Response of buffalo spermatozoa to low temperatures during cryopreservation.

This is the first detailed report on the response of buffalo spermatozoa to low temperatures during freezing. The study determined the critical temperature zone for buffalo spermatozoa and developed a suitable freezing rate for this species. Semen from four Nili-Ravi buffalo bulls diluted in Tris-citric acid was frozen in a programmable freezer. Motion characteristics, plasma membrane integrity and acrosome morphology were determined at +4, 0, -5, -10, -20, -30, -40, -50, -80 and -196 degrees C by removing semen straws from the freezer at exactly these temperatures and rewarming them at 37 degrees C. The first statistical decline in sperm motility and lateral head displacement was observed at -40 degrees C. For all other parameters, there was biphasic decline: for curvilinear velocity, at 0 degrees C and -50 degrees C; and for plasma membrane integrity and acrosome morphology, at -30 degrees C and -50 degrees C. In a second series of experiments, buffalo spermatozoa were frozen using slow (-10 degrees C min(-1)), medium (-20 degrees C min(-1)) or fast (-30 degrees C min(-1)) freezing rates, between -10 degrees C and -80 degrees C. Freezing of buffalo spermatozoa at a rate of -30 degrees C min(-1) yielded higher post-thaw motion characteristics, plasma membrane integrity and normal acrosomes. In conclusion, different sperm characteristics respond differently at low temperatures and the freezing of buffalo spermatozoa at a higher rate ensures higher post-thaw semen quality.

Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm.

The objective of the present study was to investigate the synergistic effect of DMSO and glycerol added at various temperatures on the post-thaw quality of buffalo sperm. Pooled ejaculates from four Nili-Ravi buffalo bulls were divided into 18 aliquots and extended (1:10) in Tris-citric acid extender differing in glycerol:DMSO ratios (0:0, 0:1.5, 0:3; 3:0, 3:1.5, 3:3; and 6:0, 6:1.5, 6:3, respectively; %, v:v) either at 37 or 4 degrees C. Semen was packaged in 0.5 mL French straws and frozen in a programmable cell freezer. Thawing was performed at 37 degrees C for 50s. Post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo sperm were determined using computer-assisted semen analyzer (CASA), hypoosmotic swelling (HOS) assay and phase-contrast microscopy, respectively. Glycerol (6%) in extender yielded better post-thaw sperm motility, velocities (straight-line and average path), plasma membrane integrity, and normal acrosomes (P<0.05). Post-thaw sperm motility and plasma membrane integrity declined in the presence of DMSO (P<0.01). The addition of glycerol (6%) at 37 degrees C yielded better post-thaw sperm motility, plasma membrane integrity and velocities than addition at 4 degrees C (P<0.05). In conclusion, glycerol is still an essential cryoprotectant for buffalo sperm. The addition of DMSO antagonized the cryoprotection ability of glycerol and reduced the post-thaw quality of buffalo sperm. Furthermore, 6% glycerol added at 37 degrees C, provided better cryoprotection to the motility apparatus and plasma membrane integrity of buffalo sperm.

Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa.

Motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa after different stages of cryopreservation (ie, dilution, cooling to 4 degrees C, equilibration at 4 degrees C, and freezing and thawing) were examined. Semen ejaculates from 4 buffalo bulls were pooled (n = 5), diluted in tris-citric acid extender, cooled to 4 degrees C over 2 hours, equilibrated at 4 degrees C for 4 hours, dispensed into 0.5-mL straws, and frozen in a programmable cell freezer before plunging into liquid nitrogen. Frozen semen was thawed at 37 degrees C for 15 seconds. After completion of each stage, sperm motion characteristics, plasma membrane integrity, and acrosomal morphology were determined using computer-assisted semen analysis, hypo-osmotic swelling assay, and phase-contrast microscopy, respectively. Data were presented as mean +/- standard error of the mean. Visual and computerized motility did not differ due to dilution, cooling, or equilibration (77.3% +/- 2.3% and 90.5% +/- 1.2%, respectively), but was reduced (P < .05) after freezing and thawing (53.0% +/- 4.6% and 48.6% +/- 6.5%, respectively). Linear motility of spermatozoa was lower (P < .05) after dilution or equilibration (56.2% +/- 2.4%) than after cooling or freezing and thawing (79.6% +/- 1.4%). Sperm curvilinear velocity was reduced (P < .05) from 112.4 +/- 5.3 microm/sec after dilution to 96.0 +/- 5.8 microm/s after cooling, and from 87.6 +/- 4.1 microm/s after equilibration to 69.4 +/- 2.0 microm/s after freezing and thawing. Sperm lateral head displacement differed (P < .05) after each stage (ie, dilution, 3.9 +/- 0.2 microm; cooling, 2.3 +/- 0.2 microm; equilibration, 3.1 +/- 0.3 microm; and freezing and thawing, 1.7 +/- 0.2 microm). Spermatozoa with intact plasma membranes were 80.2% +/- 3.9% after dilution, reduced (P < .05) to 60.4% +/- 5.6% after equilibration, and then to 32.6% +/- 3.8% after freezing and thawing. The percentage of spermatozoa with normal acrosomes remained higher after dilution, cooling, or equilibration (73.2% +/- 2.4%) than after freezing and thawing (61.8% +/- 2.4%; P < .05). In conclusion, the maximal damage to the motility apparatus, plasma membrane, and acrosomal cap of buffalo spermatozoa occurs during freezing and thawing followed by equilibration.

The effect of ketamine hydrochloride anesthesia on basal and N-methyl-D,L-aspartate induced plasma prolactin secretion in the adult male rhesus monkey.

The excitatory amino acids (EAAs), glutamate and aspartate, acting predominantly on N-methyl-D-aspartate (NMDA) receptor, have been shown to be involved in the central regulation of the secretion of several anterior pituitary hormones including prolactin (PRL), whereas ketamine hydrochloride (KH), a widely used anesthetic, has been reported to antagonize a variety of NMDA receptor mediated actions of these EAAs. In the present study, the effect of KH on basal PRL levels as well as on N-methyl-D,L-aspartate (NMA), an agonist of NMDA receptor, induced plasma PRL secretion was investigated in the adult male rhesus monkey. The values were compared to those obtained from the same animals restrained in primate chairs. The plasma PRL concentrations were higher in animals receiving KH administered either intramuscularly (2.5 mg/kg BW at 30 min intervals) or intravenously (10 mg/kg BW) as compared to those observed in the unanesthetized chair-restrained monkeys. NMA induced an unequivocal increase in plasma PRL concentrations in both conscious chair-restrained and KH anesthetized monkeys, but the response was greater in anesthetized animals than the conscious monkeys. The present findings suggest that KH has stimulatory effects on both basal and NMA induced plasma PRL secretion.

Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa.

This study was carried out to identify the suitable buffer for cryopreservation of buffalo semen. Semen was collected with artificial vagina (42 degrees C) from four buffalo bulls. Split pooled ejaculates (n=5), possessing more than 60% visual sperm motility, were extended at 37 degrees C either in tri-sodium citrate (CITRATE), Tris-citric acid (TCA), Tris-Tes (TEST) or Tris-Hepes (HEPEST). Semen was cooled to 4 degrees C in 2 h, equilibrated at 4 degrees C for 4 h, filled in 0.5 ml straws and frozen in a programmable cell freezer before plunging into liquid nitrogen. Thawing of frozen semen was performed after 24 h at 37 degrees C for 15 s. Sperm motion characteristics, plasma membrane integrity, and acrosome morphology of each semen sample were assessed by using computer-assisted semen analyzer (CASA), hypo-osmotic swelling (HOS) assay, and phase-contrast microscope, respectively. Analysis of variance revealed that percent post-thaw visual motility tended (P=0.07) to be higher in HEPEST (61.0+/-2.9) and lowest in CITRATE (48.0+/-2.5). Computerized motility did not vary due to buffering system. Percent post-thaw linear motility tended (P=0.09) to be higher in TCA (78.2+/-5.5) and lower in TEST (52.0+/-6.9). Circular motility (%) was significantly lower (P<0.05) in TCA (11.6+/-2.8) and higher in TEST (29.8+/-5.6). Curvilinear velocity (microm s(-1)) was lower (P<0.05) in TCA (69.4+/-2.0) than in CITRATE (79.0+/-5.8), TEST (87. 2+/-1.6) and HEPEST (82.6+/-3.0). Lateral head displacement (microm) was lowest (P<0.05) in TCA (1.7+/-0.2) and highest in TEST (3.7+/-0. 6). Plasma membrane integrity and normal acrosomes of buffalo spermatozoa did not differ due to buffering system and averaged 40. 0+/-2.7% and 61.4+/-4.6%, respectively. Based upon lower circular motility, curvilinear velocity, and lateral head displacement, it is concluded that post-thaw quality of buffalo semen can be improved using the Tris-TCA buffering system.

Supporting women's empowerment in Djibouti.

PIP: This article reports on the work carried out by the UN Population Fund (UNFPA) in the small island of Djibouti, Africa. The republic's population has been plagued with problems of high levels of unemployment, poverty, malnutrition, an almost non-existent family reproductive health care service, 100% prevalence rate of female genital mutilation and low literacy rate, especially for women. In addition, refugees from Ethiopia and Eritrea have settled in the country increasing the risks of sexually transmitted diseases (STDs), HIV/AIDS, prostitution, and other social ills. In 1983, UNFPA started funding family planning and later reproductive health projects aimed at assuring access to services for a majority of Djiboutan women. The first country population program of assistance was started in 1992. This would help the government with health care for its population and to conduct a population census. In addition, the Fund has paid for training of doctors, midwives, and traditional birth assistants in the country and for rehabilitating maternity clinics and information centers. Moreover, it has supported agencies concerned with educating people on STDs, HIV/AIDS, safe motherhood and reproductive health for men and women, and other important issues.^ieng

Enterotoxicity ofVibrio furnissii isolated from eels.

Fourteen strains ofVibrio furnissii, isolated from different ulcerated areas of eel, were tested to check their enterotoxicity in an animal model. Most strains caused fluid accumulation in ileal loop tests after serial passages and culture filtrates of most of the strains caused induration and increase in vascular permeability in rabbit skin. Production of extracellular haemolysin was also detected in all the culture filtrates. All of these observations clearly establish the enterotoxicity of these organisms.