Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae.

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

Respiratory Syncytial Virus and Human Metapneumovirus Infections in Three-Dimensional Human Airway Tissues Expose an Interesting Dichotomy in Viral Replication, Spread, and Inhibition by Neutralizing Antibodies.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two of the leading causes of respiratory infections in children and elderly and immunocompromised patients worldwide. There is no approved treatment for HMPV and only one prophylactic treatment against RSV, palivizumab, for high-risk infants. Better understanding of the viral lifecycles in a more relevant model system may help identify novel therapeutic targets. By utilizing three-dimensional (3-D) human airway tissues to examine viral infection in a physiologically relevant model system, we showed that RSV infects and spreads more efficiently than HMPV, with the latter requiring higher multiplicities of infection (MOIs) to yield similar levels of infection. Apical ciliated cells were the target for both viruses, but RSV apical release was significantly more efficient than HMPV. In RSV- or HMPV-infected cells, cytosolic inclusion bodies containing the nucleoprotein, phosphoprotein, and respective viral genomic RNA were clearly observed in human airway epithelial (HAE) culture. In HMPV-infected cells, actin-based filamentous extensions were more common (35.8%) than those found in RSV-infected cells (4.4%). Interestingly, neither RSV nor HMPV formed syncytia in HAE tissues. Palivizumab and nirsevimab effectively inhibited entry and spread of RSV in HAE tissues, with nirsevimab displaying significantly higher potency than palivizumab. In contrast, 54G10 completely inhibited HMPV entry but only modestly reduced viral spread, suggesting HMPV may use alternative mechanisms for spread. These results represent the first comparative analysis of infection by the two pneumoviruses in a physiologically relevant model, demonstrating an interesting dichotomy in the mechanisms of infection, spread, and consequent inhibition of the viral lifecycles by neutralizing monoclonal antibodies.IMPORTANCE Respiratory syncytial virus and human metapneumovirus are leading causes of respiratory illness worldwide, but limited treatment options are available. To better target these viruses, we examined key aspects of the viral life cycle in three-dimensional (3-D) human airway tissues. Both viruses establish efficient infection through the apical surface, but efficient spread and apical release were seen for respiratory syncytial virus (RSV) but not human metapneumovirus (HMPV). Both viruses form inclusion bodies, minimally composed of nucleoprotein (N), phosphoprotein (P), and viral RNA (vRNA), indicating that these structures are critical for replication in this more physiological model. HMPV formed significantly more long, filamentous actin-based extensions in human airway epithelial (HAE) tissues than RSV, suggesting HMPV may promote cell-to-cell spread via these extensions. Lastly, RSV entry and spread were fully inhibited by neutralizing antibodies palivizumab and the novel nirsevimab. In contrast, while HMPV entry was fully inhibited by 54G10, a neutralizing antibody, spread was only modestly reduced, further supporting a cell-to-cell spread mechanism.

Human metapneumovirus fusion protein triggering: Increasing complexities by analysis of new HMPV fusion proteins.

The human metapneumovirus (HMPV) fusion protein (F) mediates fusion of the viral envelope and cellular membranes to establish infection. HMPV F from some, but not all, viral strains promotes fusion only after exposure to low pH. Previous studies have identified several key residues involved in low pH triggering, including H435 and a proposed requirement for glycine at position 294. We analyzed the different levels of fusion activity, protein expression and cleavage of three HMPV F proteins not previously examined. Interestingly, low pH-triggered fusion in the absence of G294 was identified in one F protein, while a novel histidine residue (H434) was identified that enhanced low pH promoted fusion in another. The third F protein failed to promote cell-to-cell fusion, suggesting other requirements for F protein triggering. Our results demonstrate HMPV F triggering is more complex than previously described and suggest a more intricate mechanism for fusion protein function and activation.

Vitrification of in vitro-derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures.

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. 'fresh') embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.

Commensal enteric bacteria lipopolysaccharide impairs host defense against disseminated Candida albicans fungal infection.

Commensal enteric bacteria maintain systemic immune responsiveness that protects against disseminated or localized infection in extra-intestinal tissues caused by pathogenic microbes. However, as shifts in infection susceptibility after commensal bacteria eradication have primarily been probed using viruses, the broader applicability to other pathogen types remains undefined. In sharp contrast to diminished antiviral immunity, we show commensal bacteria eradication bolsters protection against disseminated Candida albicans fungal infection. Enhanced antifungal immunity reflects more robust systemic expansion of Ly6G(hi)Ly6C(int) neutrophils, and their mobilization into infected tissues among antibiotic-treated compared with commensal bacteria-replete control mice. Reciprocally, depletion of neutrophils from expanded levels or intestinal lipopolysaccharide reconstitution overrides the antifungal protective benefits conferred by commensal bacteria eradication. This discordance in antifungal compared with antiviral immunity highlights intrinsic differences in how commensal bacteria control responsiveness for specific immune cell subsets, because pathogen-specific CD8(+) T cells that protect against viruses were suppressed similarly after C. albicans and influenza A virus infection. Thus, positive calibration of antiviral immunity by commensal bacteria is counterbalanced by restrained activation of other immune components that confer antifungal immunity.

Comparative response of rams and bulls to long-term treatment with gonadotropin-releasing hormone analogs.

The objective was to compare the relative response between rams and bulls in characteristics of LH, FSH and testosterone (T) secretion, during and after long-term treatment with GnRH analogs. Animals were treated with GnRH agonist, GnRH antagonist, or vehicle (Control) for 28 days. Serial blood samples were collected on day 21 of treatment, and at several intervals after treatment. Injections of natural sequence GnRH were used to evaluate the capacity of the pituitary to release gonadotropins during and after treatment. Treatment with GnRH agonist increased basal LH and T concentrations in both rams and bulls, with a greater relative increase in bulls. Endogenous LH pulses and LH release after administration of GnRH were suppressed during treatment with GnRH agonist. Treatment with GnRH antagonist decreased mean hormone concentrations, LH and T pulse frequency, and the release of LH and T after exogenous GnRH, with greater relative effects in bulls. Rams previously treated with antagonist had a greater release of LH after administration of GnRH compared with control rams, while rams previously treated with agonist showed a reduced LH response. Bulls previously treated with agonist had reduced FSH concentrations and LH pulse amplitudes compared with control bulls while bulls previously treated with antagonist had greater T concentrations and pulse frequency. The present study was the first direct comparison between domestic species of the response in males to treatment with GnRH analogs. The findings demonstrated that differences do occur between rams and bulls in LH, FSH and testosterone secretion during and after treatment. Also, the consequences of treatment with either GnRH analog can persist for a considerable time after discontinuation of treatment.

Urine calcium excretion predicts bone loss in idiopathic hypercalciuria.

Although idiopathic hypercalciuria (IH) is associated with reduced bone mineral density (BMD), no studies to date have identified predictors of BMD change over an extended period of observation. We have studied change in femoral neck and spine BMD z-scores in men and women with IH and stone disease (IHSF) and their first-degree relatives in order to determine the predictive value of commonly made clinical measurements. Urine calcium excretion was inversely correlated with change in femoral neck z-score over 3 years, and marginally correlated with fall in spine z-score. Markers of bone turnover, serum calcitriol, and urine measurements of acid-base balance such as ammonium and sulfate had no predictive value, nor did calcium intake assessed using a well-established questionnaire. It would appear that IHSF with the highest 24-h urine calcium excretion rates are at highest risk for loss of femoral neck bone mineral over a 3-year period.

Induction of precocious puberty in heifers I: enhanced secretion of luteinizing hormone.

In beef heifers weaned between 3 and 4 mo of age and fed a high-concentrate diet, approximately 50% reach puberty before 300 d of age (precocious puberty). The objectives of this experiment were 1) to determine whether precocious puberty could be induced experimentally by weaning heifers early and feeding a high-concentrate diet, and 2) to determine the dynamics of secretion of LH associated with precocious puberty. Crossbred Angus and Simmental heifer calves were weaned at 73 +/- 3 d of age and 115 +/- 3 kg of BW and fed a high-concentrate (60% corn; HI, n = 9) or control diet (30% corn; CONT, n = 9). Heifers were fed individually, and target BW gains were 1.50 and 0.75 kg/d for the HI and CONT treatments, respectively. Heifers were weighed every 2 wk. Blood samples were collected weekly and assayed for progesterone concentration to determine age at puberty. Serial blood samples were collected at 20-min intervals for 24 h at mean ages of 102, 130, 158, 172, 190, 203, 217, 231, and 259 d and assayed for LH concentration to evaluate the dynamics of secretion of LH. Heifers fed the HI diet exhibited greater BW gain (P < 0.01) than CONT heifers (1.27 +/- 0.05 vs. 0.85 +/- 0.05 kg/d, respectively). As a result, BW in the HI treatment was greater (P < 0.01) than in the CONT treatment by 188 d of age and remained different through the end of the experiment. Precocious puberty occurred in 8 of 9 heifers fed the HI diet and 0 of 9 heifers fed the CONT diet. Age at puberty was reduced in the HI (P < 0.01) compared with the CONT heifers (262 +/- 10 vs. 368 +/- 10 d of age, respectively). Body weight at puberty was also reduced in the HI (P < 0.05) compared with the CONT treatment (327 +/- 17 vs. 403 +/- 23 kg, respectively). Heifers attaining puberty during the experiment continued with subsequent luteal phases as evidenced by cyclic patterns of progesterone concentrations. Frequency of pulses of LH (pulses/24 h) increased with age (P < 0.01) for both treatments. Heifers in the HI treatment exhibited a greater number of pulses of LH (P < 0.01) than those in the CONT treatment by 190 d of age and in all subsequent collection periods (treatment x age, P < 0.05). Mean LH concentrations also increased with age (P < 0.01) for both treatments but did not differ between treatments. In conclusion, precocious puberty induced by early weaning and feeding of a high-concentrate diet is preceded by increasing frequency of pulses of LH.

Induction of precocious puberty in heifers III: hastened reduction of estradiol negative feedback on secretion of luteinizing hormone.

Precocious puberty (<300 d of age) can be induced in beef heifers by early weaning and feeding a high-concentrate diet. The objective of this experiment was to determine whether precocious puberty occurs as a result of a hastened reduction of estradiol negative feedback on secretion of LH. Thirty crossbred Angus and Simmental heifers were weaned at 83 +/- 2 d of age and 114 +/- 3 kg of BW, blocked by BW, and randomly assigned to receive a high-concentrate (60% corn; H) or control (30% corn; C) diet and to receive ovariectomy (OVX), OVX plus an estradiol implant (OVXE), or to remain intact (INT). Residual ovarian tissue after OVX necessitated withdrawal of 6 heifers during the course of the experiment, resulting in the following treatment groups: OVX-C, n = 3; OVX-H, n = 5; OVXE-C, n = 4; OVXE-H, n = 2; INT-C, n = 5; INT-H, n = 5. To determine concentrations of progesterone and estradiol, blood samples were collected weekly beginning at a mean age of 160 d. To characterize LH concentrations, serial blood samples were collected at 12-min intervals for 12 h at mean ages of 119, 149, 188, 217, 246, 281, 323, 365, 407, and 449 d. By a mean age of 202 d, heifers fed the H diet were heavier (P < 0.05) than those fed the C diet. Heifers in the INT-H treatment attained puberty earlier (P < 0.05) than in the INT-C treatment (275 +/- 30 vs. 385 +/- 14 d of age, respectively). Overall mean concentrations of estradiol did not differ between OVXE-H and OVXE-C, between INT-H and INT-C, or between OVXE and INT treatments. The OVX treatments exhibited greater LH pulse frequency than the OVXE and INT treatments by the first serial blood collection (treatment x age, P < 0.05). The frequency of LH pulses was greater (P < 0.05) in the INT-H than the INT-C treatment by a mean age of 246 d and was greater (P < 0.05) in the OVXE-H than the OVXE-C treatment by a mean age of 281 d. In the OVXE-H treatment, LH secretion increased and subsequently "escaped" from estradiol negative feedback (detection of > or = 1 LH pulse/h) earlier (P < 0.05) than in the OVXE-C treatment (307 +/- 30 and 420 +/- 21 d of age, respectively). It is concluded that advancing the reduction of estradiol negative feedback on secretion of LH is the mechanism by which early weaning and feeding a high-concentrate diet results in precocious puberty in heifers.

Induction of precocious puberty in heifers II: advanced ovarian follicular development.

Precocious puberty can be induced in a majority of heifers weaned early and fed a high-concentrate diet. The objective of this experiment was to determine whether induction of precocious puberty is associated with an acceleration of ovarian maturation in heifers. Crossbred Angus and Simmental heifer calves were weaned at 104 +/- 2 (n = 18; early weaned) or 208 +/- 3 (n = 10; normal-weaned, NW) d of age. The early weaned heifers were fed a high-concentrate (60% corn; EWH, n = 9) or control diet (30% corn; EWC, n = 9). The NW heifers were also fed the control diet after weaning. Daily transrectal ultrasonography was performed to characterize a complete follicular wave beginning at a mean age of 126, 161, 196, 224, and 252 (EWH and EWC), or 224 and 252 (NW) d. Blood samples were collected daily during periods of ultrasonography to determine estradiol concentrations and weekly beginning at mean ages of 153 (EWH and EWC) or 216 (NW) d to be analyzed for progesterone concentrations. Heifers in the EWH treatment were heavier (P < 0.01) than EWC heifers from a mean age of 175 d through the end of the study (treatment x age; P < 0.05). Body weights did not differ between EWC and NW. At mean ages of 196 and 224 d, the maximum diameter of the dominant follicle (MaxDF) was greater (P < 0.05) in EWH than EWC heifers. At a mean age of 224 d, MaxDF was greater (P < 0.05) in EWC than NW heifers but was not different by a mean age of 252 d. All EWH, 5 of 9 EWC, and 5 of 10 NW heifers attained puberty at less than 300 d of age (precocious puberty). Age at puberty was less (P < 0.05) in EWH (252 +/- 9 d) than in EWC and NW (308 +/- 26 and 330 +/- 25 d, respectively) treatments. Across all heifers, MaxDF and duration of follicular waves increased with age (P < 0.05), mean number of follicles during follicular waves decreased with age (P < 0.05), and peak concentrations of estradiol during follicular waves increased until a mean age of 224 d. To further characterize aspects of precocious puberty, heifers were compared across treatments between those that experienced precocious puberty and those that did not. In heifers that experienced precocious puberty, BW at puberty was less (P < 0.01) and MaxDF, follicular wave duration, and peak estradiol concentrations were greater (P < 0.05) compared with heifers that did not experience precocious puberty. Ovarian maturation was accelerated in heifers that were weaned early and fed a high-concentrate diet and was associated with precocious onset of puberty.

Testicular development of Zebu bulls after chronic treatment with a gonadotropin-releasing hormone agonist.

The objective was to compare testis characteristics of Zebu bulls treated with the GnRH agonist, deslorelin, at different times and for different durations during their development. An additional objective was to determine the usefulness of a stain for the transcription factor GATA-binding protein 4 (GATA-4) as a specific marker for Sertoli cell nuclei in cattle. Bulls (54) were allocated to nine groups (n = 6) and received s.c. deslorelin implants as follows: G1 = from birth to 3 mo of age; G2 = from 3 to 6 mo; G3 = from 6 to 9 mo; G4 = from 9 to 12 mo; G5 = from birth to 15 mo; G6 = from 3 to 15 mo; G7 = from 6 to 15 mo; G8 = from 12 to 15 mo; and G9 (control) = no implant. Bulls were castrated at 19 mo of age. Paraffin sections (10 microm) were subjected to quantitative morphometry and GATA-4 immunohistochemistry. At castration, all bulls in the control group (6/6) had attained puberty (scrotal circumference > or = 28 cm), whereas a smaller proportion (P < 0.05) had reached puberty in G2 (2/5) and G6 (1/6). Bulls in G2 and G6 also had a lesser (P < 0.05) testis weight compared with the control group. Total volume of seminiferous epithelium and total daily sperm production in G2 and G6 were only half that observed in the control group. Spermatids were observed in less than 50% of seminiferous tubules in G2, G6, and G7 compared with 82% in the control group (P < 0.05). Staining for GATA-4 was specific for and abundant in the Sertoli cell nucleus in both pre- and postpubertal bulls, and no other cell nucleus inside the seminiferous tubule was positive for GATA-4. Total number of Sertoli cells was not affected by treatment (P = 0.45), but nuclear volume was smaller in G2 and G6 (P < 0.05) compared with the control group. In conclusion, treatment of Zebu bulls with deslorelin had no apparent beneficial effect on testis development and delayed puberty when treatment was initiated at 3 mo of age. Staining for GATA-4 was a useful method for identifying and quantifying Sertoli cell nuclei in both pre- and postpubertal bulls.

Regulation of frequency of luteinizing hormone pulses by magnitude of acute change in circulating concentration of progesterone of female cattle.

The hypothesis was the greater the magnitude of acute increase in circulating concentration of progesterone of female cattle, the greater the acute inhibitory effect on frequency of pulsatile LH release. From Day 0 to 4 of the treatment period, females without functional corpora lutea were treated with varying doses of progesterone to result in varying concentrations of progesterone within the typical physiological range in blood. From Day 4 to 7, cattle were treated with a single dose of progesterone to achieve a similar circulating concentration of progesterone among all females in the study. Therefore, from Day 0 to 4 relative to Day 4 to 7 of the treatment period, females had a: (1) large (3.1 ng/ml), (2) moderate (2.5 ng/ml), or (3) small (0.5 ng/ml) increase in concentration of progesterone in blood. Frequency of LH pulses was greater (P <0.10) in females with the greatest magnitude of change in concentration of progesterone during the first 24 h following the change in concentration as compared with females with the moderate or small of change in concentration of progesterone suggesting our working hypothesis should be rejected. The greater the magnitude of acute change in concentration of progesterone, however, the longer time required for re-initiation of release of LH pulses of the amplitude of pulses that preceded the change in concentration of progesterone.

Season of the year influences testosterone secretion in bulls administered luteinizing honrmone.

The objective of the present study was to evaluate the secretion of testosterone (T) in bulls in response to the administration of varying doses of bovine LH (bLH) during the four seasons of the year. Five adult bulls (4 yr of age) were treated with an amount of bLH that was estimated to induce a 5 ng/mL amplitude pulse of LH in blood serum on five consecutive days around the spring equinox, summer solstice, fall equinox, and winter solstice. Five hours after this dose, bulls were treated with bLH in amounts that were estimated to induce a 0.25, 0.5, 1, 2, or 4 ng/mL amplitude LH pulse in blood serum in a Latin square design. Blood samples were collected for 5 h after administration of a dose of bLH that was estimated to induce the 5-ng amplitude LH pulse, and for 3 h after administration of the variable doses of bLH, and were then assayed for concentrations of T. Average concentrations and amplitude of T release after doses of bLH that were estimated to induce the 5-ng amplitude LH pulses were greater during the spring and summer than during the winter (P < 0.05). The area under the release curve (AUC) was greater during the spring than during the winter (P < 0.05). During the 3 h after treatment with the variable doses of bLH, T response was affected by dose (P < 0.001) and season (P < 0.001), but there was no dose x season interaction. Testosterone response increased in a dose-dependent fashion for all variables studied. The greatest average concentrations of T and AUC were observed in the spring compared with the fall and winter (P < 0.05). These data support our working hypothesis that testes of bulls are more responsive in releasing T in response to bLH stimulation in the spring and summer compared with the winter; however, there were no changes in sensitivity of the testes to LH during different seasons of the year as indicated by the lack of a dose of bLH x season interaction.

Chronic treatment with an agonist of gonadotropin-releasing hormone enhances luteal function in cattle.

Our hypothesis was that luteal function, as determined by plasma progesterone concentrations, and corpus luteum (CL) size is enhanced in cattle administered an agonist of GnRH when the CL is developing as compared with administration of an agonist when the CL is fully functional. Cattle were chronically administered a GnRH agonist, azagly-nafarelin, from Day 3 to Day 21 (D3) or Day 12 to Day 21 (D12) or served as untreated control females (Day 0 = behavioral estrus). Blood samples were serially collected on Days 7 and 14 to evaluate LH secretory patterns and twice daily to measure plasma progesterone. Ultrasonographic examinations were conducted daily to record the area of the CL. CL size and plasma progesterone concentrations were both enhanced in the D3 group as compared with the control group. Progesterone was increased in the D12 group on Days 16 and 17 as compared with the control females. Treatment with GnRH agonist increased basal and mean LH concentrations in both D3 and D12 groups as compared with the controls. We rejected our hypothesis because chronic administration of a GnRH agonist increased plasma progesterone when administered both when the CL was developing and when it was fully functional. The enhanced luteal function was likely due to increased basal LH.

Frequency of luteinizing hormone pulses in cattle influences duration of persistence of dominant ovarian follicles, follicular fluid concentrations of steroids, and activity of insulin-like growth factor binding proteins.

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.

Effect of chronic treatment with the gonadotrophin-releasing hormone agonist azagly-nafarelin on basal concentrations of LH in prepubertal bulls.

Administration of GnRH agonist for an extended period inhibits pulsatile LH release but enhances testicular function of bulls. The mechanism whereby long-term administration of GnRH agonist enhances testosterone concentration in the blood of bulls has not been determined. The aim of this study was to determine whether chronic treatment with the GnRH agonist, azagly-nafarelin, increases blood concentrations of LH and FSH in prepubertal bulls. Two different doses of the GnRH agonist were administered via Alzet mini-osmotic pumps for 28 days. Blood samples were collected at 20 min intervals for 24 h at days 2, 13 and 25 of treatment. Agonist-treated groups had reduced testosterone pulse frequency (P < 0.05) and increased mean and basal concentrations of testosterone (P < 0.05) compared with untreated control bulls. Basal LH concentrations were higher in agonist-treated bulls during all three periods (P < 0.05) and overall (1 ng ml(-1) higher, compared with control bulls; P < 0.001). Frequency of LH pulses in the agonist-treated groups was reduced to less than one pulse in 24 h. Agonist-treated bulls tended to have (P < 0.10) or had (P < 0.05) a slight but significant increase in blood FSH concentration. In conclusion, the higher blood testosterone concentration in bulls after prolonged treatment with GnRH agonist may result, at least in part, from changes in the testes induced by enhanced basal concentration of LH.

Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles.

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.

Estradiol regulation of luteinizing hormone secretion in heifers of two breed types that reach puberty at different ages.

The working hypothesis was that 17 beta-estradiol (E(2)) negative feedback on the hypothalamic-pituitary axis in regulation of LH secretion decreases during peripuberty in heifers of 2 different genotypes. We investigated whether Bos indicus heifers had a period postpuberty, as compared with prepuberty, of greater E(2) inhibition of LH secretion at a time when heifers of this genotype have been reported to have a period of anestrus. Prepubertal heifers 9 mo of age of 2 genotypes (B. indicus and B. taurus) were assigned to 3 groups (6 animals/group) to either remain intact (control), be ovariectomized, or be ovariectomized and implanted with E(2). Variables evaluated from 10 to 28 mo of age were circulating concentrations of progesterone (P(4)), presence of corpora lutea, and pulsatile pattern of LH release. Results confirmed that B. taurus heifers attained puberty at younger ages (P < 0.001) and at lower live weights (P = 0.015) than did B. indicus heifers (507 +/- 37 days of age vs. 678 +/- 7 days of age; 259 +/- 14 kg vs. 312 +/- 11 kg; respectively). There was cessation of E(2) inhibition of LH pulses coincident with the onset of puberty in heifers of both breed types but at a much younger age in B. taurus heifers. There was no evidence of enhanced negative feedback of E(2) on LH secretion subsequent to puberty in B. indicus heifers nor was there cessation of estrous cycles in control heifers of either breed type after puberty.

Ballistic fingerprinting databases.

This article discusses a number of questions regarding the setting up of ballistic fingerprinting databases, consisting of ammunition components fired by all the firearms held in legal possession. These questions can be classified into three categories--the efficiency of the database, forensic issues, and practical issues to be dealt with. The current New York State legislation is used as an illustration of the choices to be made when setting up a ballistic fingerprinting database. Three important arguments are formulated against the installation of a ballistic fingerprinting database.

Atresia revisited: two basic patterns of atresia of bovine antral follicles.

Our observations of bovine follicles indicated that the original histological classifications of atresia were inaccurate. A detailed histological, ultrastructural and immunohistochemical study of antral follicles from bovine ovaries collected from an abattoir and from animals whose large follicles had been monitored by ultrasonography was conducted to investigate this further. Nidogen and CD68 were immunolocalized to observe the follicular basal lamina and macrophages, respectively. In randomly collected ovaries, approximately one quarter of all antral follicles were undergoing antral atresia, as designated in this study. Antral atresia was characterized by early destruction of the layers of the membrana granulosa closest to the antrum, whereas the most basal cells remained intact. Numerous pyknotic nuclei were observed in the most antral layers and in the antrum close to the membrana granulosa. This is the classic description of atretic follicles and was observed at all sizes of follicle development and almost universally in large follicles (> 5 mm in diameter), including dominant follicles. Basal atretic follicles, as designated in this study, were almost as prevalent as the antral atretic follicles, and were characterized by initial destruction of the most basal layer of granulosa cells, whereas the cells in the most antral layers remained associated with each other and were predominantly healthy. Pyknotic nuclei and the nuclei of dying basal cells budded into apoptotic bodies were observed rarely. The basal lamina of basal atretic follicles was often breached by macrophages, which were phagocytosing dying basal granulosa cells. The theca was characterized by an increased deposition of collagen, and the cells were orientated randomly, rather than lying parallel to the membrana granulosa as in healthy follicles. Basal atresia occurred in small (< 5 mm in diameter) follicles only. Importantly, these basal atretic follicles were originally identified incorrectly in the literature. Thus, on the basis of the results of this study and another on the expression of steroidogenic enzymes in atretic follicles, it is suggested that the standard biochemical methods for measuring steroid hormone concentrations in follicular fluids to assess atresia should be re-evaluated.