Analysing Motility Parameters on Fresh Bull Semen Could Help to Predict Resistance to Freezing: A Preliminary Study

Batches of straws often need to be thrown away after freezing because of a too few number of motile or progressive sperm cells after thawing. Our objective was to evaluate the possibility to predict before freezing the quality of the semen after freezing/thawing. Computer‐Aided Sperm Analysis was performed on motility parameters both before and after freezing of 20 ejaculates from different bulls. Significant variation between bulls was observed both before and after freezing for all the analysed traits (anova 2; p < 0.001): proportion of motile (%mot) and progressive (%prog) sperm, velocity on the curved line (VCL), velocity on the straight line (VSL), velocity on the average path (VAP), linearity (VSL/VCL), beat cross frequency and average orientation change of the head. Freezing significantly altered the motility parameters and correlations were found between samples analysed before and after freezing (Pearson coefficient: R = 0.43–0.72; p < 0.05). The mean VAP, VSL and the %prog obtained before freezing were highly correlated to the %mot and %prog observed after freezing (R = 0.75–0.82; p < 0.001). Applying thresholds on mean VAP and VSL values allowed us to predict respectively 6 and 7 of nine batches that would be rejected after freezing due to a too low %prog (<15%). Combining different traits did not add to the precision. In conclusion, analysis of velocity traits on fresh sperm seems more efficient than analysis of %mot or %prog to discard batches that will be of poor quality after freezing.     

Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.     

Effect of Semen Collection Methods on the Quality of Pre‐ and Post‐thawed Bali Cattle (Bos javanicus) Spermatozoa

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post‐thawed semen quality. The collection methods employed were electro‐ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM + EE). A total of 25 untrained Bali bulls (age between 2 and 4 years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer‐assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM + EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM + EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2–3 and >3–4 years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM + EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.     

解冻稀释液中添加精浆对冻融猪精子品质的影响

【目的】 探究冷冻前添加热休克蛋白A8(heat shock protein A8, HSPA8)和解冻后添加不同浓度精浆(seminal plasma, SP)对冻融猪精子的影响。【方法】 采用手握法采集长白猪精液, 添加0.5 μg/mL HSPA8到猪精液冷冻保护剂中进行细管分装, 投入液氮中保存3周后进行解冻, 解冻后添加不同浓度精浆(0、10%、30%和50%), 对冻融后长白猪精子的运动能力、质膜完整性、顶体完整性、细胞凋亡、线粒体膜电位、鱼精蛋白缺乏及体外获能水平等进行评估。【结果】 与对照组相比(无HSPA8和精浆), 添加0.5 μg/mL HSPA8处理组(无精浆)的精子直线速度(VSL)、曲线速度(VCL)、平均路径速度(VAP)和前向性运动(STR)均显著提升(P<0.05), 精子直线性运动(LIN)和运动的摆动性(WOB)均无显著差异(P>0.05);精子质量参数中活力、质膜完整性和顶体完整性均显著升高(P<0.05), 细胞凋亡水平与线粒体膜电位均显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。之后在解冻液中添加不同浓度的精浆, 与添加0.5 μg/mL HSPA8处理组(无精浆)相比, 精浆添加量达到50%时, 精子VSL、VCL、VAP、LIN、STR和WOB均显著提升(P<0.05);精子活力、质膜完整性、顶体完整性和线粒体膜电位均显著提高(P<0.05), 细胞凋亡水平显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。【结论】 在冷冻基础液中添加0.5 μg/mL HSPA8和解冻稀释液中添加50%精浆联合使用可以有效改善冻融精子质量, 将会对猪精液的冷冻保存及商业化生产提供一定的参考。     

Effects of the supplementation with an high‐polyphenols extra‐virgin olive oil on kinetic sperm features and seminal plasma oxidative status in healthy dogs

The aim of this work was to evaluate the effects of the supplementation of two extra‐virgin olive oils (EVOO) having different polyphenols content, on canine spermatozoa kinetic parameters and seminal plasma oxidative status. The study was conducted on 12 clinically healthy dogs of different breeds (2–7 years, 5–48 kg of body weight) divided into two groups: an experimental group supplemented with EVOO (Coratina cultivar) high in polyphenols (H‐P) and a control group fed EVOO (Cima di Bitonto cultivar) low in polyphenols (L‐P). The oil was daily administered per os (1 ml/3 kg BW) before meal. Semen collection was made twice at 15 days distance (D0₁ and D0₂) and then at 30 (D30), 60 (D60) and 90 (D90) days. Semen concentration and kinetic parameters were measured using computer‐assisted sperm analysis (CASA) system to evaluate: sperm total count, sperm motile (MOT%), progressive motility (PROGR%) and its fractions, straight‐line velocity (VSL, μm/s), curvilinear velocity (VCL, μm/s), average path velocity (VAP, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz), straightness (STR%) and linearity (LIN%). On seminal plasma, reactive oxygen species (ROS) and biological antioxidant potential (BAP) were tested. From findings, no differences were found for sperm MOT, VSL, VCL, VAP, ALH, BCF, STR, LIN and BAP. A gradual enhancement of PROGR% was observed in H‐P group (p < .01). The ROS levels were higher in dogs H‐P compared to the other group (p < .05). In conclusion, our results highlight the positive effects of EVOO polyphenols on sperm PROGR% in healthy dogs.     

Effects of the Addition of Autologous Seminal Plasma to Highly Concentrated Stallion Semen After 48 Hours of Cooled Storage

Insemination with chilled transported semen has become distinctly important in the horse-breeding industry. To ensure cell survival during cooled storage, semen is diluted with an appropriate extender and the concentration of seminal plasma (SP) is reduced. Nevertheless, SP plays an important immunomodulatory role in the female genital tract and supports sperm fertility. The aim of the present study was to evaluate the effect of the addition of autologous SP after cooled storage to highly concentrated stallion semen. Therefore, SP was removed by simple centrifugation of extended semen, aspiration of the supernatant, and resuspension of the sperm pellet with semen extender. Motion characteristics were evaluated after cooled storage for 48 hours at concentrations of 333 × 10⁶ sperm/mL in comparison with stored samples at concentration of 25 × 10⁶ sperm/mL (control). The highly concentrated semen samples were diluted with an extender containing 0%, 5%, 20%, and 80% SP directly before motility analysis. Dilution of the cooled semen with a fresh semen extender without SP (0%) increased kinematic parameters (curvilinear velocity [VCL] 137.3 vs. 151.8; straight-line velocity [VSL] 49.0 vs. 57.5; average path velocity [VAP] 69.5 vs. 79.4 μm/second; amplitude of lateral head [ALH] 3.1 vs. 3.3 μm; beat cross frequency [BCF] 31.6 vs. 33.5 Hz; P < .05) but not total motility (51% vs. 43%) and progressive motility (46% vs. 36%) compared with controls. The addition of SP after storage for 48 hours decreased sperm total motility and progressive motility regardless of SP concentration: 5 (38% and 34%), 20 (37% and 33%), and 80% SP (27% and 22%; P < .05). In contrast, kinematic parameters were enhanced by extenders containing 5% and 20% SP (VCL: 148.0 and 155.6; VSL: 59.2 and 60.9; VAP: 78.7 and 81.9; BCF: 33.4 and 35.7; ALH: 3.4 and 3.4; P < .05). However, using an extender containing 80% SP was detrimental to kinematic parameters (VCL: 151.2; VSL: 52.2; VAP: 76.9; BCF: 34.8; P < .05) except for ALH, which increased (3.5; P < .05). In conclusion, cooled storage at concentrations of 333 × 10⁶ sperm/mL did not affect sperm motility. The addition of a fresh extender or an extender containing small concentrations of SP to highly concentrated ejaculated sperm increased kinematic values after storage; however, increasing concentrations of SP decreased sperm motility.     

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.     

Comparison of Different Extenders with Defined Protein Composition for Storage of Stallion Spermatozoa at 5°C

To maintain the fertility of stallion spermatozoa during cooled storage, extender media are added to semen. In this study, three semen extenders were compared: EquiPro which contains defined caseinates and whey proteins instead of dried skim milk. The extender is provided in dry form and dissolved in distilled water prior to use. EquiPro TM has the same composition as EquiPro but is provided in a sterilized ready-to-use liquid form. AndroMed-E contains soybean lecithin as protein source. Semen was collected from seven stallions. Ejaculates were divided into three aliquots, diluted with the different extenders and stored at 5 degrees C for 4 days. Total motility, membrane integrity, average path velocity (VAP), curvilinear-velocity (VCL), straight-line velocity (VSL), distance average path (DAP), distance curved line (DCL) and distance straight line (DSL) were determined by computer-assisted analysis. Total motility decreased in all extenders during storage. The parameters VAP, VCL, VSL, DAP, DCL and DSL in semen diluted in EquiPro TM at most times and in semen diluted in AndroMed-E at some times were lower than in semen diluted in EquiPro (p < 0.05). Viability on days 0 and 4 was lowest in semen diluted in AndroMed-E (p < 0.05). Velocity decreased faster when semen had been diluted in the sterilized liquid extender EquiPro TM or in AndroMed-E compared with the dry formula of EquiPro. Therefore the liquid sterilized EquiPro despite no difference in its chemical composition differs from the dry, non-sterilized EquiPro extender. Heat sterilization apparently changes effects of the extender on spermatozoa.     

不同稀释液及绵羊品种对冷冻精液保存效果的影响

本文利用计算机精子辅助分析仪和姬姆萨染色方法评价无卵黄稀释液OPTIXcell和有卵黄稀释液Optidyl冷冻保存湖羊、白头杜泊、黑头杜泊和澳洲白绵羊精液的效果。选用湖羊、白头杜泊、黑头杜泊和澳洲白绵羊的种公羊各5只,通过假阴道方法采集精液,合格精液分别用OPTIXcell和Optidyl稀释液稀释并冷冻。结果表明:Optidyl冷冻保存白头杜泊羊和澳洲白绵羊精液的精子在活精子比例(MR)、直线速度(VSL)、平均路径速度(VAP)、曲线轨迹的直线性(LIN)、空间平均路径的直线性(STR)和尾部鞭打频率(BCF)上优于OPTIXcell,同样的,湖羊在MR、曲线速度(VCL)、精子头侧摆幅度(ALH)、VSL和VAP上高于OPTIXcell无卵黄稀释液,而黑头杜泊羊使用OPTIXcell在MR、VSL、VCL、ALH和BCF的平均值上效果更好。无论哪个品种使用Optidyl有卵黄稀释液,其绵羊精子畸形率的比例都低于使用OPTIXcell无卵黄稀释液。综上,冷冻保存湖羊、白头杜泊羊、澳洲白绵羊精液适合采用Optidyl,而黑头杜泊羊精液适合采用OPTIXcell。     

Influence of season on the freezability of free-range poultry semen

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.     

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.     

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.     

Implications of ram sperm rheotaxis analysed by microfluidics for fertility

Rheotaxis of sperm using a microfluidic device was explored in human, mice and bull. However, the rheotaxis of ram sperm and its role in fertility are unknown. Herein, we described the sperm rheotaxis in ram using microfluidic devices and focused on rheotaxis as potential markers of in vivo fertility. Computer-assisted sperm analysis (CASA) with controlled flow velocity was used to explore the kinematic parameters of sperm, total motility and positive rheotaxis (PR). The percentage of PR was defined as the number of PR sperm cells over the number of motile sperm cells. Then, according to the percentage of PR sperm, rams were classified into two groups; sperm with ≥40% PR and <40% PR, although the two ram groups showed similar total motility and kinematic values of sperm evaluated by CASA (p > .05). Two groups of rams mated one hundred thirty ewes naturally (10 ewes/ram). In the results, the pregnancy rate was higher in ≥40% PR (94.4%) than in <40% PR (42.5%, p < .05) after natural mating. Besides, the pregnancy loss was higher in <40% PR (33.3%) than in >40% PR group (8.1%, p < .05). In conclusion, the PR examination in semen can contribute to evaluate the reproductive performance of ram that will provide valuable insights into the semen evaluation.     

Evaluation of spermiation indices with multiple sperm collections in endangered sterlet (Acipenser ruthenus)

This study investigated the effects of multiple collections of sperm on endangered sterlet (Acipenser ruthenus) sperm functional parameters [spermatozoa motility and curvilinear velocity (VCL)] as well as on protein concentration and osmolality of seminal plasma. The average sperm volume and mean spermatozoa concentration per male were significantly altered with multiple collections. On the other hand, no significant effect of multiple collections on protein concentration of seminal plasma was observed. In all experimental groups, moderate impact of sequential collection on osmolality (p < 0.05) of seminal plasma was observed. Ninety to 100% of motile spermatozoa were observed at 15 s after activation, with an average VCL of 181.12 ± 19.10 μm/s. After 90 s, average VCL decreased to 130 ± 26 μm/s. Motility was maintained for up to 4 min. The maximum percentage of motile spermatozoa was observed after the third collection of sperm. The spermatozoa VCL increased significantly with subsequent collections. The results of this study provide new data on the effects of multiple collections on quantitative and qualitative parameters of sperm in sterlet. The data confirmed that the sequential stripping has no negative effect on the percentage of motility and spermatozoa velocity. This should be beneficial for the development of sterlet aquaculture programs.     

Retracted: Determination of some blood and seminal plasma ions in the beluga,Huso huso (Linnaeus, 1758)

Blood and seminal plasma ionic parameters are essential for monitoring health status, detecting illnesses, fish stock conservation and development of artificial propagation methods via extender improvement. In this study, comparison of blood and seminal plasma ionic parameters in beluga, Huso huso (30–45 kg, 1–2 m, n = 10), was made. The results obtained show that Na⁺ (82.54 ± 5.46), Cl^‐ (15.95 ± 0.72) and K⁺ (3.57 ± 0.15) were predominant ions in the seminal plasma (as mm ). Blood ionic values (as mm ) were determined for Na⁺ (110.2 ± 1.26), K⁺ (3.77 ± 0.081), Cl^? (60.12 ± 1.5), Ca²⁺ (2.05 ± 0.35) and Mg²⁺ (1.9 ± 0.16). Results of the comparison between ionic parameters of seminal and blood plasma indicated that the concentrations of all parameters of blood plasma with the exception of K⁺ were significantly (p < 0.05) higher than those of seminal plasma.     

Post‐thawing effects of three cryopreservation diluents on Rusa deer (Rusa timorensis) spermatozoa

The aim of this study was to evaluate home‐made and commercial extenders for the cryopreservation of Rusa deer semen. After collection by electroejaculation, six ejaculates were diluted and frozen in TES‐based, Tris‐based and Triladyl^® extenders. Subjective motility, viability, morphology, acrosome integrity and membrane functionality were assessed post‐thawing and after 1‐hr incubation at 37°C (Thermal stress test). Total and progressive motility, and kinematic parameters were also assessed through CASA system. Post‐thawing sperm progressive motility (PM), velocity according to the straight path (VSL) and linearity (LIN) showed significant differences, and higher values were detected for spermatozoa diluted with Triladyl^® and TES (p < 0.05) as compared with Tris (PM of Triladyl^® 14.7% vs. 3.2% TES and 2.5% Tris; VSL 56 for Triladyl^®, 59.2 for TES and 41.7 for Tris; LIN 45.6 for Triladyl^®, 52 for TES and 36.5 for Tris). Triladyl^® and TES extender led to better post‐thawing sperm parameters, but these preliminary results need to be verified through artificial insemination trials.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

Correlation of testicular artery Doppler velocimetry with kinetics and morphologic characteristics of epididymal sperm in dogs

Sperm quality can be affected by a reduction in testicular blood flow, which can be measured by Doppler ultrasonography. The aim of this study was to correlate the Doppler velocimetry of the testicular artery with kinetics of the epididymal spermatozoa in dogs. Twenty-two dogs (44 testicles) were evaluated by Doppler ultrasonography in five regions of the testicular artery before orchiectomy. Spermatozoa were recovered by the epididymal tail compression technique and analysed for kinetics on a computer-assisted semen analysis (CASA system). Morphology (modified Karras) and sperm membrane integrity were analysed by eosin–nigrosine staining. Data were analysed by Pearson's correlation test (p < .01). The mean total motility was 69.0% ± 17.7, progressive motility was 43.7% ± 14.7, average path velocity (VAP) was 127.0 µm/s ± 20.7, curvilinear velocity (VCL) was 221.0 µm/s ± 31.1, and sperm velocity index (SVI) was 389.9 ± 56.1. There were positive correlations between the peak systolic velocity (PSV) in the proximal supratesticular region with the SVI (r = .529), VCL (r = .555) and VAP (r = .473), and a negative correlation with the percentage of slow spermatozoa (r = −.463). The results suggest that the testicular artery blood flow velocity can positively affect the speed of spermatozoa movement. For the first time, we have correlated sperm kinetics with the Doppler evaluation of the testicular artery in dogs.     

Prostasomes of Canine Seminal Plasma – Zinc‐Binding Ability and Effects on Motility Characteristics and Plasma Membrane Integrity of Spermatozoa

Prostasomes are small lipid membrane‐confined vesicles that are involved in various fertilization‐related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross‐bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc‐affinity chromatography on a Chelating Sepharose Fast Flow – Zn^{2 +} showed that from 93 to 100% of the prostasome proteins bind zinc ions (P⁺Zn). SDS‐PAGE revealed that canine P⁺Zn comprised four protein bands, with low molecular weights (10.2–12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold‐stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.