Biomedical subjects

R Vishwanath

Publications and source records attributed to R Vishwanath.

At least 19 recordsLinked to original sources

PubMed2003

Artificial insemination: the state of the art.

The history of research into artificial insemination (AI) is over two centuries old and its commercial application now spans 75 years. It is appropriate to reflect on the contribution of this powerful method of gene dispersal. AI remains as one of the most important assisted reproductive technologies. The three cornerstones for its application are: it is simple, economical and successful. The importance of AI will be challenged in the next few decades. The remarkable progress made in other assisted reproductive technologies does have the potential to rapidly generate offspring. The challenge for any of these reproductive technologies to attain widespread use is to match AI in being simple, economical and successful. This review aims at capturing the salient advances in AI, the comparisons with natural mating and other reproductive technologies, and, whether the future of AI will be challenged. It predicts what the new horizon looks like and the role that AI will play in the overall reproductive technologies landscape.

Animals↗

PubMed2001

Effect of sperm number and oxygenation state of the storage media on in vitro fertility of bovine sperm stored at ambient temperature.

The effects of storage time and the oxygenation state of the storage medium on motility, viability, and in vitro fertility of stored diluted sperm were investigated. Oocytes collected from abattoir material were matured and fertilized in vitro on defined days with sperm stored for up to 11 days in a citrate-based commercial diluent. The proportions of oocytes fertilized and developing to the blastocyst stage were used to assess the quality of the stored semen. In vitro fertility of sperm declined with storage time. There was no significant effect of the oxygenation state of the medium on in vitro fertility of stored sperm. Increased sperm-to-oocyte ratios resulted in a significant elevation of the proportion of oocytes fertilized on day 0 of storage and the proportion of fertilized oocytes developing to the blastocyst stage on days 0 and 3 of storage, suggesting some form of sperm competition or egg selection of sperm based on the ability of sperm to induce normal development.

Animals↗

PubMed2000

Storage of bovine semen in liquid and frozen state.

This review describes the historical and current methods used for storage of bovine semen. The essential physiological differences between liquid and frozen semen, their relative advantages and disadvantages are addressed, and the current state of technology, the procedures used, their merits and future possibilities are also discussed.

Animals↗

PubMed2000

Changes in susceptibility of bovine sperm to in situ DNA denaturation during prolonged incubation at ambient temperature under conditions of exposure to reactive oxygen species and nuclease inhibitor.

Sperm were incubated for up to 9 days in the presence or absence of exogenous hydrogen peroxide, phenylalanine, catalase and aurintricarboxylic acid to assess the influence of reactive oxygen species and inhibition of deoxyribonucleases on sperm chromatin stability. The assessment of sperm DNA susceptibility to in situ acid denaturation by the sperm chromatin structure assay did not detect any difference in chromatin stability between sperm incubated for 9 days under aerobic and anaerobic conditions in a diluent called 14G. Exposure to exogenous hydrogen peroxide under both aerobic and anaerobic conditions and to phenylalanine under aerobic conditions (which produces hydrogen peroxide by a reaction catalysed by the aromatic amino acid oxidase present in sperm) was detrimental to sperm chromatin stability, increasing its DNA susceptibility to in situ acid denaturation over the incubation time. This effect was eliminated if catalase was present in the diluent. Inclusion of the general deoxyribonuclease inhibitor aurintricarboxylic acid in the diluent severely decreased sperm chromatin stability under both aerobic and anaerobic conditions. Aurintricarboxylic acid was mildly cytotoxic, as revealed by viability assessment, under aerobic, but not under anaerobic, incubation conditions. Exogenous hydrogen peroxide, either directly added to the diluent or generated through the enzymatic oxidation of phenylalanine, was detrimental to sperm motility and the integrity of the plasma membrane.

Animals↗

PubMed2000

Protein tyrosine phosphorylation during prolonged in vitro incubation of ejaculated bovine spermatozoa is regulated by the oxidative state of the medium.

Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylation, viability, and motility were studied as a function of extended incubation of bovine sperm in vitro at ambient temperature (18-20 degrees C). Fresh ejaculates were incubated after dilution for 8 days. On Days 0, 2, 5, and 8, an aliquot of sperm was incubated with or without theophylline at 37 degrees C for 30 min prior to assessing sperm viability, motility, and tyrosine phosphorylation of soluble and whole-cell proteins. There was a time-dependent decline in sperm motility, which was to some extent reversed by incubation with theophylline. The sum of the phosphotyrosine signal from two soluble proteins (M(r) 67 000 and 36 000) declined with incubation time in both theophylline-treated and untreated sperm. There were major differences in the pattern of tyrosine phosphorylation during incubation between ejaculates from different bulls. Tyrosine phosphorylation of a number of proteins from whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the medium. The oxygenation state of the incubation medium had profound effects on sperm motility, viability, and tyrosine phosphorylation of proteins from whole-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of proteins from whole-cell extracts increased considerably during anaerobic incubation, while there was no significant change during aerobic incubation. This increase in phosphorylation due to anaerobic incubation was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm incubated under aerobic conditions for 5 days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. The increase in protein tyrosine phosphorylation during in vitro incubation took place in a medium that did not contain capacitating substances such as heparin, sodium bicarbonate, or BSA. It transpired over a time scale of days and was not augmented by an increase in intracellular cAMP concentration through phosphodiesterase inhibition. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen in the medium.

Aerobiosis↗

PubMed1999

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.

Animals↗

PubMed1999

Measurements of bovine sperm velocities under true anaerobic and aerobic conditions.

Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.

Animals↗

PubMed1999

Replicate and technician variation associated with computer aided bull sperm head morphometry analysis (ASMA).

Associations of abnormal spermatozoa with bull fertility have yielded varying results. Manual methods of analysis are subjective and highly variable within and between technicians, which may account for these differences. Computer-aided sperm head morphometry appears to be a precise method of assessing sperm head dimensions; however, the effects of replication and technician on sperm head morphometry have not been assessed. The objective of this study was to determine the inter- and intra-analysis and technician variation associated with computer-aided bull sperm head morphometry analysis. Semen from 10 bulls was diluted to 200 x 10(6) sperm/mL, and slide smears were prepared and stained using haematoxylin and rose bengal. Each of two technicians analysed 250 images from each slide, 3 times, using computer-aided sperm head morphometry analysis. The morphometric dimensions of area, perimeter, length, width and width/length for individual sperm heads of each analysis were assessed by GLM-ANOVA for effects of bulls, replications and technicians. The coefficient of variation was recorded for each analysis and across replications. The mean coefficients of variation within and between analyses were compared between technicians by GLM-ANOVA. No differences (p > 0.1) between technicians were found between or among bulls for area (29.63 vs. 29.26 micron 2), perimeter (23.73 vs. 23.86 microns), length (8.73 vs. 8.71 microns), width (4.47 vs. 4.46 microns), or width/length (0.51 vs. 0.51). No differences (p > 0.1) between replicates for sperm head dimension were detected within or among bulls for either technician. No intra- or inter-analysis differences (p > 0.1) between technicians on CVs were observed. The mean intra-analysis CVs for all bulls for both technicians were area = 6.9%, perimeter = 4.9%, length = 4.5%, width = 5.6% and width/length = 6.5%. The mean interanalysis CVs for both technicians were area = 3.0%, perimeter = 2.4%, length = 2.0%, width = 2.0%, and width/length = 1.7%. The results indicate that ASMA is a repeatable and objective method of assessing bull sperm head morphometry within and between technicians. No differences between replications were detected, and hence replicate analyses are not necessary to acquire accurate morphometric data.

Animals↗

PubMed1998

Studies on aromatic amino acid oxidase activity in ram spermatozoa: role of pyruvate as an antioxidant.

Chemical antioxidants and catalase have been shown to be ineffective in improving the motility of ram spermatozoa in a chemically-defined diluent (RSD-1). In an attempt to identify the biochemical basis of this observation, the activity of aromatic amino acid oxidase (AAAO), the enzyme responsible for generation of hydrogen peroxide in ram and bull spermatozoa, has been investigated. Ram spermatozoa contained higher levels of AAAO activity than bull spermatozoa, although the physico-chemical properties of the enzyme were generally similar in both species. Components of the medium had a marked effect upon AAAO activity. In the presence of glutamate and 3-(N-morpholino)propanesulfonic acid (MOPS), AAAO activity was decreased. Pyruvate appeared to increase AAAO activity. This was due to the ability of this substance to destroy hydrogen peroxide. Pyruvate in RSD-1 works as an effective antioxidant and therefore eliminates the need for other antioxidants in the semen diluent.

Amino Acid Oxidoreductases↗

PubMed1998

Estrus detection using radiotelemetry or visual observation and tail painting for dairy cows on pasture.

The efficiency and accuracy of estrus detection using HeatWatch (DDx Inc., Denver, CO) or visual observation were compared in an autumn-calving Friesian herd (n = 48 per group) and a spring-calving Jersey herd (n = 50 per group) grazing on pasture. Cows in the group monitored by the HeatWatch system were fitted with a pressure-sensitive transmitter that signaled mounting activities associated with estrus. Visual observation was carried out for about 20 min before the morning and afternoon milkings and was aided by a strip of paint applied over the tailhead. Ovarian cyclicity was monitored with progesterone concentrations in milk samples collected twice a week. The efficiency and accuracy of estrus detection were, respectively, 98.4 and 97.6% for visual observation and 91.7 and 100% for HeatWatch detection. Autumn-calving herds differed from spring-calving herds in duration of estrus (9.7 vs. 7.3 h), number of mounts (13.6 vs. 8.5), total duration of mounts (36.8 vs. 19.9 s), and mean duration of a mount (2.6 vs. 2.3 s). There was no significant variation in the distribution of the time of onset of estrus or mounting activities at different hours of the day. Conception rate was similar for AI after estrus detection with HeatWatch (65.8%) or after visual observation (65.0%). The highest conception rate was obtained when AI was carried out between 12 and 18 h after the first mount. Both the HeatWatch system and visual observation plus tail painting can be used for estrus detection of dairy cows on pasture.

Animals↗

PubMed1997

Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers.

This experiment was designed to test whether spermatozoa encapsulated in an alginate poly-L-lysine matrix had an extended fertile life in vivo after insemination. Estrus was synchronized in 417 primiparous Friesian and Jersey heifers with a system based on a CIDR-B intravaginal device before the heifers were inseminated either during proestrus (24 h after device removal) or at estrus (48 h after device removal). Pregnancy rates to first inseminations did not differ between the 24 and 48 h inseminations (61 vs 60.6%) with liquid semen diluted in Caprogen (control) but differed with encapsulated semen (45.1 vs 68.6%). The difference in pregnancy rates between the 2 types of semen was more pronounced (P < 0.08) in the animals that were visually detected in estrus. The mean survival time of spermatozoa in the female reproductive tract following insemination at the 24-h insemination time was estimated to be 50 +/- 7.5 h. The increased pregnancy rate with insemination of encapsulated spermatozoa at 48 h could have been due to this process predisposing spermatozoa to capacitate soon after insemination.

Journal Article↗

PubMed1997

Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperature.

In a liquid environment, at high dilutions, fertility of bull sperm is maintained for 3-5 days when stored at ambient temperatures (10-21 degrees C), after which time it steadily declines at a rate of 3-6% per day. This decline in fertility occurs irrespective of whether the sperm are stored at 5 degrees C or at 15 degrees C, but the rate is greater once storage temperatures exceed 25 degrees C. Sperm motility can be maintained for extended periods in an environment where the extracellular oxidative stress is minimized by reducing the oxygen tension, by addition of antioxidants and chelating agents; however, this will not prevent a significant drop in fertility after five days of storage at ambient temperature. The requirement of energy by the sperm-motility apparatus demands a high level of respiratory activity. This system is very active and the free radicals produced in vivo during this process could lead to chromatin damage. As no internal repair mechanism exists in sperm, an extraneous supply of protectants, or an environment where damage is minimized, is essential to maintain its fertilizing potential. The lack of extended storage potential of sperm, even in the presence of antioxidants, seems to suggest that although oocyte-penetrating ability of the sperm could still be intact, the high rate of intracellular metabolic activity could lead to mitochondrial DNA damage and chromosomal abnormalities that would compromise the viability of the resulting conceptus.

Animals↗

PubMed1996

Computer automated morphometric analysis of bull sperm heads.

Morphological type classification of spermatozoa is an important component of the modern semen evaluation; however, current methods of analysis are subjective and highly variable between technicians. To reduce the subjectivity and thus variability of sperm morphology assessment, computer automated sperm head morphology analysis (ASMA) has been developed. Previous studies have shown the importance of standardizing ASMA procedures to optimize accuracy. The objective of this study was to standardize ASMA procedures for evaluating bull sperm heads. Semen from 10 fertile bulls was used to standardize procedures for optimal analysis of bull spermatozoa. Sample preparation methods, sperm staining methods and microscopic magnifications were compared. Semen samples that were diluted to a standard concentration of 200 x 10(6) sperm/ml were more efficiently analyzed than raw samples. A modified GZIN staining procedure, incorporating rose bengal as an acrosomal stain, was used for accurate ASMA at a magnification of x 60. The mean morphometric measurements for all bulls were the area (27.30 microM), perimeter (25.36 microM), length(8.65 microM), width(4.40 microM) and width/length (0.50). Within the analyses, coefficients of variation ranged from 3.45% for length to 8.52% for area. The ASMA system correctly digitized sperm heads 97% of the time. Results of this study indicate that bull sperm heads can be accurately analyzed using current standard procedures of ASMA technology.

Journal Article↗

PubMed1996

Substance P promotes lymphocyte-endothelial cell adhesion preferentially via LFA-1/ICAM-1 interactions.

Substance P (SP), an 11 amino acid peptide, is released by C and A delta sensory nerve fibers during tissue insult and inflammation. We investigated the effect of SP on the expression and avidity of adhesion molecules, on lymphocytes and endothelial cells, which are central to the inflammatory cascade. Using in vitro adhesion assays we found that pretreatment of murine endothelial cells with SP enhanced their adhesiveness to splenocytes, the murine T cell hybridoma EL4 and nylon-enriched primary murine T cells in a dose and time dependent manner, the optimum dose being 10(-10) M and the optimum time 6 h. SP at 10(-10) M was also able to stimulate the splenocytes, EL4 T cells and primary T cells to enhance their adhesiveness for endothelial cells. The increased adhesiveness was associated with enhanced expression of ICAM-1 on endothelial cells and increased avidity of LFA-1 on lymphocytes. Further SP was chemotactic for T cells. These data suggest that SP modulates lymphocyte-endothelial cell interactions by preferentially upregulating LFA-1 and ICAM-1 interactions.

Animals↗

PubMed1996

The psychoneuroimmunological system: a recently evolved networking organ system.

The phenomenon of psychological influences on immune activity and general physiology has generated a great deal of interest in the last decade. Several theories have been proposed to understand the evolution and the nature of this bi-directional communication and/or influence. It is shown that the existing theories do not fully explain most phenomena associated with this communication. A theory--the networking organ theory--is also suggested that could resolve the issue. The information obtained from recent studies on evolutionary history and the fossil record have been used as the basis for understanding the phenomenon of psychoneuroimmunology. In essence, we are not looking at the unification of the mind and the body, but at a recently evolved system that lends sophistication to the existing connection between the mind and the body.

Animals↗

PubMed1994

Anionic and cationic components from protein aggregates in bovine seminal plasma and their effects on sperm motility.

Bovine seminal plasma proteins are in an aggregated form of high molecular weight in their native state. By immobilisation on a cation exchanger with exposure to disaggregating conditions (i.e., acetonitrile and low pH), the high-molecular-weight aggregates could be dissociated to slowly release the low-molecular-weight components. The anionic component released from the cation exchanger during disaggregation was collected by adsorption on a hydrophobic interaction column. The cationic component remaining on the cation exchanger was eluted with NaOH. Both components were found on gel permeation chromatography to be < 5 kDa. SDS-PAGE of the various fractions showed that components of low molecular weight were still in an aggregated form. These components resulting from the disaggregation process have detrimental effects on sperm motility and the effects were more substantial compared with that of whole seminal plasma. All the cationic components were significantly detrimental to sperm motility, especially the fractions of low molecular weight. The anionic fractions reduced sperm motility when in an aggregated state. The isolated anionic peptide was not detrimental in its free form. In all fractions the peptides tended to re-aggregate to a higher molecular weight under neutral conditions, however, the isolated anionic peptide (molecular weight < 1,500) failed to do so.

Animals↗

PubMed1994

Low molecular weight components in bovine semen diffusate and their effects on motility of bull sperm.

Dialysis of diluted semen before cryopreservation is beneficial to sperm survival. This is due to removal of low molecular weight components from seminal plasma that are damaging to sperm. The apparent molecular weights (M(r)) of these components range between 1000 and 12,000 as estimated by gel permeation chromatography and electrophoresis. The detrimental effect on sperm motility is greatest with the components of M(r) between 5000 and 12,000 and with those of M(r) < 1500. Their effect on sperm motility was dependent on concentration. The small molecular weight components were derived from the high molecular weight components of seminal plasma through disaggregation under prescribed conditions.

Animals↗

PubMed1993

Microencapsulation of bovine spermatozoa for use in artificial insemination: a review.

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus of potential use in artificial insemination. The polymers poly-l-lysine, polyvinylamine and protamine sulfate have proven best for membranes. Encapsulation has been successful with capsules ranging in size from 0.75 to 1.5 mm, and with sperm concentrations from 45 to 180 x 10(6) cells mL-1. Successful extenders include CUE, CAPROGEN, and egg yolk-citrate-glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under ageing and physical stress) is negatively related to membrane thickness which ranges from 1.92 to 5.32 microns (depending on the concentration of polymer used) and positively related to concentration of sperm encapsulated. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when cows are inseminated at conventional times. Uterine retention of inseminates is favoured by capsules having a 'sticky' membrane. Using current procedures, preliminary homospermic fertility studies indicate that sperm encapsulated with poly-l-lysine or protamine sulfate may achieve normal fertility.

Animals↗