Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Selective and on-demand drenching of lambs: Impact on parasite populations and performance of lambs

AIM: To determine whether drenching regimes for lambs by which a proportion (10%) of the heaviest animals was selectively left untreated, or animals are only drenched ‘on demand’ when faecal nematode egg counts (FEC) exceeded a threshold level, would result in measurable increases in parasite larval challenge in the autumn and/or decreases in the performance of lambs.

METHODS: A replicated study compared three drenching strategies in which mobs of lambs (n=360 in total) received either: a five-drench preventive programme, administered to all animals (Treatment 1); a five-drench preventive programme, but the 10% heaviest animals left untreated each time (Treatment 2); or drench treatments administered only when FEC exceeded 500 eggs per gram of faeces (epg) (Treatment 3). After the five-drench programme, animals in Treatments 1 and 2 were treated according to FEC as for Treatment 3. A triple-combination drench containing ivermectin, oxfendazole and levamisole, administered orally, was used for all treatments. There were nine farmlets, allowing three replicates of each treatment, in a completely randomised design. Parasite infestations on pasture were measured in autumn by pasture plucks, and worm burdens were monitored in tracer lambs, while the performance of lambs was assessed by liveweight gains, fleece weights, and body condition and dag scores.

RESULTS: Increased numbers of Haemonchus contortus and Trichostrongylus colubriformis larvae on pasture were found in the autumn on farmlets treating selectively or on-demand (Treatments 2 and 3). No differences were detected in other parasite species. Mean liveweight gains did not differ between treatments but some differences were detected between drenched and undrenched lambs in Treatment 2. Mean body condition and mean dag scores of lambs in Treatment 3 tended to be lower and higher, respectively, than those of lambs in Treatment 1; Treatment 2 was generally intermediate.

CONCLUSIONS: Drenching strategies for lambs designed to slow the development of anthelmintic resistance, by increasing the pool of susceptible worms available to dilute resistant survivors after treatment, resulted in increased numbers of H. contortus and T. colubriformis but not other species of parasite on pasture. The increased parasite challenge to lambs in the autumn was associated with small production losses, which may be acceptable to farmers wishing to implement such strategies. It is clear that further work is required on the interaction between management practices and the population dynamics of parasites, especially with regard to creating pools of susceptible genotypes to slow the development of drench resistance.     

Ovarian follicular growth, function and turnover in cattle: a review

Studies in cattle assessing changes in number and size of antral follicles, concentrations of estradiol, androgens and progesterone in serum and follicular fluid, and numbers of gonadotropin receptors per follicle during repetitive estrous cycles and postpartum anestrus are reviewed. The rate of growth of small follicles (1 to 3 mm) into larger follicles increases as the estrous cycle progresses from d 1 to 18 (d 0 = estrus). Size of the largest antral follicle present on the ovary also increases with advancement of the estrous cycle. Most large follicles (greater than 10 mm) persist on the ovarian surface for 5 d or more between d 3 and 13 of the bovine estrous cycle. After d 13, most of these large follicles are replaced more frequently by new growing follicles (turnover) with an increased probability for recruitment of the ovulatory follicle after d 18. More research is needed to determine the time required for growth of bovine follicles from small to large antral size and evoke recruitment of the ovulatory follicle. Factors that regulate selection of the ovulatory follicle are unknown but may involve increased frequency of LH pulses in blood, altered blood flow and(or) changes in intrafollicular steroids and proteins. Quantitative evaluation of ovarian follicles indicated occurrence of consistent short-term changes in fluid estradiol and numbers of luteinizing hormone receptors in cells of large follicles only during the pre-ovulatory period. Presumably, low concentrations of follicular estradiol found during most of the estrous cycle are not due to a lack of aromatizable precursor or follicle-stimulating hormone receptors. Follicular fluid concentrations of progesterone increase only near the time of ovulation. Little is known about changes in follicular growth, turnover and function during postpartum anestrus in cattle. However, preliminary data suggest that the steroidogenic capacity of large follicles changes markedly during the postpartum period.     

Suberoylanilide hydroxamic acid,a novel histone deacetylase inhibitor,improves the development and acetylation level of miniature porcine handmade cloning embryos

Histone deacetylase inhibitors (HDACis) can change the histone acetylation and significantly enhance the developmental competence of the pre‐implantation SCNT embryo. To select a proper histone deacetylase inhibitor to improve the success rate and potentially developmental ability of handmade cloning (HMC) embryos of miniature porcine, we compared the effect of two histone deacetylase inhibitors (SAHA vs. VPA) on HMC embryo development, their histone acetylation level and the expression level of relevant genes. The blastocyst rate and number of blastocyst cells of HMC embryos treated with SAHA (SAHA‐HMC) or VPA (VPA‐HMC) were significantly higher than those of control (Control‐HMC), respectively, but there were no significant difference between SAHA‐HMC and VPA‐HMC groups. In addition, the acetylation level (AcH4K8) of Control‐HMC and VPA‐HMC embryos at the blastocyst stage, respectively, was significantly lower than that of in vitro fertilized (IVF) and SAHA‐HMC embryos. However, the acetylation H4K8 of the blastocysts had no significant difference between SAHA‐HMC and the IVF groups. The SAHA‐HMC blastocysts indicated comparative expression levels of Oct4 and HDAC1 (histone deacetyltransferase gene) with those of IVF blastocysts. In contrast, the expression levels of Oct4 were lower and those of HDAC1 were higher in the VPA‐HMC and Control‐HMC blastocysts, respectively, compared to those of the IVF blastocysts. Our results demonstrated that the HMC embryos treated by SAHA could promote the pre‐implantation development and increase the levels of histone H4K8 acetylation and the expression of the OCT4 gene, yet decrease the expression of the HDAC1 gene to the comparable level of the IVF embryos. Our results proved that SAHA may be a better histone deacetylase inhibitor for porcine HMC compared to VPA, and furthermore, it may indicate that SAHA can effectively correct the abnormal histone acetylation during the HMC embryo development and subsequently improve the full‐term developmental potential of the HMC embryos after embryo transplantation.     

Role of sulfur and chloride in the dietary cation-anion balance equation for lactating dairy cattle

Ten Holstein cows averaging 120 d in lactation were arranged in replicated 5 x 5 Latin squares with 3-wk periods to evaluate the role of sulfur (S) in the dietary cation-anion balance equation. Diets were based on corn silage in Exp. 1 and sorghum silage in Exp. 2. Supplemental S and chloride (Cl) from the double sulfate of potassium and magnesium and CaCl2 were used to manipulate dietary cation-anion balance from 0 to +30 meq when expressed as meq [(Na + K)-(Cl + S)]/100 g diet DM and from +19 to +49 meq when expressed as meq [(Na + K)-Cl]/100 g diet DM. Blood pH was not affected by cation-anion balance, although both S and Cl supplementation tended to lower pH. Blood HCO3- and urine pH decreased and plasma calcium (Ca) and urinary Ca excretion increased as anion was added to the diet. Milk fat production tended to be increased by the low S supplementation. Dietary Cl and S had similar effects on acid-base status. Therefore, we suggest that S be included with Cl in the dietary cation-anion balance equation for lactating dairy cows as follows: meq [(Na + K)-(Cl + S)]/100 g diet DM. Although response of acid-base status to S and Cl was similar, as more data comparing the acidogenicity of S vs Cl become available, it may be necessary to include a modifying coefficient for S in the equation to adjust for differences between S and Cl in acid-generating potential. This coefficient may be further dependent on the dietary source of S.     

Influence of exogenous gonadotropin-releasing hormone on ovarian function in beef cows after short- and long-term nutritionally induced anovulation

The effect of pulsatile infusion of gonadotropin-releasing hormone (GnRH) on follicular function was evaluated in nutritionally induced anovulatory beef cows. After 4 (short; n = 12) or 18 wk (long; n = 12) of anovulation, cows were randomly assigned within anovulatory group to either 2 microg of GnRH treatment or saline (control; i.v.) every hour for 5 d. Ovarian structures were monitored by daily ultrasonography. Growth rate of the largest follicle (P < 0.01) and maximal size of the largest follicle during treatment were greater (P < 0.01) for GnRH vs control cows. At exsanguination after 5 d of GnRH treatment, the size of the second-largest follicle was greater (P < 0.05) in short (i.e., 4 wk) anovulatory cows than in long (i.e., 18 wk) anovulatory cows and the largest follicle tended (P < 0.10) to be larger in long vs short anovulatory cows. Short anovulatory GnRH-treated cows had more small follicles than short anovulatory control cows or long anovulatory GnRH-treated or control cows (anovulation x GnRH; P < 0.10). Follicular fluid (FFL) concentrations of estradiol (P < 0.01) and androstenedione (P < 0.05) were greater in GnRH vs control cows. Concentrations of insulin-like growth factor-I were greater (P < 0.10) in large vs small follicles in cows that were anovulatory for 4 wk, but not in cows that were anovulatory for 18 wk. The amount of insulin-like growth factor-binding protein (IGFBP)-3 in FFL was greater (P < 0.05) in 4- vs 18-wk anovulatory cows. Amounts of IGFBP-2, -4, and -5 were greater (P < 0.001) in FFL of small (< 5 mm) vs large (> or = 5 mm) follicles regardless of treatment. We conclude that pulsatile treatment with GnRH for 5 d stimulates similar growth of the largest follicles in short- and long-term anovulatory beef cows, and that the duration of anovulation is not a major factor that limits follicular growth w hen anovulatory cowsare treated with GnRH. The primary intrafollicular factors associated with increased follicular size were increased concentrations of estradiol, progesterone, and insulin-like growth factor-I,and decreased concentrations of IGFBP-2, -4, and -5. Increased duration of anovulation was associated with decreased concentrations of IGF-I and IGFBP-3 in FFL.     

Cytopathological and histopathological diagnosis of canine splenic disorders

Objectives To determine (1) the common types of canine splenic disorders, and the breeds affected, that are diagnosed by cytopathological and histopathological examination in Sydney, Australia and (2) the accuracy of cytopathological examination compared with histopathological examination for the diagnosis of canine splenic disorders. Design 69 cytopathological and 51 histopathological diagnoses of canine splenic disorders presented to the Veterinary Pathology Diagnostic Services, The University of Sydney during 2006 and 2007 were tabulated and analysed; 17 cases examined both cytopathologically and histopathologically during 2001–07 were also analysed. Results The most common cytopathological diagnoses were benign disorders of growth, vascular disturbances and necrosis (29%), followed by no abnormalities detectable (28%), malignant neoplasms (20%), equivocal diagnoses (20%) and inflammatory disorders (3%). The most common breeds were Kelpie crosses and mixed breeds. The most common histopathological diagnoses were benign disorders of growth, vascular disturbances and necrosis (49%), followed by malignant neoplasms (43%) and inflammatory disorders (8%). The most common breeds were German Shepherd Dogs, Boxers and Maltese Terriers. Cytopathological and histopathological diagnoses were in complete agreement in 59% of cases, partial agreement in 29% and disagreement in 12%. Conclusion Benign disorders of growth, vascular disturbances and necrosis were the most commonly diagnosed canine splenic disorders, both cytopathologically and histopathologically. Kelpie crosses presented most frequently for cytopathological examination. German Shepherd Dogs were the most common breed diagnosed histopathologically with haemangiosarcoma. Although cytopathological and histopathological splenic examinations are complementary for diagnosis, this study has shown a high correlation for complete and partial agreement between the two.     

The role of endocrine insulin-like growth factor-I (IGF-I) in female bovine reproduction

Insulin-like growth factor-I (IGF-I) plays a pivotal role in cattle fertility, acting as a monitoring signal that allows reproductive events to occur when nutritional conditions for successful reproduction are reached. However, endocrine IGF-I is not a predictor of reproductive events, but rather an indirect estimator of the suitability of the animal to achieve the reproductive event in question. Although measuring circulating IGF-I concentrations might not have any clinical application in the cattle industry, endocrine IGF-I screening will continue to be important for the study of interactions between nutrition and reproduction. In addition, endocrine IGF-I screening could be used as an ancillary test for the selection of cattle for high reproductive potential, especially in herds of high genetic merit for milk production, in which a decline in fertility has been identified.     

Effect of growth hormone administration to mature miniature Brahman cattle treated with or without insulin on circulating concentrations of insulin-like growth factor-I and other metabolic hormones and metabolites

Previously, we determined that a primary cause of proportional stunted growth in a line of Brahman cattle was related to an apparent refractoriness in metabolic response to GH in young animals. The objective of this study was to determine the effect of administration of GH, insulin (INS), and GH plus INS to mature miniature Brahman cows (n = 6; 9.7 ± 2.06 y; 391 ± 48.6 kg) and bulls (n = 8; 9.4 ± 2.00 y; 441 ± 54.0 kg) on circulating concentrations of metabolic hormones and metabolites, primarily IGF-I and IGF-I binding proteins. We hypothesized that IGF-I secretion could be enhanced by concomitant administration of exogenous GH and INS, and neither alone would be effective. Animals were allotted to a modified crossover design that included four treatments: control (CON), GH, INS, and GH + INS. At the start of the study, one-half of the cattle were administered GH (Posilac; 14-d slow release) and the other one-half served as CON for 7 d. Beginning on day 8, and for 7 d, INS (Novolin L) was administered (0.125 IU/kg BW) twice daily (7:00 AM and 7:00 PM) to all animals; hence, the INS and GH + INS treatments. Cattle were rested for 14 d and then were switched to the reciprocal crossover treatments. Blood samples were collected at 12-hour intervals during the study. Compared with CON, GH treatment increased (P < 0.01) mean plasma concentrations of GH (11.1 vs 15.7 ± 0.94 ng/mL), INS (0.48 vs 1.00 ± 0.081 ng/mL), IGF-I (191.3 vs 319.3 ± 29.59 ng/mL), and glucose (73.9 vs 83.4 ± 2.12 mg/dL) but decreased (P < 0.05) plasma urea nitrogen (14.2 vs 11.5 ± 0.75 mg/dL). Compared with INS, GH + INS treatment increased (P < 0.05) mean plasma concentration of INS (0.71 vs 0.96 ± 0.081 ng/mL), IGF-I (228.7 vs 392.3 ± 29.74 ng/mL), and glucose (48.1 vs 66.7 ± 2.12 mg/dL), decreased (P < 0.01) plasma urea nitrogen (13.6 vs 10.4 ± 0.76 mg/dL), and did not affect GH (13.5 vs 12.7 ± 0.95 ng/mL). In the miniature Brahman model, both the GH and GH + INS treatments dramatically increased circulating concentrations of IGF-I in mature cattle, suggesting that this line of Brahman cattle is capable of responding to bioactive GH.