Effect of membrane stabilizers on semen quality and sperm membrane protein expression during cryopreservation of goat semen.

BACKGROUND: Semen cryopreservation is a complex process during which there is alteration in the expression of sperm and seminal plasma proteins, molecular weight of protein or loss of membrane proteins during the process. In order to compensate for these changes, different membrane stabilizers are used in freezing semen extenders. However, there is scarcity of such studies during cryopreservation of goat semen. OBJECTIVE: To investigate the effect of membrane stabilizers on sperm membrane protein expression during cryopreservation of goat semen. MATERIALS AND METHODS: A total of 36 semen ejaculates from nine Assam Hill Goat bucks aged 2 to 2.5 years was collected by artificial vagina method. Three membrane stabilizers, each at two different concentrations viz. 50 and 80 mM sucrose, 50 and 100 mM trehalose, and 100 and 150 ng per mL IGF-1 (insulin-like growth factor 1 protein) were added to Tris-citric acid fructose egg yolk glycerol (TCFEYG) extender and semen samples were cryopreserved. The sperm membrane protein profile was studied in fresh and cryopreserved semen by SDS-PAGE. RESULTS: SDS- PAGE of sperm membrane extract of fresh semen revealed the presence of 24 protein bands with molecular weights ranging from 10 kDa to 240 kDa. Samples supplemented with 50 mM sucrose and 80 mM sucrose revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. All the 21 protein bands were same as those observed in the sperm membrane of fresh spermatozoa, except that the 23 kDa, 29 kDa and 42 kDa bands were absent in frozen semen. Similarly, frozen semen extended with 50 mM trehalose and 100 mM trehalose revealed 22 protein bands with molecular weights ranging from 10 kDa to 240 kDa, but lacking the 29 kDa and 42 kDa bands. Proteins with molecular weights of 29 kDa, 130 kDa and 240 kDa were absent in frozen semen supplemented with 100 ng per mL IGF-1 and 150 ng per mL IGF-1. CONCLUSION: The present study revealed that supplementation of tris basic extender with trehalose at 100 mM and or IGF-1 at 100 ng/mL or 150 ng per mL improves the post-thaw semen characteristics and protects certain fertility related sperm membrane proteins. Doi.org/10.54680/fr23510110612.

Effect of Butylated Hydroxy Toluene and Vitamin E on the Cryosurvivability of Buck Semen.

BACKGROUND: The quality of frozen semen can be improved by supplementing Tris extender with antioxidant to prevent oxidation and maintain sperm motility. OBJECTIVE: To study the effects of adding combinations of suitable concentrations of butylated hydroxy toluene (BHT) and Vitamin E in Tris extender on the quality of frozen goat semen. MATERIALS AND METHODS: A total of 40 ejaculates collected from five Beetal bucks were used to study the effect on the quality of frozen semen of supplementing Tris extender with 200 µM BHT, 2 mM Vitamin E and 200 µM BHT + 2 mM Vitamin E. RESULTS: The sperm motility, live sperm, live intact acrosome and HOST-reacted sperm differed significantly (P<0.01) between stages and between antioxidants. There was no significant difference (P<0.05) in interaction between stages (equilibration, freezing) and antioxidants, except for HOST-reacted sperm. Critical difference test revealed that Tris extender containing 2 mM vitamin E showed significantly (P<0.05) higher sperm motility, live sperm, live intact acrosome and HOST-reacted sperm, and significantly (P<0.05) lower release of alanine transaminase (ALT) and aspartate transaminase (AST). CONCLUSION: Supplementation of Tris extender with 2 mM vitamin E maintained superior quality of frozen Beetal buck semen.

Antenatal care and skilled birth attendance in Bangladesh are influenced by female education and family affordability: BDHS 2014.

OBJECTIVES: Antenatal care (ANC) during pregnancy and skilled birth attendance (SBA) during delivery are important policy concerns to reduce maternal deaths. Bangladesh is one of the developing countries which has made remarkable progress in both services during the last couple of decades by improving the SBA service rate from 16% in 2004 to 42.1% in 2014. However, this rate remains below the targeted level (50%) of the Health Population and Nutrition Sector Development Program set by the Ministry of the Health and Family Welfare of Bangladesh. This article explored the sociodemographic factors associated with the ANC and SBA service attainment. Furthermore, the possible implication of using ANC on SBA was also investigated. STUDY DESIGN: The study followed a cross-sectional design using the Bangladesh demographic and health survey 2014, with a sample of size 4603 women with at least one live birth 3 years preceding the survey. METHODS: Following a bivariate analysis, linear mixed-effect models were used to assess the relationship between sociodemographic factors and the outcome indicators (ANC and SBA). Finally, the association between SBA and ANC was evaluated through another mixed-effect model. RESULTS: Wealth index, participation in household decisions, and partner's and respondent's education were significant predictors of ANC; whereas, residence, age at first birth, wealth index, working status, participation in household decisions, and partner and respondent's education were significant for SBA. Female education and household affordability were the strongest predictors for both ANC and SBA. ANC showed significant association with SBA as women accessing essential ANC during delivery seemed to be 4 times more likely (95% confidence interval: 3.05-5.93) to avail SBA services. CONCLUSIONS: Overall, four factors were significant: residence, wealth index, education, and ANC access. Women residing in urban areas, having higher financial solvency, completing higher education, and accessing ANC by skilled personnel were more likely to receive SBA at delivery than their counterparts. Accessibility to skilled care during pregnancy leads to increased professional care during delivery. Thus, policies to encourage women and heads of families to seek skilled care during pregnancy would be beneficial to reach the maternal healthcare targets of Bangladesh.

Pre-freezing Treatment with Butylated Hydroxytoluene and Cholesterol-loaded Methyl-ß-cyclodextrin Improves Quality of Cryopreserved Boar Semen.

BACKGROUND: Pre-freezing treatment of boar sperm with additives improves the quality of post-thaw sperms. OBJECTIVES: The study aimed to determine the efficacy of butylated hydroxy-toluene (BHT) and cholesterol-loaded methyl-ß-cyclodextrin (CLC) for the improvement of the frozen-thawed boar sperm quality. METHODS: Split samples of 30 ejaculates from six boars were cryopreserved in lactose-egg yolk-glycerol extender containing BHT (0.2 mM), CLC (5 mg/ 200-240 x 106 sperm) or BHT (0.2 mM) plus CLC (5 mg per 200-240 x 106 sperm). Semen samples were evaluated for motility, membrane integrity, acrosomal status, mitochondrial membrane potential (MMP), DNA integrity and lipid peroxidation after equilibration and after freezing. RESULTS: The addition of BHT and CLC into the extender significantly improved (P<0.05) plasma membrane integrity and decreased (P<0.05) lipid peroxidation after freezing. Post-thaw motility and live intact acrosome were significantly (P<0.05) higher in the extenders with BHT or BHT plus CLC. The post-thaw MMP of viable spermatozoa and DNA integrity were not affected. BHT plus CLC showed a significant (P<0.05) improvement on motility as compared to BHT and CLC alone. CONCLUSION: Treatment of boar spermatozoa with BHT and CLC improved post-thaw sperm quality.

Effect of Reduced Glutathione, Water Soluble Vitamin E Analogue and Butylated Hydroxytoluene on the Post Thaw Characteristics of Boar Spermatozoa.

BACKGROUND: Antioxidant in freezing extender of boar semen improved post thaw sperm function. OBJECTIVE: The study compared the effects of reduced glutathione (GSH), water soluble vitamin E analogue Trolox and butylated hydroxytoluene (BHT) on quality of cryopreserved boar spermatozoa. MATERIALS AND METHODS: Using split sample technique three different antioxidants namely, GSH (1 mM), vitamin E (0.2 mM) and BHT (0.2 mM) were added to the freezing medium of lactose-egg yolk-glycerol extender, and samples were frozen using controlled freezing rate of 40°C/min from -6 to -140°C. Samples were evaluated for sperm motility, acrosomal status, plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation and sperm DNA integrity after equilibration and after freezing. RESULTS: The supplementation of GSH, vitamin E and BHT resulted in significantly higher post thaw motility, live intact acrosome and plasma membrane intact sperm. The incidence of post thaw sperm lipid peroxidation was significantly reduced after addition of antioxidants. However, antioxidants treatment neither significantly improved mitochondrial membrane potential of live sperm sub-population nor sperm DNA integrity after freezing. There was no significant difference of the post thaw sperm characteristics among three antioxidants. Protective effect of GSH, vitamin E and BHT are comparable on cryopreserved boar spermatozoa.

Cumulus cell expansion and ultrastructural changes in in vitro matured bovine oocytes under heat stress.

Cumulus-oocyte complexes (COCs) from cows were matured under normal (38.5°C) and elevated temperatures (41°C) simulating heat stress and their maturation was assessed based on measurement of cumulus expansion in both groups. There was a significant reduction (P<0.01) in maturation rate in the heat stressed oocytes. The ultrastructural events associated with in vitro oocyte maturation and changes associated with elevated temperature were also studied by transmission electron microscopy (TEM). Normal maturation cellular events were marked by migration of Golgi and mitochondria from the cortical regions, and conversely by a migration of cortical granules from the inner regions to a sub-perivitelline zone. Heat stressed oocytes (41°C) were not only marked by a reduction in rate and less cumulus cell expansion, but also by a reduction in cortical granule migration. The mitochondria appeared swollen with cristolysis. Ribosomal disruption and an abundance of free ribosomes were also seen. Changes in the cumulus cells include nuclear chromatin margination, condensation and karyolysis, formation of nuclear and cell membrane blebs, and typical membrane bound vesicles enclosing cell fragments indistinguishable from apoptosis. Evidently, heat stress can be associated with reduced cytoplasmic events of oocyte maturation, thereby decreasing the oocyte competence and can be associated with apoptosis of the cumulus cells and therefore compromise the survival of the oocyte itself.

Effect of conventional and controlled freezing method on the post thaw characteristics of boar spermatozoa.

The objective of the present study was to evaluate the effectiveness of conventional, and controlled freezing method adopting three freezing rates 20°C, 40°C and 60°C/min for cryopreservation of boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender using 0.5 ml straws. Semen samples were evaluated for sperm motility, live sperm, acrosome integrity, plasma membrane integrity (PMI) by carboxyfluorescein diacetate plus propidium iodide (PI) staining, mitochondrial membrane potential (MMP) by combined JC-1 plus PI staining and lipid peroxidation (LPO) by BODIPY (581/591)-C11 probe after equilibration and after freezing. The results revealed that the post thaw sperm motility, live sperm, live intact acrosome and plasma membrane integrity were significantly (p<0.05) higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the controlled freezing methods yielded higher (p>0.05) mean values of live sperm with high MMP as compared to conventional freezing. However, the post thaw sperm LPO did not influence by difference in freezing methods. No significant difference on the post thaw sperm qualities was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly (p<0.05) after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, cryopreservation of boar semen with controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Effect of holding of semen and washing of seminal plasma on quality and fertility of Hampshire boar semen preserved at liquid state.

The present study was aimed to reveal the effect on keeping quality of boar semen on holding or not holding at an elevated temperature than that used for preservation when combined with washing or not washing of seminal plasma. Twenty ejaculates, four from each of five Hampshire boars were used to hold for 0 and 4h in GEPS extender at 22°C and subsequently washed (1500×g for 10min) of seminal plasma or left unwashed and preserved at 15°C for 72h after extending with the same extender. The seminal parameters in terms of sperm motility, live spermatozoa, and live spermatozoa with intact acrosome (LIA) were evaluated at 0h-(immediately after extension) and thereafter at 24h intervals. The mean percentage of sperm motility was significantly (P<0.01) higher in unwashed than washed semen at both 0h and 4h of holding irrespective of preservation period. It was significantly (P<0.01) higher in semen held for 4h than 0h irrespective of washing and significantly (P<0.01) lower in washed than in unwashed semen irrespective of holding during preservation. Irrespective of preservation period the mean percentage of live spermatozoa was significantly (P<0.01) higher with 4h than 0h of holding in both unwashed and washed semen and was significantly (P<0.01) higher in unwashed than washed semen at both 0h and 4h of holding. It was significantly (P<0.01) higher for 4h held semen irrespective of washing and was significantly (P<0.01) lower in washed than in unwashed semen irrespective of holding during preservation. The mean percentage of LIA was significantly (P<0.01) higher with 4h than with 0h holding in both unwashed and washed semen and was significantly (P<0.01) higher in unwashed than in washed semen at both 0h and 4h of holding irrespective of preservation period. It was significantly (P<0.01) higher for 4h held as compared to unheld semen irrespective of washing and was significantly (P<0.01) lower in washed than unwashed semen irrespective of holding during preservation. The mean percentage of sperm motility, live spermatozoa and LIA decreased significantly (P<0.01) in 0h and 4h holding irrespective of washing and in unwashed and washed semen irrespective of holding with increase in preservation period. Among all the treatments unwashed semen held for 4h yielded superior sperm quality on preservation. A total of 32 female pigs were inseminated using preserved semen obtained with the best processing technique found in the study. The conception rate, farrowing rate and litter size at birth were recorded to be 81.25%, 78.13% and 7.96 respectively as compared to 73.38%, 67.57% and 6.68 respectively in the control group. It could be concluded that unwashed Hampshire boar semen held for 4h, extended with GEPS and preserved at 15°C for 72h was conducive to obtain optimum fertility and fecundity in females when used for artificial insemination.

Evolution of community-based arsenic removal systems in remote villages in West Bengal, India: assessment of decade-long operation.

In Bangladesh and the neighboring state of West Bengal, India, over 100 million people are affected by widespread arsenic poisoning through drinking water drawn from underground sources containing arsenic at concentrations well above the permissible limit of 50 µg/L. The health effects caused by arsenic poisoning in this area is as catastrophic as any other natural calamity that occurred throughout the world in recent times. Since 1997, over 200 community level arsenic removal units have been installed in Indian subcontinent through collaboration between Bengal Engineering and Science University (BESU), India and Lehigh University, USA. Approximately 200,000 villagers collect arsenic-safe potable water from these units on a daily basis. The treated water is also safe for drinking with regard to its total dissolved solids, hardness, iron and manganese content. The units use regenerable arsenic-selective adsorbents. Regular maintenance and upkeep of the units is administered by the villagers through formation of villagers' water committee. The villagers contribute towards the cost of operation through collection of a small water tariff. Upon exhaustion, the adsorbents are regenerated in a central facility by a few trained villagers. The process of regeneration reduces the volume of disposable arsenic-laden solids by nearly two orders of magnitude and allows for the reuse of the adsorbent material. Finally, the arsenic-laden solids are contained on well-aerated coarse sand filters with minimum arsenic leaching. This disposal technique is scientifically more appropriate than dumping arsenic-loaded adsorbents in the reducing environment of landfills as currently practiced in developed countries including the United States. The design of the units underwent several modifications over last ten years to enhance the efficiency in terms of arsenic removal, ease of maintenance and ecologically safe containment and disposal of treatment residuals. The continued safe operation of these units has amply demonstrated that use of regenerable arsenic-selective adsorbents is quite viable in remote locations. The technology and associated socio-economic management of the units have matured over the years, generating promise for rapid replication in other severely arsenic-affected countries in Southeast Asia.

Changes in Mithun (Bos frontalis) spermatozoa during epididymal passage.

Genital organs of 10 healthy, adult Mithun bulls (6-8 years old) that were slaughtered at the dwellings of tribal people for meat were collected. Immediately after collection, spermatozoa from 3 different regions of the epididymis, i.e. the head, body and tail, were obtained to study morphological changes of the spermatozoa during passage through these regions. The prevalence of proximal cytoplasmic droplets significantly decreased from the head to the tail of the epididymis. Conversely, the percentage of distal cytoplasmic droplets increased significantly from the head to the tail region. The incidence of tailless heads rose significantly from head to body and then reduced significantly in the tail region. The percentage of total head abnormalities did, however, not change markedly, but total mid-piece and tail abnormalities differed significantly between the three epididymal regions.

Collection and characterization of semen in Mithun (Bos frontalis) bulls.

The objective of this study was to collect semen from semiwild Mithun (Bos frontalis) bulls using an artificial vagina (AV) and to determine semen characteristics. Collection of semen with an AV was attempted in five Mithun bulls using both anestrous and estrous Mithun females. No Mithun bull mounted an anestrous female Mithun during 60 trials, but satisfactory mounting, including extension of the penis, occurred in 25 trials with estrous Mithun females. In 15 of these trials, semen was successfully collected in an AV with an internal temperature of 42 to 46 degrees C. However, in 10 trials with an AV with an internal temperature of 36 to 40 degrees C, semen was not collected. Mean (+/- SEM) intervals to first mount and to ejaculation in the AV were 27.9+/-3.6 sec and 113.8+/-6.6 sec, respectively. Semen volume and pH were 3.1+/-0.35 mL and 6.59+/-0.04, and mean mass activity (scale, 0 to 4), initial sperm motility, live sperm count, sperm concentration, total number of sperm in the ejaculate, and overall sperm length were 2.2+/-0.3, 78.6+/-2.6%, 80.7+/-2.2%, 710.8+/-66.8 x 10(6)/mL, 2114+/-364.4 sperm, and 67.9+/-0.6 microm, respectively. The proportion of morphologically normal sperm was 80.6+/-0.2%, whereas the proportion with a morphologically abnormal head, midpiece, tail, and acrosome were 4.2+/-0.4%, 1.6+/-0.5%, 6.1+/-1.1%, and 7.1+/-0.9%, respectively. The mean incidence of tail-less heads and proximal and distal protoplasmic droplets were 0.5+/-0.1%, 0.3+/-0.2%, and 2.4+/-0.3%, respectively. In conclusion, we successfully collected semen from semiwild Mithun bulls with an AV maintained at 42 to 46 degrees C, and overall, the semen was within the normal range of that collected from fertile domestic bulls.

Studies on lactic dehydrogenase and sorbitol dehydrogenase release in relation to deep freezing of buffalo semen in certain extenders.

Forty seminal ejaculates from five mature buffalo bulls (n=8 from each bull), exhibiting more than 70% initial sperm motility, were frozen in the following three extenders: egg-yolk sodium citrate glycerol (EYCG); tris-egg yolk glycerol (TYG); and citric acid whey glycerol (CAWG). The extenders were evaluated for the release of intracellular enzymes, lactic dehydrogenase (LDH) and sorbitol dehydrogenase (SODH) from the spermatozoa during freezing (in fresh semen, after dilution and after equilibration) and post freezing (24 h and 7 d after freezing. It was found that the release of LDH and SODH enzymes was significantly lower in TYG than in EYCG and CAWG extenders. The most critical stage, at which the enzyme release was maximal, was between equilibration and 24 h post freezing in all three extenders.

Sialo proteins and phosphatases in estrual cervical mucus of fertile and repeat breeder buffaloes (Bubalus bubalis ).

Concentrations of sialic acid and protein and activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) were determined in estrual cervical mucus of fertile and repeat breeder buffaloes during late estrus. Significantly (P < 0.01) higher concentrations of sialic acid and ACP and AKP activities were observed in fertile buffaloes as compared with repeat breeder buffaloes. However, differences were nonsignificant in protein content between fertile and repeat breeder buffaloes, albeit protein concentration was higher in fertile buffaloes. The significance of sialoproteins and phosphatases in fertility has been clarified.

Treatment of summer anestrous buffalo (bubalus bubalis) with progesterone releasing intravaginal device plus pregnant mare serum go nadotropin.

Three experiments were conducted to treat forty-two summer-anestrous buffalo at three different villages. In all the experiments animals were randomly divided into two groups. One group was treated with a progesterone releasing intravaginal device (PRID) plus 800 IU pregnant mare serum gonadotropin (PMSG) at the time of PRID removal. The second group was retained as untreated control animals. All the treated animals were clinically examined and inseminated twice 48 and 72 h post treatment. Ovulation and fertility rates at induced estrus were 88, 90 and 100% and 50, 60 and 50% during Experiments I, II and III, respectively. None of the controls exhibited estrus during the course of the study.