d; Res). In each feeding group half of the calves received a MR with 0.24% butyrate and the other half received same MR without butyrate. Ad libitum MR feeding was performed from d 4 until wk 8 of age. From wk 9 to 10, Adl and Res calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Feed intake was measured daily and body weight weekly. Blood samples for analyzing plasma concentrations of glucose, insulin, IGF-I, and IGFBP-2, -3, and -4 were taken on d 1, 2, 4, and 7, then weekly or every other week (IGFBP) until wk 11 of life. Liver samples were taken on d 50 and at the end of the study (d 80) to measure gene expression of the growth hormone receptor 1A (GHR1A), IGF1, IGFBP1 to 4, and of the IGF Type 1 and insulin receptor in the liver. Intake of MR and body weight were greater, but concentrate intake was lower in Adl than in Res. Plasma concentrations of IGF-I and IGFBP-3 were greater and plasma concentration of IGFBP-2 was lower in Adl than in Res during the ad libitum milk feeding period. After reduction of MR in both groups to 2 L/d plasma concentrations of IGF-I and IGFBP-4 were lower and plasma concentration of IGFBP-2 was higher in Adl than in Res. Supplementation of B depressed plasma IGF-I from wk 1 to 4 and in wk 9. On d 50, mRNA abundance of the GHR1A and IGF1 was greater and of IGFBP2 mRNA was lower in Adl than in Res. At d 80, IGFBP2 mRNA was greater in Adl than in Res, and IGFBP2 mRNA increased with B supplementation. Ad libitum MR feeding stimulated the systemic and hepatic IGF system and mirrored the greater growth rate during the ad libitum MR feeding, whereas butyrate supplementation partly reduced the systemic and hepatic IGF system.