Novel genotypes of the subtropical grass Eragrostis curvula for the study of apomixis (diplospory)

The aim of this work was to obtain a series of genetically related lines of Eragrostis curvula with different ploidy levels and reproductive modes, which can be used for the discovery of genes associated to diplospory. E. curvula is a widely cultivated forage grass native to Southern Africa that reproduces naturally by obligate diplosporous apomixis (asexual reproduction by seeds) of the Antennaria type. In vitro culture of immature inflorescences of the apomictic cultivar Tanganyika led to the regeneration of a diploid plant with a particular morphology (plant UNST1122). Embryo sac analysis and progeny tests determined that plant UNST1122 reproduces by sexuality. UNST1122 was cloned by dividing its tillers and a R ₁ generation was obtained from seeds after open pollination among the diploid clones. A set of 500 seeds from a diploid R ₁ plant was treated with colchicine and two plants showing a duplicated chromosome number (2n = 4x = 40) were obtained (plants UNST1112 and UNST1131). Progeny tests using RAPDs indicated that these plants reproduce also by sexuality. The availability of sexual tetraploid genotypes of E. curvula will allow the production of hybrids with new interesting combinations of agronomic traits and the developing of mapping populations segregating for diplospory. Besides, these genetically related plants with different reproduction modes constitutes an excellent model for the identification of gen(es) involved in diplosporous apomixis by mRNA profiling as well as to study the genomic rearrangements and gene expression alterations associated to changes at ploidy levels.     

Bovine thyroglobulin gene polymorphisms and their association with sexual precocity in Guzerat bulls

Puberty is a stage of sexual development determined by the interaction of environmental factors and genetic mechanisms. Among them, thyroid function plays a key role in sexual development and spermatogenic function and is under the control of several genes, including the well‐described thyroglobulin gene (TG). Previous reports have shown genetic association between thyroid function and selected single nucleotide polymorphisms (SNPs) in taurine cattle. Therefore, the identification of genetic mechanisms involved in the regulation of this trait can assist with the selection for early pubertal bulls, thus improving genetic progress in livestock breeding. The aim of this study was to validate the association between TG SNPs and age at puberty in zebuine bulls. Three SNPs (rs110406764, rs109662686, rs109057985) were genotyped in 159 Guzerat animals using SEQUENOM technology. Results showed a significant association (p < .05) between the studied SNPs and puberty age, in agreement with our previous reports in a taurine breed. Interestingly, allele frequencies were different from those already reported, being GAT the most favourable allele for age at puberty in Guzerat (94.4 days lower). Overall, our findings corroborate previous reports and reinforce the importance of genetic influence in the regulation of sexual development and puberty through a thyroid pathway in zebuine cattle.     

Behavioural and EEG changes in sheep in response to painful acute electrical stimuli

Objective To measure acute pain in sheep, based on a human pain model, and examined changes in both electroencephalogram frequency spectrum and behavioural responses to increased electrical stimulation in sheep. Design Analysis of variance (treatment and animal effects) for stimulus intensity where each animal received each electric shock treatment given in the order 0, 5,10 and 20 mA. Procedure Eight sheep with electrodes implanted over the surface of the brain were examined for escape-avoidance and electroencephalogram responses to four levels of electrical stimulation from 0–20 mA. Results With increasing stimulus intensity at the time of feeding, the sheep were more hesitant to return to the feeder or remain near the feeder following stimulation. There was little difference between the 0 and 5 mA stimuli for any of the behaviour variables (P > 0.05). However, there were marked increases in the time taken to re-approach the feeder after receiving an electric shock of 5 mA and of 20 mA (P < 0.05; mean values 3 and 119 s, respectively) and remaining near the feeder for 5 s (P < 0.001; mean values 10 and 167 s, respectively). Following the stimulus, there was an overall increase in the electroencephalogram power spectrum in the first four seconds, which then rapidly returned to normal. In particular, the 20 mA stimulus resulted in higher absolute power values than in the control (0 mA) treatment for delta 2 (P < 0.001), theta 1 (P < 0.05), theta 2 (P < 0.05), alpha 1 (P < 0.001), alpha 2 (P < 0.001) and beta 1 (P < 0.01) band-widths. Similarly, for the 10 mA stimulus, the absolute power values were greater than the control treatment for delta 2 (P < 0.05), alpha 1 (P < 0.01), alpha 2 (P < 0.001) and beta 1 (P < 0.01) bandwidths. Conclusion The experiment suggests that a human acute pain model is applicable to sheep and that these electroencephalogram changes may provide a good measure of acute pain in sheep.     

Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus

Objective To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter nodosus (strain A).
Procedure Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded.
Results The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects.
Conclusion Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.     

Absorption Kinetics of Nitrogen Oxides using Sodium Chlorite Solutions in Twin Spray Columns

NO _X is a major pollutant that causes acid deposition and photochemical smog. A large amount of NO _X is emitted from combustion sources such as power plants. As some articles have indicated, NO _X removal with NaClO₂ solution in an absorption column is an effective control approach. In this approach, first insoluble NO is oxidized into water-soluble NO₂ under acidic conditions, then NO₂ is removed in alkaline NaClO₂ solutions. The results indicate that the reaction rate constant is 9.1×10⁴ (L)^4.4/cm⁶/s/mol^2.4 in the first absorption column with acidic conditions, and the reaction orders with respect to NO and NaClO₂ are 1.4 and 2, respectively. In the second absorption column with alkaline conditions, the reaction rate constant is 3.2×10⁷ (L)^5.2/cm⁶/s/mol^3.2 and the reaction orders with respect to NO and NaClO₂ are 1.6 and 2.5, respectively. The activation energies in the first and second absorption column are 71.8 and 139.6 kJ/mol, respectively.     

Effects of tramadol,morphine or their combination in dogs undergoing ovariohysterectomy on peri-operative electroencephalographic responses and post-operative pain

Abstract

AIM: To compare the peri-operative electroencephalogram (EEG) responses and post-operative analgesic efficacy of pre-operative morphine or tramadol with a combination of low-dose pre-operative morphine and post-operative tramadol, in dogs undergoing ovariohysterectomy.

METHODS: Dogs undergoing routine ovariohysterectomy were treated with either pre-operative morphine (0.5 mg/kg S/C, n=8), or tramadol (3 mg/kg S/C, n=8), or pre-operative low-dose morphine (0.1 mg/kg S/C) and post-operative tramadol (3 mg/kg I/V, n=8). All dogs received routine pre-anaesthetic medication, and anaesthesia was induced with I/V thiopentone to effect and maintained with halothane in oxygen. Respiratory rate, heart rate, end-tidal halothane tension (EtHal) and end-tidal CO₂ tension (EtCO₂) were monitored throughout surgery. The EEG was recorded continuously in a three electrode montage. Median frequency (F50), total power (Ptot) and 95% spectral edge frequency (F95) of the EEG power spectra were compared during different 100-second periods of surgery: prior to and during skin incision, ligation of each ovarian pedicle, ligation of uterine body and skin closure. Post-operatively, pain was assessed using the short form of the Glasgow composite measure pain scale (CMPS-SF).

RESULTS: There was no difference in F50 or Ptot of the EEG between baseline and noxious surgical events within each treatment group, or between the three groups (p>0.05). The mean F95 was higher during the first three periods of surgery for dogs administered tramadol and low-dose morphine than those that received 0.5 mg/kg morphine (p=0.001). Dogs that received low-dose morphine and tramadol had lower CMPS-SF pain scores after ovariohysterectomy than those that received either tramadol or morphine alone (p=0.001). There was no difference in pain scores between dogs in the latter two groups.

CONCLUSION AND CLINICAL RELEVANCE: Tramadol and morphine administered pre-operatively provided an equal degree of post-operative analgesia in dogs after ovariohysterectomy. A combination of pre-operative low-dose morphine and post-operative tramadol produced better post-operative analgesia than either drug administered alone pre-operatively. Administration of analgesics pre- and post-operatively could result in improved post-operative well-being of ovariohysterectomised dogs.     

Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen

Twenty‐four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4–8 years were used to assess various attributes of spermatozoa influencing the zona‐binding and zona‐penetration tests. Ejaculates from each bulls were subjected to in vitro sperm‐‐zona binding and sperm‐‐zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 ± 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 ± 0.64. The average number of zona‐bound and ‐penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin‐‐nigrosin stain and hypo‐osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 ± 2.32) was significantly (p < 0.05) higher than hypo‐osmotic swelling‐Giemsa (HOS‐G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 ± 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin‐‐nigrosin (r = 0.85, p < 0.05). The HOS‐G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin‐‐nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS‐G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.