Acrosomal damage and progressive motility of stallion semen forzen by two methods in 0.5-milliliter straws

Post centrifugation and postthaw progressive motility and postthaw acrosomal damage were used to evaluate two different freezing methods (A and B) on 11 ejaculates of one stallion and 6 of another. In Method A, extended semen was centrifuged in 50-ml centrifugation tubes in which 0.25 ml hyperosmotic, glucose-based medium were layered in the apex of the tubes to serve as a cushion. Spermatozoa were resuspended in freezing medium, packaged in 0.5-ml PVC straws and frozen at - 60 degrees C/min. Method B differed only in that the hyperosmotic cushion was omitted during centrifugation. Straws were thawed at 75 degrees C for 6 sec and then transferred into a waterbath at 37 degrees C. The postcentrifugation and postthaw progressive motility and postthaw acrosomal damage were similar for Methods A and B. Method B, however, was found to be technically easier to carry out. It also offers less chance of error or contamination because only two diluents are admixed to the semen as opposed to the three diluents used in Method A. Method B is regarded as more practical and thus preferable to Method A.     

Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and five straws (0.5 ml) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells, motility characteristics (computer-aided semen analysis; CASA), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (positive for fluorescein-labeled peanut agglutinin; PNA). Consistent between-boar variability was detected for postthaw sperm motility (P < 0.01), membrane integrity (P < 0.01), acrosome integrity (P < 0.01), and all CASA characteristics (P < 0.05). There was no significant difference between ejaculates (P > 0.05) or straws (P > 0.05) for any viability assessment. Multivariate pattern analysis of the viability data set highlighted three groups of boars producing spermatozoa with poor, average, and good postthaw recovery (42, 63, and 24 boars, respectively). DNA from Large White boars (n = 22) previously classified as good and poor freezers was screened for AFLP markers. Twenty-eight polymerase chain reaction primer combinations generated 2182 restriction fragment bands, of which 421 were polymorphic. Sixteen candidate genetic markers (P < 0.005) were identified by comparing the AFLP profile with semen freezability using logistic regression analysis. These findings support the hypothesis that there is a genetic basis for variation in postthaw semen quality between individuals, and that AFLP technology may be able to identify molecular markers linked to genes influencing this variation.     

Male-to-male differences in post-thaw motility of rhesus spermatozoa after cryopreservation of replicate ejaculates

BACKGROUND: The efficiency of controlled propagation to produce rhesus monkeys of particular genotypes can be maximized by use of cryopreserved spermatozoa collected from specific males to inseminate appropriate females. But this assumes that semen from males with different genotypes can be cryopreserved with equal effectiveness. METHODS: To investigate whether spermatozoa from different Macaca mulatta males can be effectively cryopreserved when frozen under identical conditions, we collected and froze semen specimens from 13 adult, fertile males maintained at three primate research centers. RESULTS: Survival, based on post-thaw motility normalized to the pre-freeze value, was assayed within 30 minutes after thawing; it varied from 50% to 70% but declined thereafter. To examine the response of semen from individual males, we collected and froze three to six ejaculates per male from each of seven males. CONCLUSIONS: In general, semen from a given male responded reproducibly to freezing, but there were significant differences among males. The cause of these differences among M. mulatta males in post-thaw sperm survival remains unidentified.     

Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins

The objective of this work was to look for useful predictive indicators of the potentially “good” or “poor” ability of a boar ejaculate to sustain cryopreservation by assessing both the conventional sperm quality parameters (Study 1) and the immunolabeling of three proteins involved in the physiology of the sperm cell: GLUT3, HSP90AA1 and Cu/ZnSOD (Study 2). Study 1 was carried out in three different steps during the cryopreservation process of the sperm-rich fraction of 29 Piétrain boar ejaculates (17 °C, 5 °C, and 240 min postthaw). These ejaculates were clustered based on sperm quality parameters analyzed at 240 min postthaw, obtaining 16 good freezability ejaculates (GFEs) and 13 poor freezability ejaculates (PFEs). The sperm linearity (LIN) and the straightforward (STR) indexes at 5 °C showed higher hyperactivated movement in the PFEs than in the GFEs, which suggests that analyzing these sperm kinematic parameters could be a useful tool for predicting the potential freezability of an ejaculate. This statement was demonstrated by grouping the 29 ejaculates into two clusters (A and B) based on LIN and STR values assessed after 30 min at 5 °C, which resulted in around 72% of coincidence with the GFE and PFE groups. Study 2, performed at 17 °C and 240 min postthaw, revealed no differences between GFEs and PFEs in the immunolabeling of the three proteins within a same step, in terms of location and reactivity, although reactivity was generally weaker at 240 min postthaw in both groups. Additional studies on Western blot are currently being carried out with the objective to quantify the expression of the three proteins in GFEs and PFEs in the three steps of the cryopreservation process.     

Designing of an artificial neural network model to evaluate the association of three combined Y-specific microsatellite loci on the actual and predicted postthaw motility in crossbred bull semen

The freezing of bull semen significantly hamper the motility of sperm which reduces the conception rate in dairy cattle. The prediction of postthaw motility (PTM) before freezing will be useful to take the decision on discarding or freezing of the germplasm. The artificial neural network (ANN) methodology found to be useful in prediction and classification problems related to animal science, and hence, the present study was undertaken to compare the efficiency of ANN in prediction of PTM on the basis of the number of ejaculates, volume, and concentration of sperms. The combined effect of Y-specific microsatellite alleles on the actual and predicted PTM was also studied. The results revealed that the prediction accuracy of PTM based on the semen quality parameters was comparatively lower because of higher variability in the data set. The ANN gave better prediction accuracy (34.88%) than the multiple regression analysis models (32.04%). The root mean square error was lower for ANN (8.4353) than that in the multiple regression analysis (8.6168). The haplotype or combined effect of microsatellite alleles on actual and predicted PTM was found to be highly significant (P < 0.01). On the basis of results, it was concluded that the ANN methodology can be used for prediction of PTM in crossbred bulls.     

Effect of cryopreservation methods and precryopreservation storage on bottlenose dolphin (Tursiops truncatus) spermatozoa

Research was conducted to develop an effective method for cryopreserving bottlenose dolphin (Tursiops truncatus) semen processed immediately after collection or after 24-h liquid storage. In each of two experiments, four ejaculates were collected from three males. In experiment 1, three cryopreservation methods (CM1, CM2, and CM3), two straw sizes (0.25 and 0.5 ml), and three thawing rates (slow, medium, and fast) were evaluated. Evaluations were conducted at collection, prefreeze, and 0-, 3-, and 6-h postthaw. A sperm motility index (SMI; total motility [TM] x % progressive motility [PPM] x kinetic rating [KR, scale of 0-5]) was calculated and expressed as a percentage MI of the initial ejaculate. For all ejaculates, initial TM and PPM were greater than 85%, and KR was five. At 0-h postthaw, differences in SMI among cryopreservation methods and thaw rates were observed (P < 0.05), but no effect of straw size was observed. In experiment 2, ejaculates were divided into four aliquots for dilution (1:1) and storage at 4 degrees C with a skim milk- glucose or a N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)-TRIS egg yolk solution and at 21 degrees C with a Hepes-Tyrode balanced salt solution (containing bovine albumin and HEPES) (TALP) medium or no dilution. After 24 h, samples were frozen and thawed (CM3, 0.5-ml straws, fast thawing rate) at 20 x 10(6) spermatozoa ml(-1) (low concentration) or at 100 x 10(6) spermatozoa ml(-1) (standard concentration). The SMI at 0-h postthaw was higher for samples stored at 4 degrees C than for samples stored at 21 degrees C (P < 0.001), and at 6-h postthaw, the SMI was higher for samples frozen at the standard concentration than for samples frozen at the low concentration (P < 0.05). For both experiments, acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol applied to fresh or liquid-stored semen maintained high levels of initial ejaculate sperm characteristics.     

Theoretical aspects of canine semen cryopreservation

Changes in canine sperm cells during freezing and thawing can cause damage to the cells resulting in cell death. No standardized freezing or thawing method appears to be ideal for all dogs and all ejaculates, because intrinsic variations in properties such as osmotic sensitivity between sperm cells from different dogs and ejaculates makes the cellular response to cryopreservation unpredictable according to the normal physics of cryobiology. Research in canine semen cryopreservation is difficult because the low ejaculate volume makes multiple comparisons from a single ejaculate difficult. True fertility data is also very limited on cryopreserved canine ejaculates. Despite this, the cottage industry that has evolved to cryopreserve dog sperm has been very successful using empirically derived methods that accommodate most ejaculates. Therefore, the practitioner must follow the recommendations supplied by the freezing center to achieve the best potential results.     

Effects of liner composition, cooling profile and time of glycerolization on survival of bovine spermatozoa

Bovine spermatozoa from five bulls (158 ejaculates) were collected into one-piece combination liner-collection cones in an artificial vagina. Spermatozoal motility and differential staining were evaluated at each sequential step of semen processing. Linear 30-min and nonlinear 150-min cooling profiles resulted in equivalent postthaw spermatozoan survival rates. Preglycerolation holding times of 60 and 150 min, after 30-min linear cooling, were associated with increased, but nonsignificant, postthaw survival rates for bovine spermatozoa collected in polyethylene liner-collection cones. Highly significant differences (P<0.0001) were found to be associated with the composition of the liner-collection cones used. Motility estimates made at collection and after thawing from liquid nitrogen storage indicated that at least 50% of motile spermatozoa collected in polyethylene liner-collection cones survived; whereas, not more than 30% of the cells survived when they had been collected in rubber liner-collection cones.     

Effect of cryopreservation protocol on postthaw characteristics of stallion sperm

Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; μm/s), linearity (LIN; %), intact acrosomal and plasma membranes (AIMI; %), intact acrosomal membranes (AI; %), intact plasma membranes (MI; %), and DNA quality. Eight of 10 experimental endpoints (MOT, PMOT, average-path velocity [VAP], mean straight-line velocity [VSL], LIN AIMI, AI, and MI) were affected by extender type, with egg yolk-based extender yielding higher values than milk/egg yolk-based extender (P < 0.05). Exposure of extended semen to a slow prefreeze cooling period resulted in increased values for six of eight endpoints (MOT, PMOT, VCL, AIMI, AI, and MI), as compared with a fast prefreeze cooling period (P < 0.05). As a postthaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, average-path velocity, and mean straight-line velocity (P < 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and MI (P < 0.05). In conclusion, a slow prefreeze cooling rate was superior to a fast prefreeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory postthaw diluent prior to semen analysis.     

Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process

Background

Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male.

Methodology/Principal Findings

We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature.

Conclusions/Significance

Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Post-thaw motility and longevity of motility of imipramine-induced ejaculates of pony stallions

Imipramine-induced ex copula ejaculates (11) and fractionated in copula ejaculates were collected from each of 5 pony stallions for freezing in 5-ml straws (6), using a modified Kenney glucose skim-milk extender (2). Initial post-thaw total and progressive motilities and daily post-thaw total and progressive motilities, as well as the number of days to reach 0 progressively motile spermatozoa, were also similar for the 2 methods of collection. The percentage of morphologically normal spermatozoa both before freezing and after thawing were also similar for in copula and ex copula ejaculates. Consistent with previous work (11), imipramine-induced ejaculates were of extremely high sperm concentration and low volume compared with those of in copula ejaculates. In this study, imipramine-induced ejaculates were of significantly higher concentration of sperm and lower volume than fractionated (first 2-3 jets) in copula ejaculates. These results suggest that imipramine-induced ejaculates may be suitable for cryopreservation. Breeding trials are necessary to evaluate actual fertility of semen.     

In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4 h at 5 °C, and at 0 and 2 h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4 ± 17.8 μm/sec) and high progressiveness (LIN: 65.1 ± 14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7 ± 25.6 μm/sec) but a nonprogressive trajectory (LIN: 33.1 ± 10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3 ± 24.3 μm/sec) and nonprogressive sperm (LIN: 39.6 ± 18.3%); and Subpopulation 4 included very rapid (VCL: 152.8 ± 25.7 μm/sec) and highly progressive sperm (LIN: 70.9 ± 13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P < 0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Multiple mating,sperm competition and meiotic drive

Most discussions of ‘sperm competition’ have ignored the potential for competition among the different sperm genotypes present in the ejaculate of a single male. Rivalry within ejaculates may limit cooperation among the members of an ejaculate when they compete with sperm produced by other males. A gene that gains an advantage in competition within an ejaculate (a segregation distorter) may increase in frequency even if it is associated with significant costs to organismal fitness. Therefore, selection will favor genes expressed in males that suppress competition within ejaculates. This may explain why sperm function is largely controlled by the diploid genotype of the male progenitor, rather than by the genotypes of individual haploid sperm. Females who mate with multiple males reduce the relative advantage of a segregation distorter whenever the distorter impairs the competitive effectiveness of the ejaculates in which it occurs. If the distorter is associated with costs to organismal fitness, selection will favor female mating behavior that reduces the distorter's equilibrium frequency. Competition within ejaculates may thus be one reason why females choose to mate with multiple males.     

Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity

Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P < 0.05), whereas the TPI amounts were significantly lower in GFE (P < 0.05) than in PFE. The association of ACRBP and TPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.     

Collection method, season and individual variation on seminal characteristics in the llama (Lama glama)

The objective of the present study was to evaluate the effect of semen collection method (electroejaculation "EE" as compared with the artificial vagina "AV"), the season (summer versus winter) and the male used on macroscopic and microscopic characteristics of ejaculates in llamas. A total of 110 ejaculates were collected from six males and 92 of them were analyzed. Ejaculate volume, concentration, total sperm and the following sperm characteristics were studied: motility, membrane function (HOS test), membrane integrity (CFDA/PI fluorochromes) and morphology. A mixed linear model, that considered season and collection method as the fixed variables and the male as the random variable, was used for the statistical analysis. Variability was found between males (p相似文献   

Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics

Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender with 4% glycerol. Spermatozoal quality was assessed at four different points: at less than 15 min after collecting and before processing (Step 1); after centrifugation and just before freezing (Step 2); immediately after thawing less than 3 h after freezing (Step 3); and immediately after thawing 10 to 20 d after freezing (Step 4). Acrosome-specific monoclonal antibody detected differences (P <0.05) among steps and ejaculates within stallions. All parameters of spermatozoal motion, including the percentage of motile sperm, percentage of progressively motile sperm, curvilinear velocity, straight line velocity, linearity, amplitude of lateral head displacement, and radius of the average path for circularly swimming sperm, differed (P <0.05) among steps, and most of these parameters differed among ejaculates within a stallion and among stallions. For Steps 2 and 3, 62 and 37% of the sperm were motile, and 56 and 23% of all motile sperm had a curvilinear velocity of >100 mum/sec. Most damage to sperm occurred as a result of freezing-thawing, whereas centrifugation of sperm caused only minor damage.     

Proceedings: Freezing injury to streptococcal L-phase variant

A comparative study of resistance to freezing in Streptococcus haemolyticus S.8 and its L-phase variant was made by viable counts of postthaw survival and also by morphological observation of freezing patterns with an electron microscope. It was recognized that L-phase cells are more susceptible to freezing than their original strains.