Protein and mRNA expression of follicle‐stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)

Follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real‐time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p < .01) during oestrus. By contrast, there was low expression of FSHR and LHR mRNA in the pineal gland and hypothalamus. FSHR mRNA expression was higher than that of LHR (p < .05) in the ovary, whereas LHR mRNA expression was higher than that of FSHR (p < .01) in the uterus. FSHR and LHR proteins were located in the pinealocyte, synaptic ribbon and synaptic spherules of the pineal gland and that FSH and LH interact via nerve fibres. In the hypothalamus, FSHR and LHR proteins were located in the magnocellular neurons and parvocellular neurons. FSHR and LHR proteins were localized in acidophilic cells and basophilic cells in the pituitary gland, and in surface epithelium, stromal cell and gland epithelium in the uterus. In the ovary, FSHR and LHR protein were present in the ovarian follicle. Thus, we concluded that FSHR and LHR are located in the pineal gland, hypothalamus, pituitary and gonad during oestrus in the yak. However, FSHR was mainly expressed in the pituitary gland and ovaries, whereas LHR was mainly expressed in the pituitary gland and uterus.     

Effect of Oestrus Synchronization with PGF2α/eCG/hCG on Luteal P4 Synthesis in Early Pregnant Gilts

Administration of hormones to synchronize oestrus is a useful tool in animal breeding. However, exogenous ovarian stimulation may be detrimental to reproductive function. This study was aimed to examine whether an oestrus synchronization with PGF2α/eCG/hCG could affect luteal P4 synthesis in early pregnant gilts. Corpora lutea (CLs) were collected on days 9, 12 and 16 of pregnancy from gilts with natural (n = 16) and synchronized (n = 18) oestrus and analysed for (i) the expre‐ssion of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide (CYP11A1), and 3β‐hydroxysteroid dehydrogenase (3βHSD); (ii) the concentration of P4 in the luteal tissue and blood; and (iii) the expression of luteinizing hormone receptors (LHR) and oestrogen receptors (ERα and ERβ). Additionally, the effect of LH on P4 secretion from CL slices collected from synchronized and naturally ovulated animals has been studied in vitro. PGF_2α/eCG/hCG administration increased mRNA expression of StAR, CYP11A1, 3βHSD, and LHR on day 9 and CYP11A1 and LHR on day 12 of pregnancy compared with the control group (p < 0.05). CYP11A1, 3βHSD, LHR, ERα and ERβ proteins were not affected by synchronization; only StAR protein increased in hormonally treated animals (p = 0.017). The concentration of P4 in luteal tissue was greater on day 9 (p < 0.01), but lower on day 16 (p < 0.05) in gilts with hormonally induced oestrus compared with control animals. Blood serum levels of P4 were lower in synchronized than control gilts (p < 0.001). Synchronization did not affect LH‐stimulated P4 secretion from luteal slices; however, greater basal concentration of P4 in incubation medium was detected for CLs collected from synchronized than control gilts (p < 0.05). In conclusion, synchronization of oestrus with PGF2α/eCG/hCG protocol in gilts did not impair the expression of luteal P4 synthesis system, although decreased P4 concentration in the blood.     

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon‐like peptide‐2 in pre‐weaned lambs

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon‐like peptide ?2 (GLP‐2) in pre‐weaned lambs using animal and cell culture experiments. In vivo, twelve 14‐day‐old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP‐2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin‐dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin‐like growth factor 1 (IGF‐1) in the jejunum and ileum and mRNA expression of GLP‐2 receptor (GLP‐2R) in the jejunum. In vitro, when jejunal cells were treated with GLP‐2 for 2 hr, the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) OD, IGF‐1 concentration, and the mRNA expression of IGF‐1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF‐1 receptor (IGF‐1R) inhibitor decreased (p < .05) the mRNA expression of IGF‐1, cyclin A2, cyclin D1 and CDK6 in GLP‐2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP‐2 in pre‐weaning lambs. Furthermore, GLP‐2 can indirectly promote the proliferation of jejunal cells mainly through the IGF‐1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.     

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E₂), progesterone (P₄), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E₂-active (EA) and E₂-inactive (EI) follicles were selected according to their E₂-to-P₄ ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E₂ and P₄ concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Effect of genistein on IGF‐1 and IGFBP‐1 in young and aged female rat ovary

The objective of this study was to assess the effects of genistein (GEN) on expression of insulin‐like growth factor 1 (IGF‐1) and insulin‐like growth factor binding protein 1 (IGFBP‐1) in young and aged rat ovary. Forty young female Sprague Dawley (SD) rats (200 ± 20 g) and forty aged female SD rats (490 ± 20 g) were selected and according to weight, they were divided into the following five groups with eight animals in each: negative control group (NC), low‐dose group (L), middle‐dose group (M), high‐dose group (H) and positive control group (PC). GEN group received GEN of 15, 30, 60 mg/kg respectively. It lasted 30 days. Concentrations of serum hormones, IGF‐1 and IGFBP‐1 were determined by enzyme‐linked immunosorbent assay (ELISA). Gene and protein expressions of IGF‐1 and IGFBP‐1 were determined by real‐time PCR and Western blot respectively. Compared with NC, GEN significantly increased oestradiol‐17β(E₂) level in aged rat, reduced luteinizing hormone (LH) level in young and aged rat. Serum levels of IGFBP‐1 in young rats were significantly higher in GEN groups (p < 0.05). mRNA and protein expression levels of IGF‐1 and IGFBP‐1 were positively correlated with GEN dose. GEN could significantly reduce the ratio of IGF‐1/IGFBP‐1 of aged rats. Multivariate Cox regression analysis result showed IGF‐1 and IGFBP‐1 levels significantly correlated with GEN dose. We speculate that there is an association between the addition of GEN and expression of IGF‐1 and IGFBP‐1, and the relationship between them is different in young and aged rat.     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.     

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Adiponectin is an adipocyte‐derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in‐vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin‐like growth factor I (IGF‐I) at 10 ng/ml for 48 hr after obtaining 75%–80%s confluency. Adiponectin at 10 µg/ml increased IGF‐I‐induced estradiol (E₂) and progesterone (P₄) secretion and FSH‐induced E₂ secretion from GC and also increased the abundance of factors involved in E₂ and P₄ production (cytochrome P45019A1 [CYP19A1] and 3‐beta‐hydroxysteroid dehydrogenase [3β‐HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.     

Bone Morphogenetic Protein‐6 (BMP‐6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre‐antral Follicles Cultured In Vitro

BMP‐6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP‐6) and recombinant follicle‐stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP‐6 (Experiment 2). Secondary follicles were cultured in αMEM⁺ alone (control medium) or supplemented with BMP‐6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP‐6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP‐6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non‐cultured control and after in vitro culture (MEM and 1 ng/ml BMP‐6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP‐6 at 1 ng/ml treatment. In conclusion, the low BMP‐6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase‐3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

Regulation of Anti‐Müllerian Hormone and Its Receptor Expression around Follicle Deviation in Cattle

The anti‐Müllerian hormone (AMH) is an important marker of ovarian reserve and for predicting the response to superovulatory treatments in several species. The objective of this study was to investigate whether AMH and its receptor (AMHR2) are regulated in bovine granulosa cells during follicular development. In the first experiment, granulosa cells were retrieved from the two largest follicles on days 2 (before), 3 (at the expected time) or 4 (after deviation) of follicular wave. In the second experiment, four doses of FSH (30, 30, 20 and 20 mg) or saline were administered twice a day starting on Day 2 of the first follicular wave of the cycle. Granulosa cells and follicular fluid were collected from the two largest follicles 12 h after the last injection of FSH or saline. AMH mRNA abundance was similar in granulosa cells of the two largest follicles (F1 and F2) before deviation (Day 2), but greater in dominant (DF) than subordinate follicles (SF) at the expected time (Day 3) and after (Day 4) deviation (p < 0.05). In experiment 1, AMH mRNA levels declined in both DF and SF near the expected time and after deviation when compared to before deviation. There was no difference in AMHR2 mRNA levels before and during follicular deviation (p > 0.05), but they tended to be greater in DFs than SFs (p < 0.1) after deviation. Experiment 2 showed that AMH and AMHR2 mRNA in granulosa cells and AMH protein abundance in follicular fluid were similar (p > 0.05) between both co‐dominant follicles collected from the FSH‐treated cows. These findings indicate the followings: AMH mRNA levels decrease in both DFs and SFs during follicular deviation; granulosa cells from heathy follicles express more AMH mRNA compared to subordinate follicles undergoing atresia and FSH stimulates AMH and AMHR2 mRNA expression in granulosa cells of co‐dominant follicles.     

Insulin‐like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3

The objective of our present study was to determine the effects of insulin‐like growth factor I (IGF‐I) on the development of yak (Bos grunniens) embryos after cumulus–oocyte complex (COC) vitrification and warming followed by in vitro fertilization (IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid (SOF) supplemented with four concentrations (0, 50, 100 and 200 ng/ml) of IGF‐I, while the yak COCs without vitrification or IGF‐I supplementation acted as the control group; the BAX, BCL‐2, AQP3mRNA and aquaporin 3 (AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real‐time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass (ICM) and trophectoderm (TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100 ng/ml IGF‐I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL‐2 gene expression was the highest in the control and 100 ng/ml IGF‐I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100 ng/ml IGF‐I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF‐I group (106.7 ± 4.9) and the control group (107.3 ± 4.2) was higher than that in the vitrified and warmed 0 ng/ml IGF‐I (91.2 ± 3.1), 50 ng/ml IGF‐I (92.3 ± 3.7) and 200 ng/ml IGF‐I (92.4 ± 3.7) groups. Therefore, we conclude that IGF‐I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL‐2 and AQP3 expression levels.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.     

Growth of rumen papillae in weaned calves is associated with lower expression of insulin‐like growth factor‐binding proteins 2, 3, and 6

This study aimed to characterize the relationship between the growth of rumen papillae in calves and the mRNA expression of insulin‐like growth factor‐binding proteins (IGFBPs) in the rumen papillae. The length of rumen papillae, the mRNA expression of IGFBPs in rumen papillae by quantitative real‐time PCR, and the presence of insulin‐like growth factors I and II (IGF‐I and II) by immunohistochemistry (IHC) were analyzed in nine Holstein calves divided into three groups: suckling (2 weeks, n = 3), milk‐continued (8 weeks, n = 3), and weaned (8 weeks, n = 3). The length of rumen papillae was greater (p < 0.01) in weaned calves than in suckling and milk‐continued calves, whereas the expressions of IGFBP2, IGFBP3, and IGFBP6 genes were lower (p < 0.05) in the rumen papillae of weaned calves than in milk‐continued calves. Thus, rumen papillae length and IGFBP2, 3, and 6 expressions were negatively correlated. The IHC analysis showed that IGF‐I and IGF‐II were present in the rumen epithelium of calves. These results suggested that the growth of rumen papillae after weaning is associated with the induction of IGFs by the low levels of IGFBP2, IGFBP3, and IGFBP6.