Single-Step Protocol for Isolating the Recombinant Extracellular Domain of the Luteinizing Hormone Receptor from the Ovis aries Testis.

The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptors superfamily. It participates in corpus luteum formation and ovulation in females and acts in testosterone synthesis and spermatogenesis in males. In this study, we extracted RNA from sheep testicles and synthetized the cDNA to amplify the gene lhr-bed. This gene consists of 762 bp and encodes 273 amino acids of the extracellular domain of LHR. The lhr-bed was cloned into pJET1.2/blunt, then subcloned into pCOLD II, and finally, transformed in E. coli BL21 (DE3) cells. Because the induced rLHR-Bed protein was found in the insoluble fraction, we followed a modified purification protocol involving induction at 25 °C, subjection to denaturing conditions, and on-column refolding to increase solubility. We confirmed rLHR-Bed expression by means of Western blot and mass spectrometry analysis. It is currently known that the structure stem-loop 5'UTR on pCOLD II vector is stable at 15 °C. We predicted and obtained RNAfold stability at 25 °C. We successfully obtained the recombinant LHR extracellular domain, with protein yields of 0.2 mg/L, and purity levels of approximately 90%, by means of a single chromatographic purification step. The method described here may be used to obtain large quantities of rLHR-Bed in the future.

Pooled molecular occurrence of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry: A systematic review and meta-analysis.

Worldwide, Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the main agents responsible for chronic respiratory disease in poultry. Therefore, we conducted a systematic review and meta-analysis to estimate their occurrence. We searched electronic databases to find peer-reviewed publications reporting the molecular detection of MG and MS in poultry and used meta-analysis to estimate their pooled global occurrence (combined flock and individual), aggregating results at the regional and national levels. We performed a subgroup meta-analysis for subpopulations (broilers, layers, breeders and diverse poultry including turkeys, ducks and ostriches) and used meta-regression with categorical modifiers. We retrieved 2294 publications from six electronic databases and included 85 publications from 33 countries that reported 62 studies with 22,162 samples for MG and 48 studies with 26,413 samples for MS. The pooled global occurrence was 38.4% (95% CI: 23.5-54.5) for MS and 27.0% (20.4-34.2) for MG. Among regions, Europe and Central Asia had the lowest occurrence for both pathogens, while MG and MS were highly prevalent in South Asia and sub-Saharan Africa, respectively. At the national level, MG occurrence was higher in Algeria, Saudi Arabia and Sudan, whereas China, Egypt and Ethiopia reported higher values of MS. Among the poultry subpopulations, MS and MG were more prevalent in the breeders and layers (62.6% and 31.2%, respectively) than in diverse poultry. The year of publication, the sample size and the level of ambient air pollution (measured indirectly by PM2.5) were associated with the occurrence of both mycoplasmas. Our study revealed high and heterogeneous occurrence values of MG and MS and justifies the need for early detection and improved control measures to reduce the spread of these pathogens.

Prevalence, main serovars and anti-microbial resistance profiles of non-typhoidal Salmonella in poultry samples from the Americas: A systematic review and meta-analysis.

Poultry and poultry-derived products such as meat and eggs are among the main sources of non-typhoidal Salmonella (NTS) transmission to humans. Therefore, we performed a systematic review and used random-effects meta-analyses to (1) estimate the prevalence of NTS in poultry samples from birds, products and subproducts and environmental samples, (2) examine the diversity and frequency of their serovars and (3) estimate the prevalence and profiles of anti-microbial resistance (AMR) in NTS isolates reported in studies from the Americas. We included 157 studies from 15 countries comprising 261,408 poultry samples and estimated an overall pooled prevalence of 17.9% (95% Confidence Interval: 10.8-26.3) in birds, 21.8% (17.7-26.1) in products and subproducts and 29.5% (24.2-35.1) in environmental samples. At the national level, the prevalence of NTS was heterogeneous across countries with the highest values in Mexico, the United States and Canada. In total, 131 serovars were identified from 13,388 isolates; Heidelberg, Kentucky, Enteritidis and Typhimurium were the most prevalent in the overall top 10 ranking (range 6.5%-20.8%). At the national level, Enteritidis and Typhimurium were identified in most of the countries, though with national differences in their ranks. The prevalence of AMR increased from 24.1% for 1 antibiotic to 36.2% for 2-3 antibiotics and 49.6% for ≥ 4 antibiotics. Kentucky, Heidelberg, Typhimurium and Enteritidis were the serovars with the highest prevalence of AMR. Besides, tetracycline, ampicillin, streptomycin, ceftiofur and amoxicillin-clavulanic acid were the most frequent antibiotics to which NTS showed resistance. In conclusion, NTS was distributed through the avian production chain with high and heterogeneous values of prevalence in poultry samples. Besides, there were distinctive patterns of serovars distribution across countries and an alarming prevalence of AMR among zoonotic serovars.

Systematic review and meta-analysis of the efficacy of reproductive management practices used to induce resumption of ovarian cyclical activity in anestrous does.

Reproductive management practices that use hormones, sociosexual biostimulation, nutritional management, or abiotic factors are used to induce the resumption of reproduction in anestrous does. However, their overall efficacy remains uncertain; therefore, the identification of evidence-based management recommendations to manipulate anestrous in goats is important. Electronic databases were searched to retrieve reports on studies using interventions based on hormonal, sociosexual, nutritional, and abiotic factors. Only experimental studies in which a group of anestrous does was treated and compared against an untreated group were included. Estrus, ovulation, and pregnancy were primary outcomes, whereas the onset of estrus after treatment, the ovulation rate, and the number of anovulatory days were secondary outcomes. Odds ratio (OR) and mean differences were used to synthesize pooled data, and random effects models were used to calculate them. Seventy studies involving 3974 goats met the inclusion criteria. Unclear risk of bias for random sequence generation and allocation concealment predominated across studies. Pooled data for hormonal, sociosexual, and abiotic interventions showed a significant, though variable, increase in estrus (OR range 7.15-144.80), ovulation (OR range 6.08-56.95), and pregnancy (OR range 3.94-30.8). Hormonal treatments significantly reduced the onset of estrus, whereas abiotic interventions failed to reduce the number of anovulatory days. Secondary outcomes were not assessed in trials using sociosexual approaches. Finally, except for pregnancy, no significant efficacy was observed for studies using nutritional management. In conclusion, reproductive management practices using sociosexual approaches showed the highest efficacy for restoring reproductive activity in anestrous does.

Impact of heat stress on the reproductive performance and physiology of ewes: a systematic review and meta-analyses.

High values of ambient temperature and relative humidity negatively affect livestock because they induce heat stress. In heat-stressed ewes, the reproduction is affected, although there apparently is a different effect depending on their physiological status. Therefore, we conducted a systematic review and meta-analyses to estimate the effect of heat stress on the reproductive performance and physiology of cycling or pregnant ewes. We included studies in which pregnant or cycling ewes were exposed to heat stress and included a comparison group. We assessed the risk of bias and used meta-analyses that included the period of exposure to calculate pooled estimates. We included 36 studies comprising 20,626 ewes, and the meta-analyses demonstrated that heat stress decreased 7.09 h (95% CI, - 10.75, - 3.43) the duration of estrus in cycling ewes but increased 0.57 days (95% CI, 0.23, 0.92) the length of the cycle, although differentially affecting the expression of sexual behavior. Heat-stressed cycling ewes had greater odds of embryo mortality (OR = 12.36; 95% CI, 7.10, 21.54) and a decreased chance of impregnation (OR = 0.41; 95% CI, 0.23, 0.75). In heat-stressed pregnant ewes, placental and fetal weights were reduced, respectively in 183.62 g (95%CI, - 252.25, - 114.99) and 1665.18 g (95% CI, - 2136.82, - 1193.55). Heat stress increased ewes' body temperature 1.13 °C (95% CI, 0.85, 1.42) and respiratory rate 93.53 breaths/min (95% CI, 63.5, 123.5) without affecting heart rate. In a sub-group analyses, the period of exposure was shown to cause a significant effect. In conclusion, our results revealed a differential effect of heat stress that depends on both the physiological status of ewes and the period of exposure, and identify susceptible reproductive stages during which the severe effects of heat stress can be prevented/ameliorated.

Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production.

Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

Differences in body temperature, cell viability, and HSP-70 concentrations between Pelibuey and Suffolk sheep under heat stress.

Pelibuey and Suffolk sheep were compared as to their capacity to regulate body temperature under environmental hyperthermia by measuring their differences in cellular response to heat stress (HS). In a first experiment, seven Pelibuey and seven Suffolk ewes were kept in a climatic chamber for 6 h daily during 10 days (temperatures within the 18 to 39.5 °C range). As chamber temperature rose, sheep rectal temperature increased in both groups, but to a lesser extent in Pelibuey (0.3 °C) than in Suffolk sheep (0.7 °C) (P < 0.05). In a second experiment, cellular viability was assessed using cultured blood mononuclear cells from 15 Pelibuey and 15 Suffolk sheep. They were incubated at 37 °C for 24 h (control) or 43 °C for 6 h followed by 18 h at 37 °C (HS). In a third experiment, another blood mononuclear cells culture from eight Pelibuey and eight Suffolk sheep was kept at 37 °C for 15 h; these were subsequently cultured for 6 h at 37 °C (controls) or 43 °C (HS). Next, HSP-70 concentration was determined. HS reduced the percentage of viable cells to a greater extent in Suffolk [37 °C (73.7 %) vs. 43 °C (61.9 %); P < 0.05] than in Pelibuey sheep [37 °C (74.9 %) vs. 43 °C (66.7 %); P > 0.05]. HS significantly increased HSP-70 average concentrations for both breeds at 43 °C. A significant effect was observed for the breed by temperature interaction (P < 0.05) caused by a greater difference between Pelibuey and Suffolk at 43 °C (2.85 vs. 0.53 ng/mL, respectively; P < 0.05) than at 37 °C (0.05 vs. 0.03 ng/mL, respectively; P > 0.05). In conclusion, Pelibuey sheep show more effective body temperature regulation under conditions of environmental hyperthermia. Also, cell viability after HS was higher in Pelibuey than in Suffolk, an effect that could be mediated by an HSP-70-related mechanism.