The hypoosmotic swelling test: Its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane

The objective of this study was to determine the effectiveness of the hypoosmotic swelling (HOS) test together with the supravital test as a means of evaluating the functional integrity of frozen-thawed bovine sperm membrane. A solution consisting of equal parts of fructose and sodium citrate was prepared and the osmolality varied from 50 to 300 mOsm/L. From these various solutions under study, the 100 mOsm/L solution resulted in a maximal number of clearly identifiable swollen spermatozoa. The results from the supravital test indicated that the HOS solution preserved the integrity and prevented excessive lysis of the sperm membrane during the assay. A good correlation was found between the percentage of motile spermatozoa and spermatozoa that reacted to the HOS test (r = 0.73) and between the percentage of sperm with intact membranes and HOS reactive sperm (r = 0.81). Spermatozoa showing swelling of the entire tail region accounted for more than 60% of the total swelling for the HOS solution at 100 mOsm/L. The results obtained in this study indicate that frozen-thawed bovine spermatozoa did react to the HOS test. This technique could prove useful in studies involving the function of the sperm membrane and could possibly predict the sperm's ability to fertilize.     

Four subpopulations of boar spermatozoa defined according to their response to the short hypoosmotic swelling test and acrosome status during incubation at 37 degrees C

This study was designed to confirm the previously observed relationship between response to the short hypoosmotic swelling test (sHOST) and acrosome resistance in boar spermatozoa. Ejaculates from 22 boars were incubated in a water bath at 37 degrees C for 2h. During the incubation period, samples were taken at 5, 20, 40, 60, 90 and 120 min and subjected to the sHOST. sHOST responses (positive HP-negative HN) and acrosomal status (normal or intact NA-damaged DA) were evaluated in 100 spermatozoa corresponding to each ejaculate and incubation time, and the results used to establish four subpopulations: HPNA, HPDA, HNNA and HNDA. Over the entire incubation period, the sHOST positive subpopulation with damaged acrosomes, HPDA, was significantly smaller than the sHOST negative, damaged acrosome subpopulation, HNDA (P<0.001). Further, proportions of HPDA spermatozoa remained stable throughout this period while the HNDA subpopulation showed a significant increase (P<0.001) from the start to the end of incubation. These results confirm the high resistance of the plasma membrane of HP spermatozoa allowing the persistence of a higher number of intact acrosomes over time, compared to HN spermatozoa. Characterising this HPNA subpopulation may help the evaluation of ejaculate quality.     

Simplified hypoosmotic swelling testing (HOST) of fresh and frozen-thawed canine spermatozoa

The clinical use of the hypoosmotic swelling test (HOST) to identify spermatozoa with a functional intact membrane has been reported for humans and domestic species, including the dog. Currently, it is recommended that canine spermatozoa be incubated with the hypoosmotic solution for periods that range from 30 to 60 min. In an attempt to simplify the test, it was hypothesized that the degree of the hypoosmotic response at 1 min of incubation would not be different from the response documented at 60 min after incubation in the hypoosmotic solution at 37 degrees C. The hypoosmotic response of spermatozoa from 50 fresh and 16 frozen-thawed semen samples obtained from 22 adult dogs was recorded at 1 and 60 min of incubation. There were no significant differences between the hypoosmotic response recorded at 1 and 60 min for all evaluated semen samples (P>0.10). The hypoosmotic response recorded for canine spermatozoa from fresh semen samples were greater than that recorded for spermatozoa from frozen-thawed semen, both at 1 min (86.2% compared with 65.2%; P<0.001) and 60 min (85.6% compared with 61.8%; P<0.001). Based on the results of this study, it is recommended to decrease the incubation time of the HOST for canine spermatozoa to as short a period as 1 min. This incubation time should encourage the application of this relatively simple and inexpensive test of canine sperm membrane function in a clinical setting.     

Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes

Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay consistency, accuracy, reliability and repeatability also were determined. Maximum spermatozoal plasma membrane swelling was observed in a 100 mosmol sucrose solution (P < 0.001). Incubation time (P = 0.67), temperature (P = 0.70) and total number of spermatozoa examined (P = 0.38 and P = 0.24 for 100 vs 200 and 100 vs 500, respectively) did not influence percent of HOS positive spermatozoa observed. A high degree of assay accuracy was indicated when a correlation of r = 0.998 was obtained between the HOS positive spermatozoa observed and expected when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. Assay consistency was demonstrated, as reflected by a mean coefficient of variation of 0.073 for 4 stallions. Also, the coefficient of variation from 2 analyses of variance was 0.168 and 0.096, indicating reasonably good assay reliability; estimates of repeatability from the same analyses were 0.794 and 0.968. The HOS test adapted to stallion spermatozoa in this study is a simple, highly accurate and consistent assay with good reliability and repeatability. Results observed under the conditions evaluated also permit some flexibility in adapting this assay to individual laboratory and practice settings for evaluating stallion spermatozoal plasma membranes.     

The hypoosmotic swelling test performed with coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout.

The hypoosmotic swelling test (HOS) is one of the methods used to evaluate sperm quality in mammals. This test is based on the swelling ability that functional spermatozoa have when submitted to hypoosmotic solutions. Only a slight increase in size is caused in rainbow trout spermatozoa in such conditions and it is not possible to distinguish between reactive cells (cells who were capable to increase in volume) and non-reactive cells (did not increase in volume) under light microscopy. In our approach we have used the coulter counter to verify the effectiveness of the HOS test in this species. Semen was diluted in different hypoosmotic solutions (50, 100, 150, 200, 250 and 320 mosM/kg) and cell volume measured at different times after dilution (30 s, 2, 5, 10, 20, and 30 min). The higher percentage of reactive cells was achieved with the 100 mosM/kg solution and swelling occurred before 30 s. Even with this solution, the small increase in cell size caused the overlapping of volumes from swollen and non-swollen spermatozoa. In order to analyse the data and to choose a parameter suitable for assessing cell reactivity, the test was performed in samples containing known rates of live/dead cells. Two parameters were analysed after swelling: the increase in volume and the percentage of cells over a standard volume (reactive cells). Results showed a high correlation between the percentages of reactive cells and the known rate of live cells (r2 = 0.65). This fact suggests that HOS test could be used to analyse the integrity and functionality of rainbow trout fresh sperm. To study the reliability of this test in cryopreserved sperm, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the two parameters obtained from coulter counter data. No significant correlation was observed in either case, showing that structural and functional integrity do not correlate after freeze/thaw. Consistently, the HOS test is not a reliable method to evaluate cryopreserved sperm quality in rainbow trout.     

Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity

The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.     

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.     

Effects of hypoosmotic incubation on acrosome and tail structure on canine spermatozoa

Hypoosmotic tests are widely used as valuable tests for determining sperm quality in species as varied as the human and the porcine. However, there is little information about the use of these tests in canine spermatozoa. This work evaluates the response of canine spermatozoa in hypoosmotic media in order to introduce the use of the hypoosmotic tests in the canine standard semen analysis. In this way, the incubation of canine spermatozoa in hypoosmotic media containing citrate (ORT medium, osmotic pressure = 100 mOsm) or citrate plus fructose (HOS medium, osmotic pressure = 150 mOsm) resulted in the swelling of the sperm tail. These reactions were time-dependent, reaching maximum percentages after 45 to 60 min. Optimal percentage of tail swelling with minimal effect on the viability of spermatozoa was observed at 100 to 150 mOsm. Response on sperm viability, tail swelling and acrosome detachment to hypoosmotic tests of both undiluted fresh, and 24 h-stored samples were similar. The percentage of swollen tails after both tests showed a good correlation to viability and to gross and progressive motility but not to concentration. However, acrosome detachment after both hypoosmotic tests did not correlate to any of the studied parameters. Our results indicate that the swelling observed after hypoosmotic shock could be used as a useful test in improving the standard semen analysis in the dog.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

Plasma membrane and acrosomal integrity in bovine spermatozoa with the knobbed acrosome defect

Previous studies have shown that bovine spermatozoa with the knobbed acrosome defect have a reduced ability to bind to and penetrate the bovine zona pellucida. Cryopreserved spermatozoa from a normal control bull (N) and two bulls with the knobbed acrosome defect (K1 and K2) were subjected to a hypoosmotic swelling test (HOST) to evaluate the functional integrity of the plasma membrane. A capacitation assay and a calcium ionophore challenge test was used to determine the ability of spermatozoa to undergo capacitation and acrosome reaction (AR), respectively. The mean percentage of spermatozoa responding to the HOST was significantly higher for Bull N (68.8 +/- 2.4) than for Bulls K1 (36.1 +/- 4.6) and K2 (40.2 +/- 4.7). The mean percentage of capacitated spermatozoa (54.0 +/- 1.8) was significantly higher for the treatment group (incubation in capacitating medium) for Bull N than that of the negative control group (29.5 +/- 1.8). However, there was no difference between the treatment and the negative control groups of the bulls with the knobbed spermatozoa (36.5 +/- 1.4 and 27.1 +/- 3.0 for Bull K1 and 47.5 +/- 3.8 and 35.2 +/- 6.6 for Bull K2, respectively). Although the mean percentage of acrosome-reacted spermatozoa (60.7 +/- 1.3) was higher for the treatment group (receiving calcium ionophore) for Bull N than that of the negative control (29.5 +/- 1.3), there was no difference between the treatment and the negative control groups for the bulls with the knobbed spermatozoa (47.8 +/- 3.3 and 49.3 +/- 5.0 for Bull K1 and 58.8 +/- 10 and 59.5 +/- 9.7 for Bull K2, respectively). A positive correlation existed between the proportion of spermatozoa that did not respond to the HOST and that undergoing a spontaneous AR. Results suggest that spermatozoa with the knobbed acrosome defect have impaired plasma membrane function which predisposes them to premature capacitation and spontaneous AR on incubation after thawing.     

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 degrees C and 37 degrees C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 degrees C for 30 sec or at 75 degrees C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 degrees C and 75 degrees C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 degrees C and 75 degrees C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 degrees C compared with that at 75 degrees C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 degrees C instead of 75 degrees C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.     

Effects of equex STM paste on viability of frozen-thawed dog spermatozoa during in vitro incubation at 38 degrees C

In the canine, artificial insemination with cryopreserved semen generally yields lower pregnancy rates with vaginal deposition than with uterine deposition, one of the reasons being the shortened life span of frozen-thawed spermatozoa. The incubation of spermatozoa at body temperature partially mimics the situation in vivo, and evaluation of the kinetics of viability loss under these conditions can be used to measure the damage caused by freezing and thawing procedures. In this study, 2 aliquots were separated from split ejaculates collected from 7 dogs and were frozen by lowering the straws, in 3 steps, into an LN(2) tank after dilution with egg yolk Tris-citrate-glucose extender with or without the addition of 0.5% Equex STM paste. Motility and plasma membrane integrity (evaluated with the combined fluorescent probes 6-carboxyfluorescein diacetate and propidium iodide) were assessed immediately after thawing and over the next 3 h at 38 degrees C. The addition of Equex STM paste significantly increased the proportion of spermatozoa having an intact plasmalemma immediately after thawing compared with the control. It also increased the longevity of the thawed spermatozoa, prolonging the maintenance of both motility and plasma membrane integrity.     

The potential fertility estimation capacity of the hypoosmotic swelling test, the thermal stress test and a modified cervical mucus penetration test in the bovine

In this study, hypoosmotic swelling (HOS), thermal stress (TS) and modified cervical mucus penetration (mCMP) tests have been used with routine tests for the assessment of semen quality. This is the first study in which the comparison of potential fertility estimation of fore-mention three tests was performed. Bull semen samples were divided into two fertility groups (high: n=3, low: n=3), according to their post-insemination NRR (non-return rate). Prior to the tests, post-thawed spermatological characteristics were assessed after which HOS, TS and mCMP tests were carried out. In the HOS test, the ratio of swollen cells, in the TS test the motility, and in the mCMP test the number of spermatozoa penetrating the cervical mucus, were examined. The relationship between the tests and fertility was also evaluated. HOS test was carried out according to different incubation times and temperatures (37 degrees C 60 min/41 degrees C 15 min/41 degrees C 30 min/46 degrees C 15 min/46 degrees C 30 min). For TS test, samples were subjected to various temperatures for different periods (no incubation (37 degrees C)/41 degrees C 15 min/41 degrees C 30 min/46 degrees C 15 min/46 degrees C 30 min). The mCMP test were subjected to various temperatures for the same period (37 degrees C 15 min/41 degrees C 15 min). In this study, post-thawed motility was found to be similar in high and low fertility groups. However, it has been determined that acrosomal (p<0.01) and other morphological defects (p<0.05) were low in the high fertility group. When HOS test was carried out at 37 degrees C, no difference was observed between the bulls with high and low fertility, but at 41 and 46 degrees C, results of high fertility group were significantly higher than those of low fertility group (p<0.01). Similarly in TS test, the progressive motility rates of high fertility bulls was higher after thermal practices at 41 and 46 degrees C (p<0.01). In mCMP test, at 37 degrees C, the number of cells that had penetrated was similar. However, significant differences were observed in the incubation at 41 degrees C (p<0.01). It has been concluded that for the estimation of potential fertility of bulls, HOS, TS and mCMP tests, in combination with routine spermatological tests can be used and the use of further penetration distance range (PDR2) in mCMP test and higher temperatures such as 41 degrees C instead of 37 degrees C, during the incubations in the afore-mentioned performance tests, is more determinative.     

Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo spermatozoa

In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili-Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 400 × g for 10 min at 37°C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37°C for 30 s) semen samples using a phase-contrast microscope (×400). Plasma membrane integrity (%) was determined by mixing 50 μl each of fresh and frozen-thawed semen with 500 μl of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37°C for 1 h. Viability (%) of fresh and frozen-thawed spermatozoa before and after challenging them to osmotic pressure (hypotonic treatments) was assessed using supravital stain under a phase-contrast microscope (×400). In the first experiment, the mean ± s.e. osmotic pressures of the buffalo semen and seminal plasma were 268.8 ± 1.17 and 256.0 ± 1.53 mOsm/l, respectively. In the second experiment, motility (%) decreased (P < 0.05) in frozen-thawed semen samples as compared with fresh semen (60.1 ± 1.34 v. 81 ± 1.57, respectively). The plasma membrane integrity (%) and magnitude of osmotic stress in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50, 100, 150 and 190 mOsm/l as compared with 250 mOsm/l. Loss of viability (%) in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50 mOsm/l (59% in fresh, 70% frozen thawed) as compared with other osmotic pressures, while it was lowest at 250 mOsm/l (4.1% for fresh, 9.7% frozen thawed). In conclusion, osmotic pressure of Nili-Ravi buffalo semen and seminal plasma is determined. Furthermore, variation in osmotic pressure below 250 mOsm/l is not favorable to fresh and frozen-thawed buffalo spermatozoa.     

A short hypoosmotic swelling test for the prediction of boar sperm fertility

This study was designed to explore the relationship between the ejaculate response to a hypoosmotic swelling test (HOST) and in vivo fertility in a group of 38 boars The hypoosmotic test used was a modification of the HOST that involves a shorter incubation time (5 vs 120 min) and lower osmotic pressure (75 vs 150 mOsm/kg). Ejaculates containing less than 20% abnormal spermatozoa were selected and checked for percentage of motility, percentage of normal acrosomes, percentage of short ORT and percentage of cells showing positive short HOST (sHOST) results Two hundred eightyeight sows were inseminated to obtain in vivo fertility and prolificacy data. No differences were shown between technicians in the sHOST results obtained. Significant differences were recorded between boars in sHOST results (p < 0.002). Only the sHOST result presented a significant correlation with in vivo fertility (r = 0.43, p < 0.01). Short HOST data significantly improved fertility prediction of routine semen analysis tests. Unlike motility and acrosomal status, sHOST values showed a significant decrease when fresh ejaculates (37 degrees C) were stored for 24 h at 15 degrees C, indicating possible damage due to cold shock.     

Comparison between glycerol and ethylene glycol for the cryopreservation of equine spermatozoa: semen quality assessment with standard analyses and with the hypoosmotic swelling test

The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprotectant (3% G, 3% EG, 6% EG, 9% EG). Sperm motility was assessed both by microscopy (in raw and frozen-thawed semen immediately after thawing) and with an HTM-IVOS analyzer (Hamilton-Thorne Research, MA, USA), at 0, 1, 4, 6, and 12 h after thawing and storage at 21 degrees C. Raw and frozen-thawed (0 h) semen samples for G and EG at 3% were also submitted to the HOS test with a 100 mOsm sucrose solution and were evaluated to detect the presence of swollen tails. The higher EG concentrations (i.e. 6% EG and 9% EG) significantly reduced the percentage of motile and PMS, immediately after thawing. At the same concentration, i.e. 3%, G resulted in a higher percentage of PMS than EG (36.2 vs. 30%, P < 0.05), but at 12 h after thawing and storage at 21 degrees C, no significant differences were detected between G and EG at 3%. The correlations between progressive motility (assessed by direct microscope observation or measured through the HTM analyzer) and the HOS test results for 3%G and EG were r = 0.61 and r = 0.35, respectively. The HOS test confirmed its suitability as a complementary method of analysis for stallion semen. We conclude that with the SM extender used, EG could substitute G as the cryoprotectant for stallion semen if used at the same or lower concentration.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

牛精子蛋白质组学技术平台的建立及冻融前后精子差异蛋白初步分析

本研究通过探索不同的精子蛋白制备方法、水化液成分和优化2D电泳程序以建立牛精子蛋白质组学研究技术平台,同时以牛鲜冻精为实验材料通过差异凝胶电泳寻找冻融前后精子蛋白的改变。结果表明：使用改进的热TRIzol法裂解精子细胞制备蛋白,结合优化的2D电泳技术可建立稳定的牛精子蛋白质组学研究技术平台。差异凝胶电泳揭示牛精子在冻融后有质和量的改变：冻融后缺失的蛋白点有20个,表达下调的有2个,表达上调的有10个。作为一项阶段性的实验成果,本研究建立的2D平台和所发现的冻融引起的差异表达蛋白质点为揭示冷冻损伤机理和性控精液的差异蛋白质组学研究奠定了较好的基础。     

Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa

The effect of a prolonged holding time (HT) during cooling on plasma membrane integrity (PMI), motility and in vitro oocyte penetration ability of boar spermatozoa frozen-thawed in different types of package was investigated. Boar semen was frozen in a split-sample design using 3 different HTs (3, 10 and 20 h) during cooling and three different types of freezing package: Maxi-straws, Medium-straws and FlatPacks. Assessment of PMI (SYBR-14 and propidium iodide, fluorescence microscopy) and sperm motility (visually and with CASA) was done during cooling (at 32 degrees C, 15 degrees C, 5 degrees C) and post-thaw (PT). The in vitro oocyte penetration ability of the spermatozoa was tested only PT, using a homologous in vitro penetration assay (hIVP). During cooling the HTs used had no significant (p<0.05) effect on either PMI or percentage of motile spermatozoa Post-thaw PMI was significantly higher (p<0.05) for 10 h and 20 h HT compared with 3 h, and the percentage of motile spermatozoa decreased significantly with 20 h HT as opposed to 3 h and 10 h. Regarding the freezing packages, the FlatPacks and Maxi-straws yielded significantly more PMI than did the Medium-straws (p<0.05). Post-thaw motility was significantly higher for FlatPacks than for straws, in terms of both percentage motile spermatozoa, and sperm velocity and lateral head displacement (LHD). The hIVP did not show any significant differences among the HTs, although FlatPacks yielded a significantly higher penetration rate and more spermatozoa per penetrated oocyte (p<0.05) than did the straws. Changes in motility patterns, toward a more circular motility during cooling and PT, could be noticed where individual spermatozoa showed a capacitation-like motility pattern. The changes were more obvious with 10-h and 20-h HTs than with 3-h HT.