Chitosan nanoparticles enhance developmental competence of in vitro-matured porcine oocytes

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus–oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25 and 50 µg/ml) during in vitro maturation (IVM). Oocytes treated with 25 µg/ml CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs10 and CNPs50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25-treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25-treated group was significantly higher than that in the CNPs50-treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25-treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25 µg/ml CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.     

Studies on immunocompetence status in two turkey varieties in India

(1) Two hundred and twenty-seven adult turkeys of both sexes, of two varieties (104 Black and 123 White) were used to evaluate their immunocompetence status and body weights. (2) Response to sheep red blood cells (SRBC) (humoral immunity) was measured by Haemagglutination (HA) test 5 days post immunisation (dpi) and expressed as log2 values. Mercaptoethanol resistant (MER) antibodies representing IgG were determined by Mercaptoethanol HA test and Mercaptoethanol sensitive (MES) antibodies, representing IgM as the difference in total HA titre and IgG. Serum lysozyme concentrations were estimated by 'Lysoplate assay' and expressed in log2 values. (3) Least squares analysis of variance revealed that the White variety had higher adult body weight (4.788 +/- 0.040 kg) than the Black (3.774 +/- 0.044 kg). Sexual dimorphism was apparent and meals were heavier than females in both varieties. The interaction effect of variety and sex on body weight was also significant. (4) Least squares means for immunological traits, namely, total anti-SRBC antibodies, MER, MES titres and serum lysozyme were 7.161 +/- 0.189, 0.801 +/- 0.071, 6.362 +/- 0.160 and 1.766 +/- 0.043 microg/ml, respectively. The Black variety had a higher MES antibody titre than the White. (5) Sex had an effect on all the immunological traits except on MER titres. Females generally had higher anti-SRBC, MER and MES titres and serum lysozyme. The variety x sex interaction effect was significant for MES titres and serum lysozyme. White males had the lowest MES titres.     

Association of bovine KISS1 single nucleotide polymorphisms with reproductive traits in Indian Cattle

Kisspeptins, a family of neuropeptide encoded by the Kiss1 gene, have emerged as crucial regulator of fertility and reproduction by regulating the hypothalamic–pituitary–gonadal axis. The present study was aimed to identify and associate SNPs in the KISS1 gene with reproductive traits in cattle of Indian origin. DNA samples collected from 300 individual cows of three Indian dairy breeds (Gir, Kankrej and Frieswal) of cattle were used in the study. The SNPs of KISS1 gene were identified with PCR-RFLP and sequence analysis using two sets of primer pairs. A total of 5 SNPs were identified in the targeted region of which, two were selected for screening the population and association studies. The analysis revealed that genotypes of rs442633552G>A and rs42022871C>T had a significant association with dry period. The SNP rs42022871C>T also established significant role in milk production traits, and selection of TT-genotyped animals will improve the reproduction and production potential of the animals.     

Identification of immunoreactive polypeptides of Babesia equi parasite during immunization

This study was carried out to identify immunoreactive polypeptides in Babesia equi merozoite antigen. Three fractions of killed B. equi merozoite antigen viz.; whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS) antigens were prepared from the parasite infected erythrocytes. These antigenic preparations along with ghost antigen from non-infected erythrocytes were fractionated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with sera showing high antibody titres. On SDS-PAGE, 16 polypeptides with molecular weight (Mr) in the range of 112-17kDa were obtained from the WM and CM antigens. But only six polypeptides were detected (96.5-28kDa) in the HSS antigen. On immunoblotting with high titred serum collected from donkeys following two immunizations with a killed B. equi merozoite immunogen, 11 polypeptides were observed in the WM and CM antigens (Mr 112-18kDa). Of these, four polypeptides (Mr 112, 45, 33 and 18kDa) were identified as most immunoreactive. Besides these, a 28kDa was observed as strong immunoreactive protein in WM and CM antigens. The HSS antigen showed only six polypeptides and one peptide (28kDa) was identified as immunoreactive. When high titred serum collected from immunized donkeys following challenge with B. equi infected blood and was used for immunoblotting, the protein profile of WM and CM antigens remained the same. However, three additional polypeptides (Mr 81, 54.5 and 39kDa) were detected in HSS antigen.     

Temporal changes in endogenous estrogens and expression of behaviors associated with estrus during the periovulaory period in Murrah buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The objective of this study were (1) to establish the duration of behavioral estrus signs and timing of ovulation in Murrah buffaloes (n = 10) and (2) to determine relationship between behavioral estrus signs with change in plasma estrogen concentrations. Estrus and its behavioral signs were detected at hourly intervals by visual observations, per recta examination of genitalia and bull parading four times in a day for 30 minutes each. Among the behavioral signs of estrus, swollen vulva (80%) was the best indicator of estrus followed by excitement (70%). Among the duration of behavioral estrus signs the first and longest duration of estrus signs was swollen vulva which was seen upto 19.8 ± 0.8 h after onset of estrus. The mean total duration of estrus symptoms from appearance to disappearance of all the behavioral estrus symptoms was 23.5 ± 1.7 h. All the behavioral estrus symptoms were observed during the period of estrogen surge. Endocrine profile during the periestrus period showed that the mean peak concentrations of total estrogen 23.9 ± 3.9 pg/ml occurred at 8.8 ± 1.7 h after onset of estrus. The average number of estrus symptoms observed per animal during onset of spontaneous estrus was 5.7. Ovulation occurred after 37.4 ± 1.7 h after onset of estrus and 13.4 ± 1.0 h after end of total estrogen surge respectively. In conclusion, our results suggest that all signs of behavioral estrus occurred during the preovulatory rise in estrogens. The first sign of estrus to be observed was a swollen vulva and this symptom persisted the longest.     

Experimental avian paramyxovirus serotype-3 infection in chickens and turkeys

Avian paramyxoviruses (APMV) are divided into nine serotypes. Newcastle disease virus (APMV-1) is the most extensively characterized, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two divergent strains of APMV-3, Netherlands and Wisconsin, in (i) 9-day-old embryonated chicken eggs, (ii) 1-day-old specific pathogen free (SPF) chicks and turkeys, and (iii) 2-week-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was 112 h for APMV-3 strain Netherlands and > 168 h for strain Wisconsin. The intracerebral pathogenicity index in 1-day-old chicks for strain Netherlands was 0.39 and for strain Wisconsin was zero. Thus, both strains are lentogenic. Both the strains replicated well in brain tissue when inoculated intracerebrally in 1-day-old SPF chicks, but without causing death. Mild respiratory disease signs were observed in 1-day-old chickens and turkeys when inoculated through oculonasal route with either strain. There were no overt signs of illness in 2-weeks-old chickens and turkeys by either strain, although all the birds seroconverted after infection. The viruses were isolated predominantly from brain, lungs, spleens, trachea, pancreas and kidney. Immunohistochemistry studies also showed the presence of large amount of viral antigens in both epithelial and sub-epithelial lining of respiratory and alimentary tracts. Our result suggests systemic spread of APMV-3 even though the viral fusion glycoprotein does not contain the canonical furin proteases cleavage site. Furthermore, there was little or no disease despite systemic viral spread and abundant viral replication in all the tissues tested.     

Larval and pupal toxicity effects of Plectranthus amboinicus, Bacillus sphaericus and predatory copepods for the control of the dengue vector, Aedes aegypti

The efficacy of copepod Mesocyclops aspericornis (Daday) combined with the larvicide Bacillus sphaericus (Bs) and a plant extract of Plectranthus amboinicus leaf abstract (PALE), used jointly and singly, was studied against Aedes aegypti in the laboratory. P. amboinicus leaf extract of 20, 40, 60, 80 and 100 ppm caused significant mortality against Ae. aegypti larvae. The LC₅₀ and LC₉₀ values for I to IV instars larvae and pupae were 26.12, 35.36, 45.76, 52.32 and 63.82 ppm, respectively. The LC₉₀ values for I to IV instars larvae and pupae were 82.53, 92.65, 108.06, 119.47 and 131.71 ppm, respectively. Under laboratory conditions, copepods treatment produced 7.9% predatory efficiency against 1st instar larvae of Ae. aegypti, at a copepod:larvae ratio of 1:10. When copepod treatment was combined with PALE this was increased to 8.7. The treatment of copepods combined with Bs and PALE yielded a better and more sustainable result (9.6%) than the agents used individually. This predation efficiency may be caused by detrimental effects of the P. amboinicus active principle compound (carvacrol) on the mosquito larvae. Our results suggest that the combined application of microbial insecticide (Bs), copepods and P. amboinicus leaf extract may be used to control Aedes populations.