Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics

In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen–thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen–thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1–S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1–FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1–S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.     

Assessment of Selected Quality Parameters of Epididymal Cat (Felis catus s. domestica,L. 1758) Sperm Using Flow Cytometry Method and Computer Assisted Sperm Analyser

The improvement of biotechnical methods connected with fast and precise semen quality assessment and its utilization in assisted reproductive techniques is an urgent necessity in felids. The aim of this study was to evaluate some quality parameters (i.e. the viability and share of cells with intact plasma membrane) of epididymal sperm of cats using the flow cytometry method and computer‐assisted sperm analysis (CASA) examination. The material consisted of epididymal spermatozoa flushed from 22 pairs of epididymes after routine neutering procedures obtained from domestic cats aged between 8 and 36 months. The epididymes were cut and incubated with an extender without egg yolk. The samples were assessed for sperm viability (Live/Dead Sperm Viability Kit^®), percentage of subtle membrane changes (Apoptosis Detection Kit^®) and motility using FACScalibur flow cytometer and assisted sperm analyser htm ivos version 12.2. The flow cytometry method revealed 71.3% and 84.4% of live sperm using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit respectively. The population of early‐apoptotic and late‐apoptotic sperm were 0.8% and 1.1% respectively. The CASA examination found 51.5% of motile sperm. However, the motility examination under light microscope revealed 69.5% of motile sperm. The data revealed an indistinctive per cent of apoptotic cells and 18.9% and 15.6% of dead cells using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit, respectively, which indicate that the sperm obtained after flushing the epididymis possess potential properties for further assisted reproduction techniques.     

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation

The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 +/- 12.7% vs. 66.8 +/- 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 +/- 5.8% in May and 15.6 +/- 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 +/- 43.9 vs. 164.4 +/- 66.4 microm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) > stallion (P < 0.001) > season (P < 0.05).     

Cholesterol-loaded cyclodextrins added to fresh bull ejaculates improve sperm cryosurvival

Damage occurring to spermatozoa during cryopreservation results in a loss of motile cells and cells that are functionally normal, compared with fresh sperm samples. Treating bull sperm with cholesterol-loaded cyclodextrins (CLC) before cryopreservation results in increased sperm cryosurvival. However, in previous studies, CLC were always added to sperm samples that had been highly diluted. The aim of this study was to develop a procedure for adding CLC to whole bull ejaculates that would optimize sperm cryosurvival. Adding 2 or 4 mg of CLC/120 x 10(6) sperm to sperm samples ranging in concentration from 120 to 2,000 x 10(6) sperm/mL resulted in greater (17 to 28 percentage points; P < 05) numbers of live cells compared with control samples (no CLC treatment), regardless of the sperm concentration, except for samples at 120 x 10(6) sperm/mL treated with 4 mg of CLC. Incubating sperm with CLC at 23 or 37 degrees C before cryopreservation resulted in similar sperm cryosurvival. The cooling rate used to cryopreserve CLC-treated cells did not affect sperm cryosurvival. Finally, adding CLC to undiluted ejaculates (2 mg of CLC/120 x 10(6) sperm) resulted in greater percentages of live sperm compared with the control samples (62 vs. 45%; P < 0.05), although the percentages of motile sperm were similar for both CLC-treated and control samples (58%). In conclusion, bull sperm cryo-survival can be improved if spermatozoa are treated with CLC before freezing. In addition, CLC can be added to fresh ejaculates at either 23 or 37 degrees C. This technique is simple, practical, and can be easily integrated into current cryopreservation protocols.     

Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct Subpopulations

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.     

Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa

The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.     

Motility Characteristics and Fertilizing Capacity of Boar Spermatozoa Stained with Hoechst 33342

Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer‐aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35°C with 9, 18, 27, 60 and 90 μM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 μM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 μM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 μM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 μM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 ± 0.32 vs 10.47 ± 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X‐ and Y‐bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.     

Effects of pH, sodium bicarbonate, cryoprotectants and foetal bovine serum on the cryopreservation of European eel sperm.

The main objective of the present study was to evaluate the influence of pH and bicarbonate concentration in the activation or inhibition of European eel (Anguilla anguilla) spermatozoa and to evaluate the effect of different cryoprotectants: dimethyl sulphoxide (DMSO), acetamide, ethylene glycol, propanol, glycerol and methanol (MeOH). The effect of these factors was evaluated comparing the percentage of motile cells, the percentage of alive cells (by Hoechst staining) and the spermatozoa morphometry pre- and post-cryopreservation (by computer-assisted morphology analysis). Based on the above findings, three cryoprotectants (DMSO, MeOH and glycerol) were chosen and evaluated in two media (P1 and P1 modified) with different concentrations of NaHCO(3) and in the presence or absence of foetal bovine serum (FBS). The effect of these factors was evaluated comparing the percentage of alive and motile cells post-cryopreservation. DMSO was the cryoprotectant showing better results in relation to the percentage of spermatic alive cells post-freezing and caused a smaller modification of the head spermatozoa morphology. The combination of P1-modified medium with DMSO and containing FBS increased slightly but significantly the percentage of motile spermatozoa post-cryopreservation.     

Effect of Cryopreservation on the Sperm DNA Fragmentation Dynamics of the Bottlenose Dolphin (Tursiops truncatus)

Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species‐specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post‐thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post‐thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax^®). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES‐TRIS‐fructose buffer (TTF), an egg‐yolk‐free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen–thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen–thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1^? buffer had higher levels (p < 0.05) of DNA fragmentation after 24‐ and 48‐h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen–thawed samples.     

Retracted: Effect of Post‐Thaw Storage Time on Motility and Fertility of Cryopreserved Beluga Sturgeon (Huso huso) Sperm

The aim of this study was to test the influence of post ‐ thaw storage time on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30 and 60 min at 4°C post‐thawing. After being frozen in liquid nitrogen and then thawed, the percentage of motile sperm and duration of motility were not affected by 30 min of storage at 4°C, whereas a significant decline in these parameters was observed after 60 min of storage. Similarly, fertilization and hatching rates were significantly affected within 60 min of storage at 4°C, and the fertility of frozen‐thawed sperm was significantly lower than that of fresh sperm. We conclude that cryopreserved sperm of beluga sturgeon could be stored for 30 min without the loss of sperm quality. This described procedure for beluga sturgeon cryopreservation is reliable and efficient and therefore can be recommended for hatchery practice after scaling up this technique.     

Impact of Ultra‐Rapid Freezing on the Motility,Morphology, Viability and Acrosome Integrity of Epididymal Cat Sperm Diluted in Sucrose‐Based Extenders

The objective of the present study was to investigate the influence of different sucrose‐based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra‐rapid freezing method. Nine cats were castrated, and collected semen was diluted 1 : 1 with Dulbecco`s phosphate‐buffered saline‐BSA1%‐based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6 m ). After ultra‐rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p < 0.01) and morphologically normal (p < 0.01) sperm was higher in the sucrose‐supplemented groups than in the sucrose‐free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4 m sucrose, spermatozoa viability showed higher values (57.0 ± 4.7; p < 0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra‐rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4 m of sucrose was used. This is the first report on sperm ultra‐rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.     

Electroejaculation and semen cryopreservation of free-ranging Japanese black bears (Ursus thibetanus japonicus)

The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and cryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) x 10(6), 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by freezing the thus collected electroejaculates.     

Use of Cholesterol‐Loaded Cyclodextrin in Donkey Semen Cryopreservation Improves Sperm Viability but Results in Low Fertility in Mares

The use of cholesterol‐loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S‐MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10⁶ spermatozoa. Semen was frozen, and thawed samples were evaluated by computer‐assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen‐thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC‐treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC‐treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC‐treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S‐MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S‐MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.     

Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrode's Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca²⁺-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca²⁺ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca²⁺ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrode's complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrode's complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.     

Effect of Different Extenders on In Vitro Characteristics of Feline Epididymal Sperm During Cryopreservation

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS–egg yolk–based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25‐ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0‐ and 6‐h post‐thaw. Experiment 1 compared the effect of three non‐feline commercial extenders – based on TRIS–egg yolk (Triladyl), egg‐yolk‐free medium (AndroMed) and skimmed milk‐egg yolk (Gent) – on the quality of frozen‐thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen‐thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS–egg yolk–based extenders, one non‐feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing‐thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing‐thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS–egg yolk–based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.     

Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa

Metformin is clinically used to treat diabetes. Given its role‐impacting metabolism, metformin has been also added to semen cryopreservation media showing specie‐dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin‐including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ‐population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.     

European Eel Sperm Diluent for Short‐term Storage

The sperm of European eel shows a high density and the time of spermatozoa motility is very short after activation with sea water. These characteristics make difficult the sperm handling and its quality assessment. Several diluents were previously described for the Japanese eel obtaining over 3 weeks’ conservation times under refrigeration, but they rendered bad results in the European species. In the present study, several diluents were developed taking as basis the P1 medium, and using different dilution ratios (1 : 50, 1 : 100) and two pH (6.5, 8.5). The effect of the addition of bovine serum albumin (BSA, 2% w/v) was also evaluated. At 24 h, undiluted samples already showed significant lower motility and viability than sperm samples diluted in the different media. The results for diluents with pH 6.5 and 8.5 were different. Spermatozoa diluted in media at pH 6.5 cannot be activated at 24 h, while samples diluted in the diluents with pH 8.5 and added with BSA did not show significant differences with respect to the fresh sperm motility until 48 h. The viability (percentage of alive cells) did not show differences until 1 week, independent of the dilution ratio. After 1 week, the motility was approximately 30% in the media containing BSA, which presented no differences for head size of the spermatozoa (perimeter and area) until 72 h and 1 week, respectively. In conclusion, the combination of one medium having similar physico‐chemical characteristics to the seminal plasma, including pH 8.5, and supplemented with BSA can be used in different dilution ratios for the sperm’s short‐term storage, preserving its motility capacity.     

A comparison of bovine seminal quality assessments using different viewing chambers with a computer-assisted semen analyzer

Computer-assisted sperm analysis of fresh and frozen-thawed bovine sperm requires proper handling and preparation, and the type of slide used in the assessment is critical if the resultant data are to be useful quality control measurements. In the present study, 4 different slide viewing chambers, a Makler chamber, a clean slide-coverslip, or a 2- or 4-cell chamber Leja slide, were compared with assess their utility in providing reliable measurements of sperm motility variables. A Hamilton-Thorne IVOS Computer-Assisted Semen Analyzer (CASA) was the instrument used to determine sperm measurements utilizing the 4 different chambers. Fifty-eight different freeze batches of bovine semen that had been collected from 47 bulls at 7 sites that sex-sort sperm using Sexing Technologies sorting criteria were incorporated into the trial. Neither the percentage of motile sperm nor the percentage of progressively motile sperm differed for the Makler chamber vs. slide-coverslip comparisons. Similarly, total and progressively motile sperm did not differ between the 2- and 4-cell chambered Leja slides. However, total and progressive motility of sperm determined with the Makler chamber and slide-coverslip were greater (P < 0.0001) than motilities recorded by the 2- or 4-cell chambered Leja slides. Based on the results, the type of viewing chamber can affect the range of sperm motility values when CASA is used for quality control evaluations of thawed, cryopreserved sex-sorted sperm samples.     

A Scanning Electron Microscopy Study of Frozen / Thawed Dog Sperm During In Vitro Gamete Interaction

The aim of this work was to study the effects of cryopreservation on the binding and penetration of dog spermatozoa to the zona pellucida (ZP) by scanning electron microscopy (SEM). The sperm-rich fraction of six ejaculates from five dogs was divided into two aliquots and washed by centrifugation. One aliquot was processed as fresh control sample and the other aliquot frozen in Tris-fructose extender. Gamete interaction was assessed using in vitro matured bitch oocytes, which were co-incubated for up to 3 h. At hourly intervals after the start of co-incubation, in vitro fertilized (IVF) oocytes were processed by SEM. The results were analysed statistically using the anova test. Differences in binding and penetration of the spermatozoa to the ZP occurred; a lower proportion of oocytes with spermatozoa bound to ZP was observed using frozen sperm (p < 0.05) than with fresh sperm (61%, 57% and 53% vs 42%, 40% and 44% at 1, 2 and 3 h, respectively). The percentage of ZP penetration by fresh sperm was directly proportional to the time of co-incubation (9%, 25% and 34%; p < 0.05); in contrast, no differences were observed in the penetration rate with frozen-thawed sperm (21%, 17% and 21%). More acrosome reacted sperm were observed in frozen sperm than in fresh sperm on the surface of the ZP. The differences in the percentage of binding and penetration between fresh and frozen sperm during the co-culture could indicate that the time course of penetration is faster in frozen-thawed dog spermatozoa than in fresh sperm, but that fresh spermatozoa can penetrate more oocytes over a given period of time, which may be related to their reacted or non-reacted initial status.     

Short‐Term Storage and Swim‐Up Selection Do Not Affect the X/Y Ratio in Equine Spermatozoa

The standard procedure of artificial insemination with fresh equine spermatozoa involves short‐term storage (to 48 h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short‐term storage and the swim‐up procedure. We used a standard protocol, for short‐term storage (0, 24 and 48 h at 5°C) of stallion semen diluted in the commercial extender EquiPro? (Minitüb GmbH, Tiefenbach, Germany). After each set‐up storage period, the motile fraction of sperm cells was selected by the swim‐up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non‐selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1 : 1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48 h and the swim‐up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species‐related differences.