Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.     

Effects of linear cooling rate on motion characteristics of stallion spermatozoa

Three experiments were designed to analyze the effects of cooling rate on survival of stallion spermatozoa in a milk-based extender, at 0 to 96 hours after reaching the desired temperature. The samples were warmed to 37 degrees C and were evaluated by computer-assisted analysis of sperm motility. In Experiment 1, rate of cooling between 37 and 20 degrees C was evaluated. Sperm motion was not affected by cooling at plunge, -0.42 or -0.28 degrees C/minute. However, storage of spermatozoa at 5 degrees C after slow cooling below 20 degrees C was superior to storage at 20 degrees C. In Experiment 2, 3 cooling rates from 37 degrees to 5 degrees C were evaluated. Cooling at either -0.05 or -0.7 degrees C/minute was superior (P<0.05) to plunging spermatozoa to 5 degrees C. Cooling at -0.05 degrees C/minute rather than -0.7 degrees C/minute maximized the percentage of motile spermatozoa and their curvilinear velocity. In Experiment 3, cooling rates from 20 to 5 degrees C were evaluated, with all samples cooled at -0.7 degrees C/minute from 37 to 20 degrees C. Sperm motion was similar (P>0.05) after cooling below 20 degrees C at -0.012, -0.05 or -0.10 degrees C/minute, and the 2 slower rates were superior (P<0.05) to cooling at -0.3 degrees C/minute. It was concluded that stallion spermatozoa can be cooled rapidly from 37 to 20 degrees C, but should be cooled at 相似文献   

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Cooling rates,storage temperatures and fertility of extended equine spermatozoa

Two experiments were designed to evaluate cooling rates and storage temperatures for stallion spermatozoa extended in caprogen (CAP), Cornell University extender (CUE), heated skimmilk (SM) and a nonfat-dried milk solids glucose extender (NFDMS-glucose). In Experiment 1, each extender was evaluated in a separate but similar 4 × 4 × 6 factorial trial using two ejaculates from each of six stallions. Aliquots of 66 × 10⁶ spermatozoa were transferred to each of 16 coded tubes and extended to 6 ml with SM, CAP, CUE or NFDMS-glucose. Four tubes of extended semen were either plunged into 5C water or cooled at a rate of ?1.0, ?0.5, or ?0.2C/min. Within each treatment, one tube of extended semen was maintained at 20C, 15C, 10C or 5C. Progressive spermatozoal motility was estimated immediately after dilution (0 h) and at 4, 8, 12, 24 and 36 h. Regardless of extender, all three slower cooling rates were superior (P<0.05) to plunging to 5C; storage temperatures of 20C and 15C were superior (P<0.05) for maintaining spermatozoal motility. Experiment 2 was designed so that all extenders could be evaluated simultaneously. Since CUE resulted in an immediate depression of spermatozoal motility, it was not evaluated further. Semen was collected from 12 stallions and each ejaculate was extended in SM, CAP and NFDMS-glucose. Semen was cooled at ?1.0C/min and maintained at either 20C or 15C. Spermatozoal motility was assessed as in Experiment 1. Overall, the CAP and NFDMS-glucose extenders were superior (P<0.05) to SM for maintenance of spermatozoal motility. Storage at 20C or 15C resulted in similar (P>0.05) spermatozoal motility. Two fertility trials compared the use of SM and NFDMS-glucose extenders. Embryo recovery 6 days post-ovulation (Experiment 3) and pregnancy rate 50 days postestrus (Experiment 4) was similar (P>0.05) for mares inseminated with spermatozoa extended in SM or NFDMS-glucose.     

Seasonal functional relevance of sperm characteristics in equine spermatozoa

A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed clear seasonal-based differences (P < 0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period, animal, and fertility were evident (P < 0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus, it may be advisable to adjust the use of stallion semen according to seasonal variations.     

Effect of antibiotics on motion characteristics of cooled stallion spermatozoa

The control of bacteria in semen of stallions has been most effective with the use of seminal extenders containing suitable concentrations of antibiotics. However, the detrimental effect of antibiotics on sperm motility may be greater in stored, cooled semen due to the prolonged exposure to the antibiotic. Therefore, a study was conducted to determine the effect of various antibiotics on sperm motion characteristics following short term exposure and during cooled storage of semen. Reagent grade amikacin sulfate, ticarcillin disodium, gentamicin sulfate and polymixin B sulfate were added to a nonfat, dried, skim milk - glucose seminal extender at concentrations of 1000 or 2000 mug or IU/ml. Aliquots of raw semen were diluted with extender-antibiotic combinations to a concentration of 25 x 10(6) spermatozoa/ml. An aliquot was also diluted with extender without antibiotic. Aliquots were incubated at 23 degrees C for 1 h. In addition, portions of the aliquots were cooled from 23 to 5 degrees C and stored for 48 h. During 1 h of incubation of extended semen at 23 degrees C, there was a significant (P<0.05) reduction in the percentage of progressively motile spermatozoa for samples containing gentamicin sulfate. After 24 h of storage at 5 degrees C, 2000 mug/ml of gentamicin and levels equal to and greater than 1000 IU/ml of polymixin B in seminal extender resulted in significant (P<0.05) reductions in the percentages of motile and progressively motile spermatozoa. After 48 h of cooled storage, a level of 1000 mug/ml of gentamicin sulfate. resulted in significant (P<0.05) reductions in the percentages of motile and progressively motile spermatozoa. Levels equal to or greater than 1000 IU/ml of polymixin B sulfate also resulted in a significant (P<0.05) reduction in mean curvilinear velocity. Levels up to 2000 mug/ml of amikacin sulfate and ticarcillin disodium had no significant effect on sperm motion characteristics during short-term incubation at 23 degrees C or storage for 24 h at 5 degrees C. Overall, the addition of antibiotics to extender did not significantly (P>0.05) improve motion characteristics of spermatozoa over control samples. However, levels of gentamicin sulfate greater than 1000 mug/ml and of polymixin B sulfate equal to or greater than 1000 IU/ml should be avoided in seminal extenders used for cooled semen.     

Effects of elevated testicular temperature on morphology characteristics of ejaculated spermatozoa in the bovine

A mild thermal insult to the testes was studied with respect to ejaculated sperm motility and morphology. A 48-h scrotal insulation was administered to 6 young Holstein bulls whose semen was collected by artificial vagina twice in succession at 3-d intervals for 7 wk. For assessment of results, collection days were grouped in the follwing way: Period 1 (control) = Days -6, -3 and 0, where Day 0 = initiation of scrotal insulation after semen collection; Period 2 = Days 3, 6 and 9 (spermatozoa presumed to be in the epididymis or rete testes during scrotal insulation); and Period 3 = Days 12 and 15 through 39 (spermatozoa presumed to be in spermatogenesis during scrotal insulation). Daily sperm output per collection day was 5.3±0.7 × 10⁹ and did not differ across the experimental periods. Moreover, Periods 1 and 2 did not differ in the mean percentage of motility or in the mean percentage of abnormal spermatozoa (69.1±2.5 and 19.6±5.7%, respectively, for Period 1). Morphological change was first noted on Day 12 (47.5±27.4% abnormal) and peaked on Day 18 (86.3±24.3%). Motility depression began on Day 12 and reached a nadir on Day 15 (42.0±9.8%). Bulls varied in the type of abnormal spermatozoa produced and in magnitude of response; however, specific abnormalities appeared in ejaculates in a predictable chronological sequence following scrotal insulation (Day 0). The sequence was: tailless, (Days12 to 15); diadem, (Day 18); pyriform and nuclear vacuoles, (Day 21); knobbed acrosome, (Day 27); and Dag defect, (Day 30).     

Effects of angiotensin II on the acrosome reaction in equine spermatozoa

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.     

Effect of ambient temperature storage on potable water coliform population estimations

The effect of the length of time between sampling potable water and performing coliform analyses has been a long-standing controversial issue in environmental microbiology. The issue is of practical importance since reducing the sample-to-analysis time may substantially increase costs for water analysis programs. Randomly selected samples (from those routinely collected throughout the State of Wisconsin) were analyzed for total coliforms after being held at room temperature (20 +/- 2 degrees C) for 24 and 48 h. Differences in results for the two holding times were compared with differences predicted by probability calculations. The study showed that storage of the potable water for up to 48 h had little effect on the public health significance of most samples containing more than two coliforms per 100 ml.     

Effects of ambient temperature on avian incubation behavior

Ambient temperature is commonly thought to influence avian incubation behavior. However, results of empirical studies examining correlationsbetween ambient temperature and bout duration are equivocal.We propose that these equivocal results can be partly explainedby developing a conceptual understanding of how we should expecttemperature to influence incubation. We demonstrate why linearcorrelation analyses across a wide range of temperatures canbe inappropriate based on development of an incubation model for small birds that incorporates how ambient temperature influencesboth embryonic development and adult metabolism. We found supportfor predictions of the model using incubation data from orange-crownedwarblers (Vermivora celata) in Arizona. Both off- and on-boutduration were positively correlated with ambient temperaturebetween 9° and 26°C, but unrelated to ambient temperature<9° and 26-40°C. Bout durations declined as ambienttemperature approached or exceeded 40°C. Incubating orange-crowned warblers appeared to avoid bouts off the nest <7 min andbouts on the nest <20 min. Time of day, duration of theprevious bout, and variation among nests all explained variationin both on- and off-bout duration. Although we found supportfor the general shape of the incubation model, temperature still explained only a small portion of the overall variation in on-and off-bout duration. Results of previous studies were generallyconsistent with the model for off-bout duration; most studiesin colder environments reported positive correlations withtemperature, and the one negative correlation reported was from a hot environment. However, the relationships between on-boutduration and temperature reported in previous studies wereless consistent with our model and our data. Although somediscrepancies could be explained by considering our model,some studies reported negative correlations in cold environments.The effect of ambient temperature on duration of on-bouts probablydiffers among species based on the amount of fat reserves females typically carry during incubation and the extent of male incubationfeeding. Additional studies of the effects of temperature onavian incubation will help improve the general model and ultimatelyaid our understanding of energetic and ecological constraintson avian incubation.     

Effects of peristaltic and longitudinal wave motion of the channel wall on movement of micro-organisms: application to spermatozoa transport

A mathematical model is presented to study the motion of the spermatozoa in the cervical canal by considering the transverse waves along its tail and the transverse and longitudinal motions of the cervical wall. In an attempt to control fertility by reducing the speed of sperm, the transverse waves have been considered in the direction opposite to the motion of the spermatozoa. It has been shown that by having appropriate transverse wave motion and longitudinal velocity, the sperm may not be able to move towards the oviduct even if it could continue to have its own propelling velocity. A particular case of the motion of a thin plane sheet in a channel under peristaltic motion of its walls has also been obtained and studied.     

Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa

The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.     

Effect of ambient temperature storage on potable water coliform population estimations.

The effect of the length of time between sampling potable water and performing coliform analyses has been a long-standing controversial issue in environmental microbiology. The issue is of practical importance since reducing the sample-to-analysis time may substantially increase costs for water analysis programs. Randomly selected samples (from those routinely collected throughout the State of Wisconsin) were analyzed for total coliforms after being held at room temperature (20 +/- 2 degrees C) for 24 and 48 h. Differences in results for the two holding times were compared with differences predicted by probability calculations. The study showed that storage of the potable water for up to 48 h had little effect on the public health significance of most samples containing more than two coliforms per 100 ml.     

Desiccation tolerance of spermatozoa dried at ambient temperature: production of fetal mice

Long-term preservation of mouse sperm by desiccation is economically and logistically attractive. The current investigation is a feasibility study of the preservation of mouse sperm by convective drying in an inert gas (nitrogen). Mouse sperm from the B6D2F1 strain isolated in an EGTA-supplemented Tris-HCl buffer were dried using three different drying rates and were stored for 18-24 h at 4 degrees C. The mean final moisture content was <5% for all the protocols. After intracytoplasmic sperm injection (ICSI), the mean blastocyst formation rates were 64%, 58%, and 35% using the rapid-, moderate-, and slow-drying protocols, respectively. The slow-drying protocol resulted in a rate of development significantly lower than that observed using rapid- and moderate-drying protocols and indicated that a slower drying rate may be detrimental to the DNA integrity of mouse sperm. The transfer of 85 two- or four-cell embryos that were produced using rapidly desiccated sperm resulted in 11 fetuses (13%) on Day 15 compared with the production of 34 fetuses (40%) produced using the transfer of 86 two- or four-cell embryos that were produced using fresh sperm (P < 0.05). The results demonstrate the feasibility of using a convective drying protocol for the successful desiccation of mouse sperm and identifies some of the important parameters required for optimization of the procedure.     

Effects of ambient temperature steps on thermal comfort requirements

The purpose of this study was to determine the thermal comfort requirements for steps in temperature. Thirty male subjects were exposed for 50 min to a 34 or 37°C condition, and then quickly transferred to a cooler environment of 31, 28, 25, and 22°C for 50 min. Mean skin temperature was continuously measured, and the subjects reported their thermal sensation and comfort sensation every 2 min. Just after the step changes, the mean skin temperature immediately decreased, while the thermal sensation overshot and gradually rose again. Both the skin temperature and the thermal sensation seemed to reach a constant level within about 20 min. However, there were differences in the mean skin temperature and the neutral temperature derived from the correlation between the ambient temperature and the thermal sensation even 50 min after the steps, due to the thermal environmental condition before the changes of temperature. The change in the neutral temperature with time was expressed as two attenuating equations. These equations indicate that there is an obvious difference between the neutral temperatures due to the thermal condition before step changes, and that it takes >50 min after the step changes to reach the steady state. It is expected that these equations predict in quantitative terms the thermal comfort requirements within a given experimental condition.     

Long-term storage of somatic embryogenic white spruce tissue at ambient temperature

The effeet of long-term storage on the viability and regeneration eapacity of somatic embryogenic white spruce tissue (Picea glauca) was investigated. It was found that by keeping white spruce embryogenic tissue in serum-capped flasks at ambient temperatures, a viability of 80% could be maintained without subculturing for a period of over one year. Growth characteristics of the embryogenic tissue on solid medium and suspension cultures derived therefrom were essentially identical to the culture from which they originated. Ethylene and carbon dioxide accumulation peaked around days 8 to 10 in serum-capped cultures and declined thereafter during the short-term (20 days). When ethylene and carbon dioxide levels were measured over a period of five months, in flasks varying in their degree of gas tightness, it was found that those cultures that maintained higher carbon dioxide concentrations after five months remained viable and could be recultured under normal conditions. Development to cotyledon stage embryos from immature embryos could be obtained by exposure of the long-term storage material to 10 M ABA, and the number of maturing embryos was found to be similar to that of the original culture.     

Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection

The objective was to develop a sperm freezing procedure suitable for use in the propagation of valuable founder animals by assisted reproductive technologies. Here, we report a comparison of processing methods by measuring the motility of fresh and frozen-thawed rhesus monkey spermatozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed post-thaw with or without a density gradient centrifugation step. Among the four study series, motility post-thaw was improved with density gradient centrifugation (17-24% versus 75%, P<0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total n=377) were co-cultured on BRL cells and observed for fertilization and development. With spermatozoa frozen in straws in liquid nitrogen vapors, the fertilization rate after ICSI was lower than with fresh spermatozoa (40-44% versus 77-86%, P<0.05), even with the Percoll-enriched fraction that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date. Additionally, a 3-4 h incubation after thawing improved the fertilization rate with spermatozoa from a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate after ICSI with motile, frozen-thawed primate spermatozoa was observed comparable to that obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used with ICSI to facilitate propagation of desirable primate genotypes.