Comparison of Apoptotic Cells Between Cryopreserved Ejaculated Sperm and Epididymal Sperm in Stallions

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5°C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5°C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computer-assisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.     

Artificial insemination with frozen epididymal sperm in cats

Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 x 10(7) sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 x 10(7) sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats.     

Effect of Sperm Cryopreservation on the European Eel Sperm Viability and Spermatozoa Morphology

The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed^® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed^®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed^®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.     

Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.     

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.     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

Development of Procedures for Sex-sorting Frozen–Thawed Bovine Spermatozoa

Dairy bull sperm may be sex‐sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well‐managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex‐sorting from frozen–thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex‐sort and re‐freeze frozen–thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm^® or BoviPure?, followed by staining in one of three diluents (Androhep^®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen–thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm^® rather than BoviPure? gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep^® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen–thawed bull sperm to be sex‐sorted with high resolution between the sexes, then re‐frozen and thawed with retention of motility and acrosome integrity.     

Caspase Activation,Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Evaluation of a portable device for assessment of motility in stallion semen

In horse breeding, quality assessment of semen before insemination is often requested. Non‐laboratory‐based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer‐assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 10⁶ sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 10⁶ sperm/ml when compared to 25 × 10⁶ sperm/ml (p < 0.05) but not when compared to 50 × 10⁶ sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland–Altman plot. Intra‐assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 10⁶ sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 10⁶ sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 10⁶ sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably.     

In Vitro Evaluation of Liquid‐stored Buffalo Semen at 5°C Diluted in Soya Lecithin Based Extender (Bioxcell®), Tris‐Citric Egg Yolk,Skim Milk and Egg Yolk‐Citrate Extenders

This study was designed to compare the quality of liquid‐stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell^® (BIOX), milk (MILK), tris‐citric egg yolk (TEY) and egg yolk‐citrate (EYC) extender at 5°C. Semen was collected from five Nili‐Ravi buffalo (Bubalus bubalis) bulls of 6–7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10⁶ motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX^® extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p < 0.05) in BIOX^® compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell^® were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell^®, milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell^® were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.     

Comparison of Computer-assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein–Friesian Bulls

Subjective microscopic sperm motility results have recently been demonstrated to differ between Holstein-Friesian (HF) and Belgian Blue (BB) bulls. However, such assessments are rather imprecise. In the present study, sperm motility was assessed objectively by means of the Hamilton Thorne CEROS version 12.2c computer-assisted sperm motility analyser (CASA), and differences between the BB and HF breed could also be demonstrated. Higher percentages of both totally (p < 0.0001) and progressively (p < 0.0001) motile spermatozoa were encountered in the HF breed compared with the BB breed. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross-frequency (p = 0.0007) combined with a higher lateral head displacement (p = 0.0015), was the basis for the lower velocity of BB sperm cells. Additionally, BB spermatozoa move less straight forward, resulting in a lower straightness (p < 0.0001). No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at AI centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls. However, these bull populations are selected for fertility. A similar pattern was observed in an unselected bull population of both breeds, although these differences were mostly non-significant for the different CASA parameters. Nevertheless, these data suggest that a genetic component might be responsible for the observed sperm motility breed differences.     

Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality

This study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8°C), the other in a styrofoam box with ice (+3°C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4°C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 ± 0.6°C and 8.6 ± 2.5°C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3°C) or higher (+8°C) temperature than +5°C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions.     

Does single-layer centrifugation with Bovicoll improve sperm quality of frozen-thawed semen in Fleckvieh bulls?

The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl^® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.     

Effect of Organic Selenium in the Diet on Sperm Quality of Boars

The effect of a diet supplemented with organic selenium (Se) on sperm production and quality of boars was investigated. Sixty mature boars from a commercial artificial insemination centre were randomly allocated at Day (D) 0 into Group A and B. Group A received the regular ration supplemented with inorganic Se (0.4 mg/kg feed as Na₂SeO₃) whereas Group B was switched to the same diet but with organic Se (0.4 mg/kg fed as Se‐yeast). The sperm was investigated during 4 months (D0, D30, D60, D75, D90, D105 and D120). Sperm concentration and motility were objectively measured using a photometer and Computer Assisted Semen Analysis (CASA) respectively. Morphology of the sperm was assessed using eosin–nigrosin staining and the resistance to induction of oxidative stress (production of malonaldehyde, MDA) through thiobarbituric acid reagent species analysis. Additionally, the Se concentration in sperm and blood plasma were measured. Repeated measures analysis of variance (anova) from D60 to 120 (spermatogenesis of approximately 2 months) or anova at D120 (Se concentrations) were used for statistical analysis. The total number of ejaculated sperm was not significantly different between both groups, but boars of Group B had a significantly higher sperm concentration (434.6 vs 514.1 × 10⁶ sperm/ml; p < 0.05). Small differences (p < 0.05) were observed between both groups for some CASA parameters, namely straight line velocity (μm/s) (Group A: 48.3, Group B: 45.1), straightness (%) (Group A: 65.6, Group B: 62.2) and linearity (%) (Group A: 32.2, Group B: 29.3). The sperm of Group B showed more oxidative stress (4.1 vs 4.9 μmol MDA/l; p < 0.05) compared with those of Group A. No significant differences (p > 0.05) were observed for the other parameters. Under the present study conditions, changing from inorganic Se to organic Se in the diet of boars increased sperm concentration but reduced some motility parameters and resistance to oxidative stress.     

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes

Routinely, swim‐up method is used to separate high‐quality sperm; however, long processing time and close cell‐to‐cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex^? and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus–oocyte complexes (COC s) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO ₂ incubator at 38.5°C and 5% CO ₂. Matured COC s were rinsed twice in fertilization TALP and placed in the pre‐warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex^?, glass wool filtration and swim‐up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15–20 min in CO ₂ incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co‐incubation with sets of 10–15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA , while in vitro fertilizing rates were compared by chi‐squared test using SPSS ‐20. Least significant difference (LSD ) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex^? filtration improved (p  <  .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim‐up (control). In conclusion, cryopreserved Nili‐Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.