Bovine reproductive immunoinfertility: pathogenesis and immunotherapy.

Infertility is one of the primary factors for cattle reproduction in the present scenario. Reproduction-related immunoinfertility mainly involves immunization against the antigens related to reproductive hormones (LHRH, GnRH, Gonadal steroids, PGF2α and oxytocin), spermatozoa, seminal plasma and ovum. Anovulation, delayed ovulation, sperm immobilization, failure of fertilization, prolonged uterine involution, extended calving interval, prolonged post-partum estrus and reduced conception rate could be a result of immunoinfertility that occur due to the blockage of receptor site by antibodies formed against hormones, sperm and ovum. Immunoinfertility can be treated in the animal by giving sexual rest to females, by using various reproductive technologies such as in-vitro fertilization, gamete intra fallopian tube transfer, and intracytoplasmic sperm injection, sperm washing and by treating the animals with immunomodulators such as LPS, Oyster glycogen, etc. This review summarizes the different causes of bovine reproductive immunoinfertility and amelioration strategies to overcome it.

New dimensions on maternal and prepubertal nutritional disruption on bull fertility: A review.

Earlier, it was said that a bull is half of the herd because of its half contribution towards the genetic makeup in each subsequent generation. Nowadays, bulls are considered more than half of the herd because of the extensive use of frozen semen samples in artificial insemination. Bull's low fertility accounts for a major economic loss to livestock farmers. It is well known that fertility is a low-heritable trait governed by many factors such as genetics, epigenetics, climate, stress, and physical soundness. Apart from all these factors, the nutritional status of the bull also affects the semen quality. It has been seen that a bull given undernutrition at an early age is affected by androgen synthesis and semen quality. The nutrition given to the pregnant dam also affects the male progeny's postnatal semen quality. However, more studies are needed to elucidate the effect of periconception nutrition on the fertility of progeny as far as bulls are considered. This review focused on the effect of maternal undernutrition during the periconception period and undernutrition during the early growth phase of bull calves on the postnatal fertility of bulls.

Effect Of The Flavonoid 6-Aminoflavone In Aspirin Induced Gastric Ulcer In Sprague-Dawely Rats: A Histomorphological Study.

Background: The analgesic drugs are the main cause of gastric ulcer. The objective of this study was to determine the gastroprotective ability of flavonoid, 6-aminoflavone in a rat pyloric ligation model of aspirin associated gastro-ulcerogenesis. Methods: A laboratory based experimental study was conducted in the animal house and research laboratory at Khyber Medical College, Peshawar from July to November 2019. A total of 42 adult male Spargue-Dawely rats were divided into seven groups. Flavonoid, 6-aminoflavone was administered orally in doses of 10, 25 and 100 mg/kg with misoprostol, as standard at 50 µg/kg orally for 4 days. On the last day aspirin was given orally at 200 mg/kg and the pyloric ligation surgery was performed. After 4 hours all animals were killed by cervical dislocation. The gastric tissues were collected for histomorphological study. The obtained data were expressed as mean±SEM. Analysis was carried out by using ANOVA. p value ˂0.05 was considered significant. Results: The animals treated with the different doses of 6-aminoflavone showed a marked protective effect in the histological observations. The 10 mg/kg dose had a mild protective effect as occasional ulcerative changes were observed. However, doses of 25 and 100 mg/kg significantly caused the reduction in the ulcer score. These effects produced were equipotent to the gastroprotective effectiveness inherent in the misoprostol. . Conclusion: These findings conclude that 6-aminoflavone as like other flavonoids has a significant gastroprotective propensity with significant effect produced at doses of 25 and 100 mg/kg and can be used as a part of therapy management for the treatment of gastrointestinal disease particularly ulcerative condition.

Introduction to the pathways involved in the activation and regulation of sperm motility: A review of the relevance of ion channels.

To participate in sperm-oocyte fusion, spermatozoa need to be motile. In the testes, spermatozoa are immotile, although these gametes acquire the capacity for motility during the transit through the epididymis. During the period of epididymal transport from the male genital tract to the female genital tract, spermatozoa exhibit various types of motility that are regulated by complex signalling and communication mechanisms. Because motility is very dynamic, it can be affected by small changes in the external or internal environment of spermatozoa within a very short time. This indicates that regulatory membrane proteins, known as sperm ion channels, are involved in the regulation of sperm motility. Research results from studies, where there was use of electrophysiological, pharmacological, molecular and knock-out approaches, indicate ion channels are possibly involved in the regulation of sperm membrane polarisation, intracellular pH, motility, energy homeostasis, membrane integrity, capacitation, hyperactivity, acrosome reaction and fertilisation processes. In this review, there is summarisation of the key functions that ion channels have in the regulation, initiation, maintenance, and modulation of sperm motility. In addition, in this review there is highlighting of novel insights about the pathways of ion channels that are activated in spermatozoa while these gametes are located in the oviduct leading to the fertilisation capacity of these cells.

Nuclear Disaster Preparedness Level of Medical Responders in Pakistan.

Pakistan is a country with nuclear capability in a region sharing borders with 2 other nuclear powers-that is, India to the east and China to the northeast. This geographic positioning makes the area a nuclear flash point. It is therefore imperative to have commensurate health-care facilities to meet any eventualities. Although Pakistan is trying to improve medical facilities for the people, health-care facilities are already overwhelmed with patients because of the large population of the country. The current study was conducted to evaluate the level of awareness and preparedness of medical responders for nuclear and radiological disasters in public hospitals in the major cities of Pakistan. Methods: A detailed questionnaire covering all aspects of the study was designed and discussed with the people most concerned, including health-care workers in the field of medicine, nuclear sciences, and disaster management in Pakistan. It was adopted on the basis of early studies on the subject, with necessary modifications to fulfill the requirements at Pakistan's level. Semistructured interviews were also conducted with key personnel of different response agencies in Pakistan. Results: Of 880 surveys, 554 were completed and collected from medical providers in different hospitals across the country. The respondents included doctors, nurses, and medical assistants from emergency and nonemergency departments. They are aware of the catastrophic consequences of nuclear disaster and are willing to respond to these kinds of disasters voluntarily, but they are not satisfied with their level of preparedness and awareness regarding nuclear and radiological disasters. Conclusion: Although medical responders have good educational backgrounds and knowledge in their specific fields and are quite active and energetic in their response to conventional injuries and natural disasters, they feel a dire need to enhance their level of knowledge about and preparedness for unconventional disasters and injuries such as those caused by nuclear or radiological events.

Quantifying movement patterns and vaccination status of high risk mobile populations in Pakistan and Afghanistan to inform poliovirus risk and vaccination strategy.

BACKGROUND: Stopping serotype 1 wild poliovirus transmission in Pakistan and Afghanistan requires ensuring all children <5 years of age are repeatedly vaccinated, including the large proportion living in mobile groups. Vaccinating children living in high-risk mobile populations (HRMPs) remains a priority for the polio programme. METHODS: In 2017-2018, group-level censuses were conducted in 43 districts of Pakistan, gathering information for all HRMP children <5 years of age residing in settlements. Demographic and mobility information was collected, including HRMP type, ethnicity, language, mode of transportation and movement patterns. Vaccination status was recorded for the most recent polio campaign. Proportion of HRMP children by demographic factors and mode of transportation was determined and the magnitude of movement was quantified based on the origin, previous and next locations. Magnitude of cross-border movement with Afghanistan was evaluated, as was primary crossing point. Vaccination status was evaluated for each district by demographic and mode of transportation information. RESULTS: In total, 188,130 HRMP children <5 years of age were assessed. The predominant HRMP type, ethnic group, language and mode of transport was Afghan refugees (27%), Pashtun (69%), Pashto (69%) and bus (52%). Overall, 84% of children originated outside of their current district, including 29% from Afghanistan. Previous and next locations, were reported outside of current location by 34% and 77% of children. Afghanistan was previous and next location for 5% and 11% of children, with 5.5% and 3% of children crossing the Afghanistan border in the past 6-months and next 3-months. Primary crossing route was Torkham (79%). Overall vaccination coverage was 98% (IQR: 96%-99%) and consistently >90% across HRMP type, ethnic group, language and mobility means. CONCLUSION: Large numbers of HRMPs were found across Pakistan, with substantial links throughout the country and with Afghanistan. While vaccination coverage of HRMPs was high, ensuring these populations are consistently vaccinated remains a priority.

Modification of French mini-straw plug position for cryopreservation of small doses of bull sperm.

Greater than optimal diluting of semen for producing a large number of doses containing relatively small numbers of sperm can lead to compromised quality of sperm, post-thawing. In the present study the French mini-straw plug position was modified and the effect of re-positioning was evaluated on the quality of sperm after thawing subsequent to cryopreservation of small doses of sperm. Four types of mini-straws were used based on the position of cotton plug including no plug displacement (Type 1; Manufacturers location for plug-placement in 0.25 mL French mini-straws), and Type II, III, and IV with re-positioning the cotton plug being 2.5, 5, and 7.5 cm, respectively, further from the manufacturer's placement location. Each ejaculate was proportioned into four Aliquots (I, II, III, and IV) and diluted to 80, 60, 40, and 20, million sperm/mL, respectively. Aliquot I was placed in all types of straws, while Aliquots II, III, IV were placed only in Type I straws. Semen straws were equilibrated, cryopreserved and sperm kinetic and functional variables were evaluated post-thawing. The results indicate that in Aliquots III and IV there were lesser (P < 0.05) values for sperm kinetic and function variables compared with sperm from Aliquot I (i.e., unmodified mini-straw). In conclusion, cryopreservation of small doses of sperm in modified French mini-straws resulted in acceptable values for kinetic and function variables, post-thawing.

Biodegradation potential of MTBE in a fractured chalk aquifer under aerobic conditions in long-term uncontaminated and contaminated aquifer microcosms.

The potential for aerobic biodegradation of MTBE in a fractured chalk aquifer is assessed in microcosm experiments over 450 days, under in situ conditions for a groundwater temperature of 10 degrees C, MTBE concentration between 0.1 and 1.0 mg/L and dissolved O2 concentration between 2 and 10 mg/L. Following a lag period of up to 120 days, MTBE was biodegraded in uncontaminated aquifer microcosms at concentrations up to 1.2 mg/L, demonstrating that the aquifer has an intrinsic potential to biodegrade MTBE aerobically. The MTBE biodegradation rate increased three-fold from a mean of 6.6+/-1.6 microg/L/day in uncontaminated aquifer microcosms for subsequent additions of MTBE, suggesting an increasing biodegradation capability, due to microbial cell growth and increased biomass after repeated exposure to MTBE. In contaminated aquifer microcosms which also contained TAME, MTBE biodegradation occurred after a shorter lag of 15 or 33 days and MTBE biodegradation rates were higher (max. 27.5 microg/L/day), probably resulting from an acclimated microbial population due to previous exposure to MTBE in situ. The initial MTBE concentration did not affect the lag period but the biodegradation rate increased with the initial MTBE concentration, indicating that there was no inhibition of MTBE biodegradation related to MTBE concentration up to 1.2 mg/L. No minimum substrate concentration for MTBE biodegradation was observed, indicating that in the presence of dissolved O2 (and absence of inhibitory factors) MTBE biodegradation would occur in the aquifer at MTBE concentrations (ca. 0.1 mg/L) found at the front of the ether oxygenate plume. MTBE biodegradation occurred with concomitant O2 consumption but no other electron acceptor utilisation, indicating biodegradation by aerobic processes only. However, O2 consumption was less than the stoichiometric requirement for complete MTBE mineralization, suggesting that only partial biodegradation of MTBE to intermediate organic metabolites occurred. The availability of dissolved O2 did not affect MTBE biodegradation significantly, with similar MTBE biodegradation behaviour and rates down to ca. 0.7 mg/L dissolved O2 concentration. The results indicate that aerobic MTBE biodegradation could be significant in the plume fringe, during mixing of the contaminant plume and uncontaminated groundwater and that, relative to the plume migration, aerobic biodegradation is important for MTBE attenuation. Moreover, should the groundwater dissolved O2 concentration fall to zero such that MTBE biodegradation was inhibited, an engineered approach to enhance in situ bioremediation could supply O2 at relatively low levels (e.g. 2-3 mg/L) to effectively stimulate MTBE biodegradation, which has significant practical advantages. The study shows that aerobic MTBE biodegradation can occur at environmentally significant rates in this aquifer, and that long-term microcosm experiments (100s days) may be necessary to correctly interpret contaminant biodegradation potential in aquifers to support site management decisions.

The effects of forest clearance for peatland restoration on water quality.

Recognition of the importance of peatlands has led to increased efforts to protect and restore these environments but there are concerns about the impact of restoration on water quality, particularly in terms of sediment delivery, nutrient transport and carbon losses. In this study we present the results of almost 10 years of water quality monitoring in 3 catchments at a lowland raised bog that was afforested with conifers in the 1960s and 1970s and cleared over the 2010s. Phosphate concentrations increased after clearfelling with the main peaks seen in the summer after forest clearance; the use of low ground impact harvesting methods, removal of forest residues and especially phased felling tempered phosphate losses. Annual mean phosphate concentrations returned to pre-felling levels 3-5 years after felling finished. Nitrate concentrations increased slightly from baseline levels during and after felling. DOC concentrations increased at two sites with mean annual concentrations still elevated at one site after 4 years; little increase in DOC was recorded with phased felling. Colour levels increased and remain elevated at all sites relative to pre-felling. In only one stream, pH increased improving the WFD water body status from 'poor' to 'high' in the space of 3 years. Our results show that forest clearance for peatland restoration can impact negatively upon water quality due to phosphate, DOC, colour and suspended sediment releases. The mechanisms by which the releases occur require further investigation but are thought to be driven by nutrient leaching from forest residues, soil disturbance by machine trafficking and indirect effects of forest clearance, such as water table rise following cessation of forest water use. Climatic effects also play a significant role, particularly drying-rewetting cycles and periods of extended drought. To prevent negative impacts, phased felling and less intensive forest harvesting techniques should be employed.

Modulation of voltage-gated sodium channels induces capacitation in bull spermatozoa through phosphorylation of tyrosine containing proteins.

In our previous study, we have reported the molecular presence of Nav 1.8 in bull spermatozoa and its potential involvement in regulation of sperm functions. With the selective blocking of Nav 1.8 using A-803467, alterations in sperm functions were observed, therefore, we envisaged of investigating the involvement of Nav in regulating sperm function and the mechanism(s) involved in it using veratridine, a selective opener of Nav channels. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the non-significant variations between the different ejaculates. Treatment of sperm cells with veratridine (6, 8, and 10 µM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 6 h. However, hyperactive motility was induced by veratridine at higher concentrations (8 and 10 µM) and after 2 h of incubation, which was confirmed by subjective assessment followed by chlortetracycline staining showing the increased B-pattern spermatozoa, and thereby suggesting the involvement of Nav in regulation of capacitation in spermatozoa. To substantiate the functional study observations especially veratridine-induced capacitation, immunoblotting and indirect immune fluorescence assays were performed for detection of the tyrosine-phosphorylated proteins. The immune blot study revealed the presence of five tyrosine phosphorylated proteins, namely-p17, p30, p54, p90 and p100. The p17 protein showed the highest band intensity compared to other protein bands indicating its potential involvement in the process of capacitation. Immunolocalization study revealed positive immunoreactivity for tyrosine phosphorylated proteins in the middle piece, post acrosomal region (high fluorescence) and tail of the spermatozoa (low fluorescence). From the results of present study, it is evident that activation of NaV by veratridine, especially at higher concentrations, induced capacitation which is evidently mediated through phosphorylation of the tyrosine containing proteins localized in the post acrosomal regions, middle piece and tail of the spermatozoa. However, further studies will help in unraveling the involvement of Nav and other ion channels regulating different physiological functions of sperm.

Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa.

The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 106 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at -196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P < 0.05) significantly in Group II as compared to Group I, III, and IV. Immunoblot demonstrated a 32 kDa (p32) protein showing differential variation in the band intensity in all the four groups being lower in Group II. Further, the immunolocalization study revealed positive immune reactivity for tyrosine phosphorylated proteins at middle piece and neck (high fluorescence), post-acrosomal region (medium fluorescence), and principal piece (low fluorescence) of spermatozoa. Addition of CLC significantly increased (P < 0.05) the percentage of spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 106 in semen extender significantly decreased (P < 0.05) spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes.

Functional and molecular characterization of voltage gated sodium channel Nav 1.8 in bull spermatozoa.

The aim of our study was to characterize the voltage gated sodium channel Nav 1.8 in bull spermatozoa. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the nonsignificant variations between different ejaculates. Functional characterization was undertaken using A-803467, a selective blocker of Nav1.8, and veratridine as an opener of the voltage gated sodium channels while molecular characterization was done using western blotting and indirect immunofluorescence assays. In vitro capacitation was induced using heparin, and to study the functional involvement of Nav 1.8 in regulation of capacitation induced hyper sperm motility, A-803467 was used. Selective blocking of NaV 1.8 by A-803467 at 6 and 8 µM concentration significantly (P < 0.05) decreased the forward progressive sperm motility in a time-dependent manner, while, blocking at higher concentrations (10 and 15 µM) resulted in fast forward motility in spermatozoa after 2 h of incubation and it was observed up to 3 h. Treatment of sperm cells with veratridine (6, 8, 10, 15, 20 µM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 4 h. However, hyperactive motility was induced by veratridine at higher concentrations (10 and 15 µM) after 2 h of incubation. In vitro capacitated spermatozoa treated with A-803467 revealed significant (P < 0.05) reduction in forward progressive motility after 2 h of incubation. Both A-803467 and veratridine altered the percentage of spermatozoa showing high mitochondrial transmembrane potential in concentration- and time-dependent manner. High concentrations (10 and 15 µM) of A-803467 and veratridine resulted in bent neck condition in spermatozoa along with significant (P < 0.05) reduction in membrane integrity (HOST negative). Immunoblot revealed the presence of a single protein band of 260 kDa molecular weight along with positive immunoreactivity (IR) in head, neck, middle piece and tail of the spermatozoa. Strongest IR was observed in the neck and middle piece whereas weak IR was observed in tail and acrosomal region of the spermatozoa. Results of our present study evidently revealed the presence of voltage gated sodium channel Nav1.8 in bull spermatozoa and its functional involvement in regulation of spermatozoa dynamics in terms of motility, membrane integrity, acrosome integrity, capacitation and mitochondrial transmembrane potential. Further studies are warranted to unravel their mechanistic pathways and/or their interaction with other ion channels in regulating sperm dynamics.

Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa.

To provide new insights into the mechanisms through which reduced glutathione (GSH) is able to protect spermatozoa, we tested the hypothesis that cryocapacitation and apoptosis like changes can contribute to the negative effect of freezing and thawing on bull spermatozoa, and that GSH prevent this damage. Having known protective effects of GSH in terms of a potent antioxidant, we evaluated capacitation, tyrosine phosphorylation and apoptosis like changes in bull spermatozoa after freezing and thawing in egg yolk tris glycerol extender containing (0.5m M-GSH-T1 & 1mM GSH-T2) and without GSH serving as the control (C). Forty ejaculates were collected from four Hariana bulls and were pooled due to non significant variations among the bull ejaculates for the evaluation of sperm attributes. Capacitation like changes, tyrosine phosphorylation, localization of tyrosine phosphorylated proteins, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA fragmentation after final dilution, 4h of equilibration at 4°C and 24h after freezing and thawing were evaluated. GSH supplementation at 0.5mM showed significant reduction in B- and AR- pattern spermatozoa during all stages of semen freezing and thawing. Immunoblot revealed six proteins which were tyrosine phosphorylated and protein of 30 and 75kDa (p30, p75) were the major tyrosine phosphorylted proteins. On further analysis, the p30 showed differential variation in intensity in all the three groups after freezing and thawing. Positive immune reactivity for tyrosine phosphorylated proteins was found in neck, middle piece and post-acrosomal regions of spermatozoa. Addition of 0.5mM GSH decreased percentage of spermatozoa showing fragmented DNA and increased the percentage of spermatozoa having high transmembrane mitochondrial potential (P<0.05). This study demonstrates that GSH favours survival of bull spermatozoa by interfering with apoptotic and cryocapacitation pathways, and thereby protects the spermatozoa from deleterious effects of cryopreservation. The findings of the study indicated that GSH at 0.5mM can be effectively used as an additive in bull semen extender for freezing and thawing.