Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.     

Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 10⁶ sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1 mg/ml papain or 5 U/ml bromelain were used (n = 10 straws per treatment). Immediately after thawing (38°C for 40 s), sperm concentration of each straw within treatment was readjusted to 15 x 10⁶ sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p < .05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p < .05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p < .05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.     

Low‐density lipoproteins and milk serum proteins improve the quality of stallion sperm after vitrification in straws

Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low‐density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25‐ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post‐warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p < 0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification.     

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.     

Variation in sperm cryosurvival is not modified by replacing the cryoprotectant glycerol with ethylene glycol in bulls

Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programmes. This study hypothesized that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl^®, 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh – 0 hr, pre-freeze – 4 hr and post-thaw). Tuli bulls produced larger (11.8 ± 0.31 ml vs. 8.5 ± 0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70 ± 0.09 × 10⁹ sperm/ml vs. 1.37 ± 0.10 × 10⁹ sperm/ml), sperm motility index (SMI, 35.0 ± 3.4 % vs. 67.8 ± 2.1 %) and viability (69.7 ± 1.1 % vs. 75.7 ± 1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.     

Stallion Sperm Integrity After Centrifugation to Reduce Seminal Plasma Concentration and Cool Storage for 4 days

The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 10⁶ sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator for the remainder of the cooling period. At day 0, 2, and 4, total motility (TM), progressive motility (PM), and plasma (PLM) and acrosomal membrane integrity were assessed. Centrifuged groups had higher TM and PM at day 4 than the control group (P < .05). Likewise, centrifuged groups had higher intact PLM in day 4 (P < .05). A single centrifugation cycle to reduce seminal plasma concentration will suffice to preserve sperm integrity acceptable for an artificial insemination dose up to 4 days of cool storage.     

Method agreement between three different chambers for comparative boar semen computer‐assisted sperm analysis

The computer‐assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 10⁶ sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 μm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non‐progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.     

Total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs

The aim of this study was to evaluate the total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs. The study was conducted on 10 infertile and 10 fertile dogs of various breeds. Infertility was defined as conception failure at least three matings with different bitches. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using CASA Hamilton Thorne, Vers. IVOS 12.3. The morphology of spermatozoa and the percentage of live and dead sperm cells were assessed microscopically, total antioxidant capacity and the content of SH‐groups in seminal plasma were determined spectrophotometrically, the contents of protein peroxidation markers in seminal plasma, bityrosine and formylokinurenine, were determined using spectrofluorimetric methods. Sperm concentration and total sperm count were significantly (p < 0.05) lower in infertile dogs than in fertile dogs (99.92 ± 3 0.05 × 10⁶/ml vs. 282.07 ± 48.27 × 10⁶/ml; 214.19 ± 114.74 × 10⁶ vs. 747.57 ± 210.94 × 10⁶, respectively). The percentage of spermatozoa with normal morphology and the most determined motility parameters differed significantly (p < 0.05) between both groups. The mean values of total antioxidant capacity in the seminal plasma were significantly (p < 0.05) lower (19.95 ± 20.94 vs. 25.66 ± 23.18 µmol/g protein), whereas the mean contents of bityrosine and formylokinurenine in seminal plasma were significantly (p < 0.05) higher in infertile dogs than in fertile dogs (3.71 ± 4.83 µg/mg protein vs. 1.55 ± 2.00 µg/mg protein and 0.37 ± 0.45 µg/mg protein vs. 0.14 ± 0.08 µg/mg protein, respectively). In conclusion, the obtained results suggest that the poor semen quality and infertility in dogs could be associated with lowered total antioxidant capacity and increased protein peroxidation in seminal plasma as a consequence of oxidative stress.     

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non‐permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.     

The Effect of Sperm Concentration and Storage Vessel on Quercetin‐Supplemented Rabbit Semen During Chilled Storage

Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris‐based extender supplemented with 100 μm quercetin to a concentration of 15, 30 or 60 × 10⁶ spermatozoa/ml, packaged into plastic tubes or 0.5‐ml straws and stored at 15°C. Sperm quality was assessed by computer‐assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H₂O₂ production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10⁶ spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD‐DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10⁶ spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10⁶ spermatozoa/ml may provide a suitable balance between motility and H₂O₂ production to best maintain overall sperm function and should be evaluated in a large‐scale AI trial.     

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.     

Assessment of the accuracy of testicular dysfunction detection in male donkey (Equus asinus) with the aid of colour-spectral Doppler in relation to plasma testosterone and serum nitric oxide levels

This study aimed to determine the usefulness of colour and pulsed Doppler modes for the accurate diagnosis of donkeys suffering from subfertility to determine whether testicular vascularity assessment could be an indicator for sperm functionality. The study sample was composed of 10 male donkeys with normospermia (control group) and 10 donkeys with hypospermia. Animals underwent scrotal circumference measurement, testicular Doppler examination, seminal evaluation, blood sampling and hormonal assay. Semen volume and concentration were significantly (p ≤ .05) lower in the subfertile group (30.25 ± 1.22 ml and 89.44 ± 2.55 × 10⁶/ml) as compared with the control group (82.76 ± 1.65 ml and 452.78 ± 1.25 × 10⁶/ml), and total sperm/ejaculation was significantly (p ≤ .05) higher in the normal donkeys (28.30 ± 2.32 × 10⁹/total ejaculated) as compared with the subfertile group. Intratesticular coloured area showed a marked decline in the hypospermic males. There was no significant difference between the two groups in testosterone level, although the normal group showed an increase in nitric oxide metabolites. Both Doppler indices of the three branches of the testicular artery were elevated significantly (p ≤ .05) in abnormal donkeys, whereas Doppler peak systolic and end-diastolic velocities were increased in the normal group. Male donkeys with subfertility demonstrated lower arterial vascularity parameters in the form of intratesticular coloured area and blood flow rate; therefore, the most optimal parameters for differentiating subfertile hypospermic from normospermic donkeys were found to be the two Doppler indices, velocities parameters, testicular blood flow rate and nitric oxide levels.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Exogenous cholesterol prevents cryocapacitation-like changes,membrane fluidity,and enhances in vitro fertility in bubaline spermatozoa

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10⁶ spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca²⁺, capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca²⁺ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10⁶ spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10⁶ spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10⁶ spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca²⁺); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca²⁺, low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca²⁺, medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10⁶ spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing–thawing-associated damages in bubaline species.     

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.     

Estimation of genetic parameters and season effects for semen traits in three pig breeds of South China

The economic profitability of a boar station largely depends on semen quantity and quality traits. However, genetic analysis of semen traits has not yet been done in the boar population in China. In this study, we aimed to estimate genetic parameters for semen traits and the influence of seasons on these traits by using data of Duroc, Landrace and Yorkshire boars in South China. The following four semen traits were analysed: semen volume (ml; VOL), sperm concentration (10⁶/ml; DEN), sperm motility (MOT) and percentage of abnormal sperm (ABN). Genetic parameters and season effects were estimated simultaneously for each breed by using a multiple‐trait (4 × 4) repeatability animal model. The four traits had a moderate heritability with average estimates of 0.23, 0.28, 0.26 and 0.17 across the three breeds, respectively. The estimates of genetic correlations among four traits differed in the three breeds. In particular, in Yorkshire, the four traits were nearly genetically independent. The season of collecting semen had a significant impact on these four semen traits except ABN in Duroc (Bonferroni adjusted p < 0.05/6). The moderate heritabilities indicate the possibility of effective selection of boars for semen traits. Different genetic correlations for the three breeds suggest that the selection strategy for the four traits should be investigated separately for each breed. Some necessary actions should be taken to reduce the influence of seasons on semen traits.     

Effect of frame rate capture frequency on sperm kinematic parameters and subpopulation structure definition in boars,analysed with a CASA‐Mot system

Motility is the most widely used indicator of sperm quality. Computer‐Assisted Semen Analysis (CASA) allows the objective evaluation of sperm motility parameters. CASA technology is a common tool to predict semen doses in farm animal reproduction. The kinds of video cameras used until now for image acquisition have presented limited frame rates (FR), which have a negative influence on the quality of the obtained data. The aim of the present work was to define the optimal frame rate for a correct evaluation of boar sperm motility and its subpopulation structure. Eighteen ejaculates from nine mature boars of the Pietrain breed were used. Using the ISAS^®v1 CASA‐Mot system, with a video camera working up to 200 Hz, six FRs (25, 50, 75, 100, 150 and 200 fps) were compared. ISAS^®D4C20 counting chambers, warmed to 37°C, were used. FR affected all the kinematic parameters, with curvilinear velocity (VCL) and BCF the most sensitive ones. All the parameters showed differences among animals. Non‐linear correlation showed the asymptotic level for VCL at 212 fps, being the highest FR for all the parameters. For future studies based just on progressive motility, almost 100 fps FR for 0.5 s must be used, while when kinematics must be considered, almost 212 fps for one‐second should be analysed. Three principal components were obtained (velocity, progressivity and oscillation), being similar at 50 and 200 fps. Cells were grouped in four subpopulations but with different kinematic and cellular distribution at both FRs.     

Thymus satureioides and Origanum majorana essential oils improve the quality of Beni Arouss buck semen during storage at 4°C

This study aims to investigate the effects of essential oils (EOs), extracted from Thymus satureioides (TS) and Origanum majorana (OM), on Beni Arouss buck semen quality stored in skimmed milk at 4°C. EOs were extracted by hydro-distillation, and the chemical compounds were determined. Ejaculates were collected from six Beni Arouss bucks, once a week for 10 weeks, and they were pooled, divided into five equal aliquots and diluted to 400 × 10⁶ sperm/ml with skimmed milk supplemented with 0.01% of OM EO, 0.01% of TS EO, 0.05% of OM EO and 0.05% of TS EO. Non-supplemented skimmed milk was considered as a control. Semen motility, kinematic parameters, viability, abnormality, membrane integrity and lipid peroxidation were evaluated at 0, 4, 8, 24, 28, 32 and 48 hr of liquid storage at 4°C. The main EO components were carvacrol (31.7%), thymol (28.0%) and borneol (14.4%) for TS, and terpinene-4-ol (31.2%), γ-terpinene (17.4%) and α-terpinene (12.7%) for OM. The results highlighted a dose-dependent effect of TS and OM EOs on all semen quality parameters. 0.01% of both EOs had a beneficial effect on the sperm preservation stored at 4°C compared with control (p < .05) excepted for the straight-line velocity. The 0.05% EO addition had harmful effects during storage particularly for TS EO. In conclusion, 0.01% of TS and OM EOs are recommended to improve the Beni Arouss buck semen preservation at 4°C.     

Addition of Cholesterol‐Loaded Cyclodextrins to the Thawing Extender: Effects on Boar Sperm Quality

The aim of the present study was to evaluate the effect that the addition of cholesterol‐loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen‐thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg‐yolk‐based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 10⁶ sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 10⁶ sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 10⁶ sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing.