Agrobacterium-mediated Cry1A(b) gene transfer in Punica granatum L. cv. Kandhari Kabuli using different In Vitro regeneration pathways

Three different regeneration systems, viz. regeneration through callus cultures using embryonic explant, direct regeneration using shoot bud/nodal segments as explant and regeneration through cell suspension culture using cotyledonary explant (for the induction of transgenic callus for suspension culture) were evaluated to see their effect on transfer of Cry1A(b) gene to Punica granatum L. cv. Kandhari Kabuli through Agrobacterium mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency of different explants. Out of different explants used (embryo, shoot bud, and cotyledon) for different regeneration systems cotyledonary explant showed highest putative transformation frequency (13.54%) inducing callus on selective medium for carrying out cell suspension culture to regenerate transgenic shoots. Despite of the highest transformation frequency obtained from the cotyledon explant, the plating efficiency of the transgenic cells generated through the transgenic callus (callus formed from the cotyledonary explant) during cell suspension culture was found to be very low (0.7%). Thus the plating efficiency has also played worth mentioning role in the regeneration of transformants following cell suspension culture. Among the three regeneration systems, regeneration through callus cultures using embryonic explant was found to be best for regeneration of transformants. The highest per cent regeneration of 23.33 was obtained from the putative transgenic embrogenic calli. Successful genetic transformation in the transformed plantlets was confirmed by PCR analysis. The transformation system thus developed is valuable and may be used to produce insect resistant trees.     

Linseed oil in boar's diet improved in vivo fertility and antioxidant status

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0 h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography–mass spectrometry (GC–MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0 h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.     

Exogenous cholesterol prevents cryocapacitation-like changes,membrane fluidity,and enhances in vitro fertility in bubaline spermatozoa

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10⁶ spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca²⁺, capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca²⁺ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10⁶ spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10⁶ spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10⁶ spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca²⁺); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca²⁺, low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca²⁺, medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10⁶ spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing–thawing-associated damages in bubaline species.