Motile sperm subpopulations in bull semen using different clustering approaches

There are sperm subpopulations (SPs) with different kinematic characteristics in various species, however, biological relevance of these SPs is still uncertain. The objective of the present study was to investigate associations of motile sperm SPs with sperm characteristics determined by evaluations with flow cytometry and assessment of bull fertility, using multiple approaches for sperm clustering. Semen from 24 bulls was evaluated concomitantly using computer-assisted sperm analysis (CASA) and flow cytometry before freezing and after thawing. Motile SPs were determined utilizing two acknowledged clustering methods (TwoStep and K-Means) and one customized method. With the customized method, there was utilization of mean values of sperm velocity and linearity as thresholds for direct assignment of motile spermatozoa into four SPs. Regardless of approach for identifying SPs, sperm quality, as determined using flow cytometry, was correlated particularly with the subpopulation (SP) of fast and linear spermatozoa immediately after thawing and with the SP of fast and nonlinear spermatozoa before freezing and 3 h after thawing. Furthermore, there was a positive correlation between proportion of spermatozoa with fast and nonlinear movements before freezing and bull non-return to estrous rates. These results indicate that with different sperm SPs, there are different biological implications which can be evaluated to gain useful information concerning semen quality as determined using flow cytometry and fertility. Furthermore, determining SPs by assigning motile spermatozoa into clusters based on a combination of “below and “above” threshold values for sperm velocity and linearity might be considered a practical alternative to otherwise intricate clustering procedures

Effects of different freezing protocols on motility, viability, mitochondrial membrane potential, intracellular calcium level, and DNA integrity of cryopreserved equine epididymal sperm

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous

Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen

This study was designed to investigate the effects of an equilibration period up to 96 hours and three extenders (AndroMed, OPTIXcell, and Triladyl) on the quality of cryopreserved bull semen and to evaluate, whether an extension of the equilibration time to 72 hours does affect fertility in the field. One ejaculate of 17 bulls was collected and divided into three equal aliquots and diluted, respectively, with the three extenders. Each aliquot was again divided into five parts and equilibrated for 4, 24, 48, 72, and 96 hours before freezing in an automatic freezer. Sperm motility, plasma membrane and acrosome integrity (PMAI), and DNA fragmentation index (% DFI) were measured during equilibration. In addition to the parameters measured during equilibration, the percentage of viable sperm cells with high mitochondrial membrane potential (HMMP) was measured immediately after thawing, and after 3 hours of incubation at 37 °C. Sperm motility was assessed using CASA, and PMAI, HMMP, and % DFI were measured using flow cytometry. Equilibration time did affect all parameters before freezing (P 0.05). In conclusion, extension of the equilibration time from 4 hours to 24-72 hours can improve motility and viability of cryopreserved semen after thawing. The extent of improvement in semen quality is dependent on the extender used. Prolongation of the equilibration period from 4 hours to 72 hours had no effect on fertility in the field

Comparison of the effects of five semen extenders on the quality of frozen-thawed equine epididymal sperm

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1-4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5)

Mitoquinone does not improve sperm cryo‐resistance in bulls

Oxidative stress is associated with impaired post-thaw sperm quality. As mitochondria are the main source of reactive oxygen species (ROS) in sperm, the goal of this study was to evaluate effects of the mitochondria-targeting antioxidant Mitoquinone (MitoQ) during cryopreservation of bull sperm. Semen was collected from 11 Simmental bulls (two ejaculates per bull) and diluted in Triladyl® supplemented with various concentrations of MitoQ (0, 0.2, 2, and 20 nM) to a final concentration of 65 × 106 sperm/ml. After thawing (0 and 3 hr), we assessed the following sperm traits: sperm motility by computer-assisted sperm analysis (CASA), DNA fragmentation index by SCSA® and plasma and acrosome membrane integrity, intracellular calcium concentration, esterase activity, mitochondrial membrane potential and synthesis of ROS using two multicolour flow cytometric assays. After 3 hr of incubation, 20 nM MitoQ increased (p .05). Therefore, we concluded that addition of MitoQ to semen extender before cryopreservation of bull sperm was unable to improve post-thaw sperm quality. Furthermore, 20 nM of MitoQ increased frozen-thawed sperm ROS synthesis, without apparent negative effects on the evaluated sperm traits

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting.

BACKGROUND: The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRR$_{conv}$ and NRR$_{ss}$, respectively) was recorded for each bull. RESULTS: In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21-5p were significantly correlated to NRR$_{ss}$ but not to NRR$_{conv}$. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRR$_{ss}$. CONCLUSIONS: A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire's suitability for the production of sex-sorted sperm

Multicolor flow cytometric analysis of cryopreserved bovine sperm: a tool for the evaluation of bull fertility

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; nHF = 10 bulls) or low fertility bulls (LF; nLF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC1(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (Cpos), intact plasma membrane (PIneg), unstained acrosome (PNAneg), low intracellular Ca2+ levels (Fneg), and high mitochondrial membrane potential (Mpos). In both fertility groups, Mpos cells comprised more than 90 and 84% of PInegPNAneg sperm at 0 and 3 h, respectively. The percentage of CposPInegPNAnegFnegMpos sperm did not differ between HF and LF ejaculates; however, the percentage of Fneg cells within the PInegPNAneg and PInegMpos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of Fneg sperm within the PInegMpos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at -196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing-thawing

Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm

The aim of this study was to investigate the effects of different concentrations of catalase in a TRIS-egg yolk extender on sperm quality and embryonic development after in vitro fertilization of frozen-thawed bull sperm. For this purpose, from each of 7 bulls 2 ejaculates were collected and diluted with a TRIS-egg yolk extender containing 0, 5, 10, 15 or 20 IU catalase/mL. Sperm quality was evaluated 0, 3, 6, 12 and 24 h after thawing by using computer assisted analysis of motility and by flow cytometric assays. Embryonic development was determined after in vitro fertilization of bovine oocytes. Semen diluted with TRIS-egg yolk extender containing different concentrations of catalase showed more motile sperm, more sperm with intact plasma membranes, acrosomes and DNA, a high mitochondrial membrane potential, a high esterase activity, a low calcium level, a lower amount of synthesis of reactive oxygen species and lower degree of lipid peroxidation of sperm compared to semen frozen without catalase (P < 0.05), but not before 3 h after thawing. There was a dose-response relationship with the most prominent effect of 20 IU catalase/mL. However, the improvement of sperm quality had no effect (P ≥ 0.05) on embryonic development after in vitro fertilization with 20 IU catalase/mL. In conclusion, the addition of catalase to the sperm extender improved sperm quality with no obvious effect on in vitro fertility