Relationships of growth hormone gene and milk protein polymorphisms to milk production traits in Simmental cattle.

The importance of milk proteins and the positive effect of administration of growth hormone (GH) on milk production, and the presence in some dairy cattle lines of greater GH concentrations prompted us to examine the presence of restriction fragment length polymorphism at the GH gene using the restriction enzyme TaqI and to investigate associations between this polymorphism in Simmental cows and bulls, as well as milk protein variants in Simmental cows, and milk production traits. Blood and milk were sampled from 279 Italian Simmental cows and semen was collected from 148 bulls of the same breed. Two fragment bands, denoted A and B, of 6200 and 5200 bp respectively, were examined and three patterns, AA, AB and BB, were found in both animal samples. All variants previously reported in other studies, for kappa, beta, and alpha s1-caseins, and beta-lactoglobulin, were found in the cows' samples. For the cows' samples, a BLUP (Best Linear Unbiased Predictor) analysis of results was performed using a REML (Restricted Maximum Likelihood) program and known heritabilities, whereas for bulls we have performed a General Linear Model analysis. The effect of GH gene polymorphism, using TaqI restriction enzyme, on milk production traits was not significant, but bulls of BB pattern had a higher breeding value for milk yield than AA bulls (P < 0.05). For the kappa-casein genotypic effects, cows of AB genotype gave milk with 1.53 +/- 0.70 g/kg less fat than cows of AA genotype. In addition, breeding values for milk protein content were significantly higher in BB bulls, with 0.87 +/- 0.32 and 0.71 +/- 0.34 g/kg more milk protein than AA and AB bulls respectively. Thus, our results revealed a GH gene polymorphism and indicated significant effects of milk protein polymorphisms on milk production traits in the Italian Simmental breed.

Relationships of polymorphisms for growth hormone and growth hormone receptor genes with milk production traits for Italian Holstein-Friesian bulls.

Allelic variation in the structural or regulatory sequences of growth hormone and its receptor genes might directly or indirectly affect milk traits. This possibility prompted us to investigate the eventual relationships of restriction fragment length polymorphisms at the locus of bovine growth hormone (using TagI and MspI restriction enzymes) and its receptor (using TaqI restriction enzyme) to PTA of milk production traits of bulls. Ninety-one Italian Holstein-Friesian bulls were used in this experiment, and data were analyzed with a fixed linear model. The restriction fragment length polymorphisms at the growth hormone locus did not affect the milk traits studied. Six restriction enzyme TaqI bands of 7.1, 6.2, 5.7, 5.4, 4.2, and 3.3 kb with nine patterns were observed after hybridization by a cDNA probe containing the coding sequences for the intracellular C-terminal part of the receptor. The effect of this polymorphism on PTA for milk protein percentage was highly significant and was favorable for the rare (6.6%) 5.7- and 5.4-kb pattern. Our results indicate that further study is needed to explain the DNA polymorphism and to obtain more definite conclusions about effects on milk traits.

Characterization of growth hormone-binding protein in cattle plasma: prolactin-binding activity and 24-hour profile.

The purpose of this study was to characterize circulating growth hormone-binding proteins (GHBP) and prolactin-binding proteins (PRLBP) in cattle blood plasma. In particular, the 24-hr profile of these molecules was investigated. The preincubation of bull plasma with iodinated bovine growth hormone (bGH) or bovine prolactin (bPRL), followed by gel filtration chromatography (Superdex 200; 1.6 x 60 cm column), resulted in the formation of essentially two complexes. The majority of [125I]bPRL eluted with the first one (M(r) approximately 600 kDa), whereas [125I]bGH mainly appeared in the second one (M(r) approximately 70 kDa). The fractions corresponding to these two peaks were analyzed by western ligand blotting (WLB), under reducing conditions. WLB revealed, respectively, 190-, 56-, 52-, and 28-kDa bands for the first peak and only 52- and 28-kDa bands for the second one. The nature of the 600-kDa peak is at present undetermined, but the 70-kDa one was previously identified as high-affinity GHBP. Displacement studies demonstrated that bGH and bPRL were both able to bind to this GHBP, because the bGH- and bPRL-binding activities of this protein could be saturated by an excess of either of these two hormones. This was indirectly confirmed by the close correlation (r = 0.615; P = 0.0001; n = 155) observed between plasma bGH- and bPRL-binding activities, because this correlation could suggest that both ligands are bound to the same proteins. The temporal concentrations of plasma GHBP were measured in samples collected at 20-min intervals for 24 hr from 8 young bulls. The evaluation of GHBP was realized by WLB, followed by densitometric analysis. Some fluctuations were observed, but these were not correlated with bGH release, even with a +/- 2-hr lag period. In summary, we found that bovine high-affinity GHBP binds not only bGH, but also bPRL. A second type of protein, of higher molecular weight, also binds these two hormones, but further investigations are needed to determine its nature. Finally, GHBP concentrations in cattle blood plasma apparently show fluctuations over a 24-hr period, but no correlation was found between these fluctuations and plasma growth hormone concentrations.

Dissociation of increases in plasma insulin-like growth factor I and testosterone during the onset of puberty in bulls.

The present study was conducted to examine the relationship between plasma concentrations of testosterone, insulin-like growth factor I (IGF-I) and IGF-binding proteins (IGFBPs) during puberty, in male calves treated with GnRH or testosterone propionate. Twelve male Holstein calves (10 weeks old) were assigned to the control group (n = 6), the GnRH-treated group (n = 3) or the testosterone-treated group (n = 3). For 8 weeks, the GnRH-treated group received a single i.v. injection of GnRH (0.5 microgram kg-1 body mass) each day while the testosterone-treated group received an i.m. injection of testosterone propionate (0.5 mg kg-1 body mass) twice a day. The calves were studied until they were 200 days old. Hormone treatments were stopped one month after puberty was reached in the control group. Blood samples were collected every 30 min for 8 h every third day. Hormone concentrations were determined by radioimmunoassay. Western ligand blotting and immunoblotting, using monoclonal antibodies against IGFBP-2 and IGFBP-3, were used to characterize the IGF-binding proteins. In the control group, puberty occurred at about 120 days of age and was associated with an increase in concentrations of testosterone, IGF-I and IGFBP-3 and a decrease in concentration of IGFBP-2. In the GnRH-treated group, plasma testosterone remained low until 8 weeks after establishment of puberty in the control group (4 weeks after the end of treatment). In the testosterone-treated group, testosterone was high during the treatment period and then decreased to prepubertal values when treatment was stopped. Testosterone values increased again to reach postpubertal values 5 weeks after the end of hormone treatment. Nevertheless, independent of testosterone status, the profile of IGF-I and the IGFBPs in the GnRH- and testosterone-treated groups were parallel to that reported for the control group with the transition from prepubertal to adult values at about 120 days of age. In conclusion, concentrations of testosterone, IGF-I and IGFBP-3 increase together, but probably independently, during the onset of puberty in male calves.

TaqI restriction fragment length polymorphisms for growth hormone in bovine breeds and their association with quantitative traits.

The objectives of the present study were (1) to reveal GH-TaqI RFLPs in different bovine breeds and (2) to look for association between quantitative traits and GH-TaqI RFLPs in bulls highly selected for growth and meat production. Blood was sampled from 269 Holstein heifers, 48 Italian and 45 Hungarian Simmental heifers, 30 Normande heifers and 41 bulls and 13 heifers of the double-muscled Belgian White Blue breed. Bulls were weighed from 7 until 13 months of age and the average daily gain, the feed efficiency, the height and the phenotypic index were determined at the end of the experiment. GH-TaqI RFLPs revealed 4 DNA fragments of 6 (A), 5.2 (B), 4.4 (C) and 4.2 (D) kbp. The distribution of genotypes was different between Normande and the other breeds (P < 0.001) and between Belgian White Blue and Holstein (P < 0.001) or Hungarian Simmental breed (P < 0.005). The Normande heifers were also characterized by a high frequency of the AB genotype (53%) compared with other experimental breeds (range values: 7-24%). In the statistical analysis of the relationships between quantitative traits and GH-TaqI RFLPs in double-muscled Belgian White Blue bulls, the AC genotypic class was not used because of its low distribution. The AA class showed greater records of weight at 7 (P < 0.07) and 13 (P < 0.06) months of age than the AB class. In conclusion, this study has shown a correlation between the GH-TaqI RFLPs and weight at 7 and 13 months of age in Belgian White Blue bulls.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the hypophysial-gonadal axis during the onset of puberty in young bulls.

The objectives of this study were to determine the extent of changes in concentrations of testosterone, growth hormone (GH) and insulin-like growth factor I (IGF-I) concentrations in blood plasma and to characterize the respective plasma-binding proteins of these two peptides during the onset of puberty in male calves. The jugular veins of six male Holstein calves (11-weeks-old) were catheterized and blood was collected every 3 days (one sample every 30 min for 8 h). Hormone concentrations in plasma were determined by specific radioimmunoassay. After incubation with [125I]IGF-I, IGF-I-binding proteins (IGFBPs) were separated by gel filtration; radioactivity was determined in each fraction. Western ligand blotting using radiolabelled hormones as ligand was also used to characterize the IGF-I- and GH-binding proteins in plasma. Puberty was characterized by a rapid (in 1 or 2 days) increase in mean concentrations of testosterone in plasma (from 0.5 to > 2 ng ml-1) and a pulsatile release of the hormone. During puberty, IGF-I concentrations also increased rapidly in 8-10 days from +/- 50 ng ml-1 to > 150 ng ml-1, whereas concentrations of GH in plasma remained relatively stable during the experimental period. A significant correlation was observed between IGF-I and testosterone concentrations (r = 0.77; P < 0.001) throughout the experimental period. Three different IGF-I-binding protein fractions with apparent molecular masses of > 200, 150-170 and 45-65 kDa were found in plasma using gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of growth hormone-binding proteins in cattle plasma and milk.

The presence of GH-binding proteins (GHBPs) in the plasma of adult cattle was investigated using Sephadex G-200 filtration, Western ligand blotting and Western blotting. The changes in the concentration of GHBP in the plasma of dairy half-sister heifers during the first year of life as well as the presence of GHBP in milk were also investigated. When analytical chromatography (on a 1.6 x 100 cm column) was performed, five peaks of recombinant bovine GH (rbGH)-associated radioactivity were revealed in cattle plasma; the first peak, which appeared near the void volume, was presumed to represent aggregates, the second (M(r) 290 kDa) and the third peaks (M(r) 75 kDa) corresponded to specific rbGH-GHBP complexes; the last two peaks representing free 125I-labelled rbGH and Na[125I]. Western ligand blotting revealed multiple GHBPs. Three major bands were observed at approximately 190, 58 and 31 kDa; an excess of unlabelled hormone blocked the binding of 125I-labelled rbGH. Minor non-specific binding bands were also detected in cattle plasma with molecular weights between 40 and 136 kDa. One monoclonal antibody (8H7) produced against synthetic peptide (amino acids 54-63 of the extracellular domain of the bovine GH receptor) specifically interacted with 190 and 58 kDa bands while the 31 kDa band was not recognized. Finally, Western ligand blots were performed to evaluate the changes in plasma GHBP during the first year of life in 55 dairy half-sister heifers and to identify GHBP in milk. In plasma, the intensity of the 31 kDa band varied greatly between animals while the other specific bands remained stable.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of a cDNA encoding the beta-subunit of the bovine insulin-like growth factor-1 receptor.

This communication reports the sequence of the beta-subunit of the insulin-like growth factor-1 receptor. It shares 91.1% homology at the nucleotide level and 97.7% at the amino acid level with the equivalent human receptor subunit.

Cloning and characterization of a cDNA encoding the bovine insulin-like growth factor binding protein 1 (bIGFBP-1).

This communication reports the complete amino acid sequence (263 amino acids) for the insulin-like growth factor binding protein 1 in the bovine (bIGFBP-1). It is 70% homologous with the equivalent IGFBP-1 in human and rat.

[Evolution of plasma concentrations of testosterone and insulin-like growth factor I during the peripubertal period of cattle catheterized at the hepatic level]. / Evolution des concentrations plasmatiques de testostérone et d'insulin-like growth factor I en période péripubertaire chez le bovin cathétérisé au niveau hépatique.

To study endocrinal control of growth development in cattle during puberty, major hepatic vessels were cannulated in 8-13 week old Holstein male calves. In our experimental conditions, chronic hepatic cannulation with "grey" polyurethane catheters was performed for a long period (more than 65 d) without any rejection problems. A physiological washing solution containing at least 5 x 10(5) IU/l heparin-lithium was required to prevent cannula occlusion. Moreover, echography was utilized for rapid and accurate introduction of catheters into the vessels. With chronic hepatic cannulation, we have observed that plasma testosterone and insulin-like growth factor I (IGF-I) levels in cattle were lower in portal and in portal and cava veins respectively than in the other vessels. Our experiment has also shown that average plasma testosterone and IGF-I concentrations sharply increased at puberty to reach adult values. However, no relation was observed between these two hormones during the pubertal period. In conclusion, both testosterone and IGF-I plasma levels can be used as predictive factor of age at puberty but more investigations are needed to establish standard values for each breed.

Production and characterization of monoclonal antibodies to bovid herpesvirus-4.

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test.

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen. A sandwich ELISA procedure using the chosen monoclonal as "capture and detecting" antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.

Effects of an anabolic treatment before puberty with trenbolone acetate-oestradiol or oestradiol alone on growth rate, testicular development and luteinizing hormone and testosterone plasma concentrations.

Scrotal circumference, growth and hormonal status after prepubertal anabolic treatments were studied in 18 conventional Belgian White Blue bulls from 3 to 13 mo of age. Young bulls were assigned into three groups: six untreated (control) bulls, six bulls implanted with 140 mg trenbolone acetate + 20 mg oestradiol (Revalor; TBA-E2) and six bulls treated with 45 mg oestradiol (Compudose; E2). Mean scrotal circumference was similar in the three groups at Day O (between 13.0 +/- 0.3 cm to 13.4 +/- 0.7 cm). From Days O to 230, scrotal circumference was strongly inhibited in implanted bulls, 23.2 +/- 1.4, 21.7 +/- 1.0 cm, respectively, for TBA-E2 and E2 at Day 210, as compared with 29.5 +/- 2.2 cm in control bulls (P < 0.001). Afterwards, differences lessened gradually and no significant divergence was observed between the three groups from Day 310. Average plasma luteinizing hormone (LH) concentrations were similar in the three groups throughout the assay. Mean testosterone levels remained extremely low upto Day 150 in TBA-E2 and E2 groups (0.6 +/- 0.6, 1.2 +/- 0.7 ng/ml, respectively) before they increased abruptly and reached values observed in control bulls at Day 180 (4.0 +/- 1.9 ng/ml). The pulsatil character of LH and testosterone profiles was abolished by the anabolic treatments. Luteinizing hormone-releasing hormone (LHRH) injection was followed by an immediate and sharp increase in plasma LH concentrations in all groups at Day 0. Anabolic treatments strongly reduced LH and testosterone responses to LHRH in treated groups.