Cryopreservation Of Nili-Ravi Buffalo Bull Sperm in Cryodiluent Supplemented with Lolium perenne Protein Preparations.

BACKGROUND: Semen from the Nili-Ravi buffalo bull, Bubalus bubalis, shows poor survival after freeze storage compared to bovine (Bos taurus and Bos indicus) semen. Freeze-susceptibility distinctions in these two genera have been attributed to differences in sperm membranes. MATERIALS AND METHODS: We measured the impact of protein preparations derived from a frost-resistant perennial grass, Lolium perenne, with ice recrystallization inhibition activity on the low temperature storage of B. bubalis semen. RESULTS: When the L. perenne preparations (0.1, 1, 10 µg/mL) were added to buffalo semen [2 ejaculates per bull (N=3) per replicate (r=3)] in Tris-citrate extender (50×106sperm mL-1), there was no impact on semen quality, as measured by sperm motility and plasma membrane integrity, after storage at 4 degree C (P>0.05). However, when semen supplemented with the grass proteins (0.1 and 1 µg mL-1) was evaluated after freezing and storage in liquid nitrogen for 24 h, post-thaw sperm progressive motility and plasma membrane integrity was higher (P<0.05) than in control samples. Post-thaw sperm viability and sperm acrosome integrity was similar (P > 0.05) to controls. CONCLUSION: The improvement in cryopreserved buffalo sperm progressive motility and plasma membrane integrity suggests that the use of these easily-made preparations may improve fertility after cryopreservation and offers the prospect of improved conception rates after artificial insemination with cryopreservation.

A novel extracellular glycosidase activity from Rhodotorula mucilaginosa: its application potential in wine aroma enhancement.

UNLABELLED: The aim of the work was to evaluate the application potential of a glycosidase extract of one indigenous non-Saccharomyces strain in wine aroma enhancement. The isolate was selected from a local winemaking region in China for its high ß-glucosidase level and was identified as Rhodotorula mucilaginosa. The tolerance of the glycosidase extract to the typical winemaking conditions was assessed using the activity of its ß-glucosidase. After that, the hydrolysis capacity of R. mucilaginosa glycosidase for liberation of grape aroma glycosides was characterized in comparison to commercial enzyme preparations. Results of this work revealed that glycosidase extract from R. mucilaginosa proved to be active in the presence of 0-20% (w/v) glucose, 0-20% (v/v) ethanol and at pH 3·0-5·0. In the hydrolysis of aroma precursors, enzymes obtained from different origins possessed various levels of specificity and activity, showing high origin dependence (α = 0·05). Compared to commercial enzymes, the indigenous R. mucilaginosa glycosidase extract presented better catalytic preference for the 'fruity and floral' glycosides of benzenic compounds and C13 -norisoprenoids, but less sensitivity to the glycosides of C6 compounds and volatile phenols. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents a novel extracellular glycosidase preparation from an indigenous Rhodotorula mucilaginosa strain selected from a local winemaking region in China. This enzyme extract exhibits strong tolerance towards winemaking conditions. It shows hydrolysis specificity for glycosides of benzenic compounds and C13 -norisoprenoids, proving a potential candidate for improving floral and fruity aroma characteristics of wine.

Arachidic acid in extender improves post-thaw parameters of cryopreserved Nili-Ravi buffalo bull semen.

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post-thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post-thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili-Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10(6) spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5-ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose-dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome-intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome-intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post-thaw quality parameters of cryopreserved Nili-Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post-thaw semen quality parameters was observed at 20.0 ng/ml.

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa.

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.

Size at sexual maturity estimation for 36 species captured by bottom and mid-water trawls from the marine habitat of Balochistan and Sindh in the Arabian Sea, Pakistan, using maximum length (Lmax) and logistic (L50) models.

The aim of this study was to estimate the size at first sexual maturity (Lm) for 36 species belonging to 24 families from the marine habitat of Balochistan and Sindh in Arabian Sea, Pakistan through maximum length based an empirical and logistic model using commercial catch during June 2020 to May 2021. Individual total length (TL) was noted up to 0.1 cm using measuring broad. The Lm was calculated using two formulae; (i) log (Lm) = - 0.1246 + 0.9924 * log (Lmax) for Elasmobranchs and (ii) log (Lm) = -0.1189 + 0.9157* log (Lmax) for ray-finned fishes. The minimum Lm was recorded as 10.27 cm TL for Caranx malabaricus and 108.38 cm TL for Isurus oxyrinchus, respectively. Around Lm with 58.33% species were ranges from 19.00 cm to 25.00 cm TL. This study was estimated 16 newly Lm which is globally absent and rest 20 Lm are absent in the Arabian Sea (Pakistan coastal habitats). Therefore, the results will be helpful for the sustainable management and conservation of these marine fishes through the establishment of mesh size of trawl nets based on the size at sexual maturity (Lm).

siRNA-mediated GABA(B) receptor at early fetal rat brain upon acute and chronic ethanol exposure: down regulation of PKA and p-CREB expression.

To observe the modulatory role of GABA(B1)R upon ethanol's effect during early brain development, we studied the effects of chronic maternal (10% ethanol during pregnancy) and acute (in vitro) ethanol exposure on the neuronal protein kinase A (PKA-α) and phosphorylation of cAMP-response element binding protein (p-CREB), using a system where GABA(B1)R were specifically knocked down in the primary cells cultured at gestational day (GD) 12.5. The results showed that upon acute and chronic ethanol treatment the GABA(B1)R expression was decreased and further decreased when GABA(B1)R was transfection with siRNA, while increased upon exposure of baclofen, and baclofen plus phaclofen treatment. PKA expression was also decreased with acute and chronic ethanol treatment, whereas it showed increase upon exposure of baclofen and baclofen with phaclofen. Furthermore, intracellular Ca(2+) concentration was increased upon ethanol, baclofen, phaclofen exposure but showed decrease in GABA(B1)R siRNA group. Finally, these effects could lead to changes of p-CREB expression, which showed same expression pattern as PKA. We speculate that GABA(B)R activity upon ethanol exposure could modulate intracellular calcium homeostasis and the expressional changes of PKA and p-CREB, which cause various negative effects on fetal brain development and modulation of GABA(B)R upon ethanol exposure may underlying cause of ethanol's effects.

Vitamin C protects against ethanol and PTZ-induced apoptotic neurodegeneration in prenatal rat hippocampal neurons.

Exposure to alcohol during brain development may cause a neurological syndrome called fetal alcohol syndrome, characterized by pre- and postnatal growth deficiencies, craniofacial anomalies, and evidence of CNS dysfunction. The objective of this study was to evaluate pentylenetetrazol (PTZ) and ethanol effects on Bax, Bcl-2 expression, which further induced activation of caspase-3, release of cytochrome-c from mitochondria, and to observe the protective effects of vitamin C (vit-C) against PTZ and ethanol-induced apoptotic neurodegeneration in primary-cultured neuronal cells at gestational day 17.5. Apoptotic neurodegeneration and neuroprotective effect of vit-C were measured by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay, Western blot analysis, which further conformed by the measurement of mitochondrial membrane potential using JC-1 detection kit and immunofluorescence analysis. The results showed that PTZ and ethanol produced extensive Bax-dependent caspase-9 and caspase-3 activation and caused neuronal apoptosis. Furthermore, the cotreatment of vit-C along with ethanol and PTZ showed significantly decreased expression of Bax, caspase-9, caspase-3, cytochrome-c, and significantly increased expression of antiapoptotic Bcl-2 protein when compared with control group. Our findings indicate that PTZ and ethanol activate an intrinsic apoptotic death program in neurons that is likely to contribute to the neuropathologic effects in fetal alcohol exposure, and vit-C can prevent some of the deleterious effects of PTZ and ethanol on the developing brain. The available experimental evidence and the safety of vit-C in pregnancy suggest the experimental use of ascorbic acid as a new and effective protective agent ethanol and PTZ-induced apoptotic neurodegeneration.

In vitro evaluation of liquid-stored buffalo semen at 5°C diluted in soya lecithin based extender (Bioxcell®), tris-citric egg yolk, skim milk and egg yolk-citrate extenders.

This study was designed to compare the quality of liquid-stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell(®) (BIOX), milk (MILK), tris-citric egg yolk (TEY) and egg yolk-citrate (EYC) extender at 5°C. Semen was collected from five Nili-Ravi buffalo (Bubalus bubalis) bulls of 6-7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10(6) motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX(®) extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p<0.05) in BIOX(®) compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell(®) were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell(®) , milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell(®) were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.

Serological prevalence of hepatitis B virus (HBV) in Mardan district, Khyber Pakhtunkhwa, Pakistan.

Hepatitis B virus infection is perilous among the five types of Hepatitis, as it remains clinically asymptomatic. The present study draws up-to-date prevalence of Hepatitis B virus (HBV) in the general population of Mardan, Khyber Pakhtunkhwa Pakistan. The blood samples from 4803 individuals including 2399 male and 2404 females were investigated. All the suspected samples were analyzed for hepatitis B surface antigen using Immuno-chromatographic test (ICT), Enzyme-linked immunosorbent assay (ELISA), and followed by Reverse transcription-polymerase chain reaction (RT-PCR). Results showed that 312 (13.00%) out of 2399 individuals contained antibodies in their blood against HBV, while among the different age groups, the highest incidences of HBV antibodies were found in the age of 21-30 groups (10.73%). Furthermore, the ICT positive samples were screened by nested polymerase chain reaction to detect the existence of active HBV-DNA. It was observed that 169 (7.04%) out of (2399) male of the total population (4803) tested was positive. On the other hand, the female 463 (19.25%) possessed antibodies in their blood against HBV. Accumulatively, our results showed a higher percentage of HBV prevalence in males than females in the age group 21-30 years. The total HCV infected in Mardan general population was recorded at 5.7% comprising both male and female.

Comparative brain analysis of wild and hatchery reared Mahseer (Tor putitora) relative to their body weight and length.

The present study was aimed at comparing the brain size of mahseer (Tor putitora) in relation to their body weight and standard length, to investigate the potential impact of rearing environment on brain development in fish. The weight of the brain and three of its subdivisions cerebellum (CB), optic tectum (OT), and telencephalon (TC) were measured for both wild and hatchery-reared fish. The data was analysed using multiple analysis of covariance (MANCOVA), analysis of covariance (ANCOVA), and discriminate function analysis (DFA). We found the fish reared under hatchery conditions exhibit smaller brain size related to body weight, when compared to the wild ones. A significant (p<0.5) difference was observed in the length of CB and OT concerning the standard body length while no significant difference was found in TC of the fish from both the origins. The results of the current study highlight a logical assumption that neural deficiency affects the behaviour of fish, that's why the captive-reared fish show maladaptive response and face fitness decline when released to the natural environment for wild stock enhancement. The current study concluded that hatchery-reared fish exhibit variations in gross brain morphology as compared to their wild counterpart.

Prevalence of active HCV infection and genotypic distribution among the general population of district Mardan, Pakistan.

Hepatitis C virus (HCV) is the serious global public health burden of liver disease. Approximately 170 million people in the world are infected with (HCV). In Pakistan, where the disease has high occurrence rate. The present study envisages an up-to-date prevalence of HCV and genotypic distribution in the general population of Mardan District, Khyber Pakhtunkhwa (KP), Pakistan. The blood samples from 6,538 individuals including 3,263 males and 3,275 females were analyzed for hepatitis C surface antigen by Immuno-chromatographic test (ICT), Enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (PCR). It was found that 396 (12.13%) out of 3263 individuals contained antibodies in their blood against HCV, while among the different age groups, the highest incidences of HCV antibodies were found in the 31-40 age group (11.01%). The ICT positive samples were further screened by nested PCR to determine the existence of active HCV-RNA. It was identified that 7.11% (3263) of the total population (6538) tested was positive, among which the 461 (14.07%) females possessed antibodies in their blood against HCV. Our data showed total HCV infection in the investigated population was 5.78%. Higher percentage of HCV prevalence was detected in males than females in the age group 31-40 and 41-50. To compare the prevalence of HCV genotypes age-wise in male and female genotype 3a was found most prevalent genotype followed by 1a, 2a and 3b, respectively.

Expression analysis of transcription factors in sugarcane during cold stress.

Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Bioaccumulation of heavy metals in the tissues of Schizothorax plagiostomus at River Swat.

Snow trout (Schizothorax plagiostomus) is an economically important freshwater fish, mostly found in northern areas of water reservoirs of Pakistan. The current study was conducted in River Swat to analyze the bioaccumulation of heavy metals (Pb, Cr, Ni, and Zn) in tissues of Schizothorax plagiostomus. Tissues were extracted and dissolved in perchloric acid (HClO4) and nitric acid (HNO3) along with hotplate. The heavy metals, zinc (Zn), lead (Pb), chromium (Cr), and Nickel (Ni) were determined using Perkin Elmer 2380 atomic absorption spectrophotometer. Results shows great variation in the content of the metal related to tissue type and sampling sites. A high concentration of bioaccumulation was reported at Charbagh, whereas lowest at Odigram: Charbagh>Landakai>Odigram. In the same way, Cr was the most accumulated heavy metal followed by lead, nickel, and Zinc.

Episode of Hepatitis C viral infection in the people of Swat, Pakistan.

Infectious agents cause serious diseases in humans worldwide and are responsible for the high rate of morbidity and mortality. The prevalence and epidemiology of infectious disease (HCV) in the hospital visited patients referred by the physicians through the initial findings and their associated risk factors were studied in Swat. The data of 174 infected patients were collected during the period of 2015 to 2017 from two clinical laboratories of Tehsil Matta Swat. Inform consent form was taken before blood collection. After taking informed consent blood samples were collected and ICT test was performed and then ICT positive cases were conform through PCR. A total of 174 ICT positive samples [106 male and 68 females] were included in this study. Age was considered from 10 to 72 years. Of the 174 ICT strip positive, 99 [63 males, 36 females] were confirmed through PCR. The prevalence rate was recorded 56.89%. I.V/I.M injection was recorded in 100% of the individuals. Visits to the barber shop was reported in (58%) of the individuals, married individuals were (81.0), surgical operation was reported in (44.8%), sharing toothbrush was observed in (29.9%), piercing was reported in (39.7%), family history was reported in (26.4%), dental treatment was observed in (21.8%), jaundice were (13.2%) and tattooing was (1.7%). Blood transfusion, surgical operations, Jaundice, family history and dental treatment were found significant risk factors for acquiring HCV infection. It was concluded that proper implementation of precautionary measures should be needed to control the spread of HCV in far near future.

Effect of different protein based feed on the growth of mahseer.

For the proper growth of fish, it is necessary to feed the fish with a proper and balanced diet. A study was conducted to find out the effect of different protein-based diets on fingerlings of Tor putitora (mahseer). A feed with dietary protein levels of 35%, 40%, 45%, and 50% were prepared. The effect of different protein-based feed on weight gain, standard growth rate (SGR), food conversion ratio (FCR), percent weight gain, food conversion efficiency (FCE), and protein efficiency ratio (PER) was studied. An increase was observed in the growth rate with an increase in protein concentration up to 45%. The fingerlings fed a 45% protein diet shown the highest growth, followed by 50%, 40%, and 35%. The SGR value was greatest for 45% protein diet (8.56) followed by 50% and 40%, while the least values were observed for 35% protein feed (1.57). The same trend was observed for FCE. The highest PER values was observed in fishes fed 45% protein-based feed (0.65) followed by 50% (0.56), 40% (0.38) and35% (0.17). The food conversion ratio was lowest for 45% protein diet (3.41), while the greatest for 35% protein feed (16.85). It was concluded that a 45% protein-based diet was the best feed formulation for higher production of Tor putitora. However, research on the same percentage of protein diet is recommended for yearlings.

Ethanol and PTZ effects on siRNA-mediated GABAB1 receptor: down regulation of intracellular signaling pathway in prenatal rat cortical and hippocampal neurons.

GABA(B) receptors (R) are widely expressed and distributed in the nervous system, and have been implicated in variety of neurodegenerative and pathophysiological disorders. However, the exact molecular mechanism regarding responsibility of GABA(B1)R in downstream signaling pathway is not well understood. The present study was undertaken to explore the downstream signaling and role of GABA(B1)R upon acute ethanol and pentylenetetrazol (PTZ) exposure for (20 min) in cortical and hippocampal neuronal cell cultures by using GABA(B1)R RNA interference (i) (30 nM, 48 h) at gestational days 17.5. The results showed that GABA(B1)R and protein kinase A-alpha (PKA) showed decreased expression upon ethanol and PTZ exposure in cortical and hippocampal neurons during transfected and nontransfected conditions, whereas these effects could lead to significant changes in phosphorylation of cAMP-response element binding protein (p-CREB) expression where GABA(B1)R was knocked down. Furthermore, intracellular Ca(+2) concentrations were also reduced in some groups after transfection with GABA(B1)R RNAi. These results showed a critical role of GABA(B1)R upon ethanol and PTZ exposure by modulating downstream signaling pathway. Finally, these findings suggested that inhibition of GABA(B1)R results in the modulation of PKA, p-CREB pathway may play a role in long-term changes in the nervous system, and may be an underlying cause of ethanol's effects.

Morphology and ectoparasite spectrum of Pteropus vampyrus (the flying fox) in different parts of Malakand Division, Pakistan.

Three districts viz. Lower Dir, Swat and Shangla in Khyber Pakhtunkhwa province, were surveyed for the collection of Pteropus vampyrus (the Indian flying fox) in Malakand division, the north western region of Pakistan from February to November 2018. A total of 35 specimens were captured. Out of the examined specimens 22 were found to be infected with ectoparasites. Three types of ectoparasites were observed including flies 20% (n=7/35), mites 28.5% (n=10/35) and bugs 14.2% (n=5/35). Male bats were more infected 57.1% (n=20/35) as compared to females 42.8% (n=15/35), however, no significant difference (p>0.05) was noted. Flies were recovered from wings membrane and head region, mites were found on wings, ears and around eyes while bugs were found attached on body surface and neck region. Body weight, circumference and wing span of male bats were greater as compared to female bats and were considered a key factor in clear cut identification of male and female Pteropus vampyrus (the Indian flyingfox). It was concluded that Pteropus vampyrus from north western part of Pakistan were parasitized by a varied parasite fauna with high infestation rates. We assume that in male bats the number of parasites is generally higher than in females. Our results revealed new insights into parasite fauna of Pteropus vampyrus.

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa.

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili-Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n=5) with artificial vagina (42 degrees C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 degrees C. Extended semen was cooled to 4 degrees C in 2 h and equilibrated for 4 h at 4 degrees C. Cooled semen was then filled in 0.5 ml straws at 4 degrees C and frozen in programmable cell freezer. Thawing of semen was performed at 37 degrees C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 degrees C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P<0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest (P<0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P<0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.

Effect of antibiotics in extender on bacterial and spermatozoal quality of cooled buffalo (Bubalus bubalis) bull semen.

The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5 degrees C. Semen collected from Nili-Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 microg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 microg/ml, tylosin 100 microg/ml, lincomycin 300 microg/ml and spectinomycin 600 microg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5 degrees C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco-spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5 degrees C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5 degrees C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5 degrees C.

Expression analysis of transcription factors in sugarcane during cold stress / Análise de expressão de fatores de transcrição em cana-de-açúcar sob estresse por frio

Abstract Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Resumo Fatores de transcrição (FT) são uma ampla classe de genes em plantas e podem regular a expressão de outros genes em resposta a vários estresses ambientais (estresses bióticos e abióticos). No presente estudo, a atividade do fator de transcrição na cana-de-açúcar foi examinada durante o estresse pelo frio. Inicialmente, as leituras de transcrição de RNA de duas cultivares de cana-de-açúcar (ROC22 e GT08-1108) sob estresse frio foram baixadas do banco de dados SRA NCBI. As leituras foram alinhadas em um genoma de referência e as análises de expressão diferencial foram realizadas com o pacote R / Bioconductor edgeR. Com base em nossas análises no cultivar ROC22, 963 genes TF foram significativamente regulados positivamente sob estresse pelo frio entre um total de 5.649 genes regulados positivamente, enquanto 293 genes TF foram regulados negativamente entre um total de 3.289 genes regulados negativamente. No cultivar GT08-1108, 974 genes TF foram identificados entre 5.649 genes regulados positivamente e 283 genes TF foram encontrados entre 3.289 genes regulados negativamente. Os fatores de transcrição, em sua maioria, foram anotados com categorias GO relacionadas à ligação de proteína, ligação de fator de transcrição, ligação específica de sequência de DNA, complexo de fator de transcrição, atividade de fator de transcrição em RNA polimerase II, atividade de fator de transcrição de ligação de ácido nucleico, atividade de corepressor de transcrição, sequência específica da região reguladora, atividade do fator de transcrição da RNA polimerase II, atividade do cofator do fator de transcrição, atividade do fator de transcrição do promotor do plastídio, atividade do fator de transcrição do promotor da RNA polimerase I, polimerase II e RNA polimerase III. As descobertas dos resultados acima ajudarão a identificar fatores de transcrição expressos diferencialmente durante o estresse pelo frio. Ele também fornece uma análise abrangente da regulação da atividade de transcrição de muitos genes. Portanto, este estudo fornece base molecular para melhorar a tolerância ao frio em cana-de-açúcar e outras gramíneas economicamente importantes.