145 ProAKAP4 concentrations in semen as a predictive tool of bull fertility: A preliminary study

2020 ◽  
Vol 32 (2) ◽  
pp. 199 ◽  
Author(s):  
I. Ruelle ◽  
N. Seregeant ◽  
D. Bencharif ◽  
F. Charreaux ◽  
C. Thorin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sperm Quality ◽  
Intrauterine Insemination ◽  
Human Reproduction ◽  
Sperm Parameters ◽  
Enzyme Linked Immunosorbent Assay ◽  
Computer Assisted ◽  
Predictive Tool ◽  
Progressive Motility ◽  
Preliminary Study

Recently, ProAKAP4 has been described as a pertinent indicator of sperm quality in humans, pigs, and stallions. In knockout mouse models lacking AKAP4 expression, the male mice were infertile. As high proAKAP4 levels were significantly correlated with a lower proportion of abortions in intrauterine insemination settings in human reproduction, proAKAP4 could be considered a pertinent new sperm parameter for assessing embryo quality. Our main goal was to assess the proAKAP4 concentrations in Holstein bull semen for comparison with the motility sperm parameters and fertility outcomes in post-thawed conditions. Straws issued from 52 ejaculates from 13 bulls, retrospectively identified with known nonreturn rates (NRR) as a fertility indicator, were provided by Evolution XY. Expression of ProAKAP4 and AKAP4 was assessed using enzyme-linked immunosorbent assay, western blotting, flow cytometry, and microscopy methods. Using the Bull 4MID kit (4BioDx), striking variations in proAKAP4 concentrations were observed independently of the classic sperm parameters that were measured using computer-assisted semen analysis. A mean proAKAP4 concentration of 44.42ng per 10 million spermatozoa was obtained through all our series. Interestingly, the variations in proAKAP4 concentrations were positively correlated with progressive motility and with the linearity coefficient parameter. Furthermore, the post-thawed concentrations of proAKAP4 were significantly higher in bulls with a higher NRR in a field study of more than 190 000 AI. We then demonstrated for the first time a correlation between the semen concentration of proAKAP4 and NRR (P=0.05) in bulls. Threshold values of proAKAP4 were then determined, with good values being between 25 and 60ngmL−1. Below 25ngmL−1, the sperm were of poor quality. The proportion of functional spermatozoa (i.e. spermatozoa expressing proAKAP4 in ejaculates) was assessed using flow cytometry. We observed that the cell debris and dead spermatozoa were never immunolabeled with proAKAP4 antibodies. On testis tissue sections, proAKAP4 was expressed only from the spermatids stages up to the ejaculated spermatozoa, being influenced by external factors and reflecting good spermatogenesis. Our preliminary study highlighted the pertinence of proAKAP4 in assessing sperm quality in bulls. It could be interesting to further analyse the effect of proAKAP4 level of expression on capacitation and IVF. As high levels of proAKAP4 were significantly correlated with fertility rates and with progressive motility, proAKAP4 could be proposed as a predictive marker of bull fertility and could be further investigated to evaluate the quality of invitro-produced embryos.

scholarly journals Improvement in Sperm Parameters With Traditional Iranian Remedy

2016 ◽  
Vol 22 (2) ◽  
pp. 223-226 ◽  
Author(s):  
Farnaz Sohrabvand ◽  
Somaye Mahroozade ◽  
Sodabe Bioos ◽  
Seyed Mohammad Nazari ◽  
Fataneh Hashem Dabaghian
Keyword(s):  
Male Infertility ◽  
Traditional Medicine ◽  
Assisted Reproductive Technology ◽  
Intracytoplasmic Sperm Injection ◽  
Sperm Quality ◽  
Intrauterine Insemination ◽  
Sperm Count ◽  
Reproductive Technology ◽  
Sperm Parameters ◽  
Medicinal Treatment

Introduction. Idiopathic male infertility is a global problem with almost no definite medicinal treatment. Most patients have to go through intrauterine insemination or assisted reproductive technology for achieving fertility. Unfortunately, success rates are low in cases with very low sperm count. Therefore it seems that improvement in sperm quality can have beneficial effects on assisted reproductive technology outcome. Case Report. A 39-year-old man with history of infertility for 6 years was referred to the traditional medicine clinic with a recurrent unsuccessful intracytoplasmic sperm injection trial. His sperm analysis showed severe oligoasthenoteratozoospermia. After taking a traditional remedy he had a remarkable improvement in his sperm parameters, which led to the formation of 8 embryos in the following intracytoplasmic sperm injection cycle. Conclusion. Traditional medicine presents various food and remedy options for treating male infertility. It seems that combination therapy can be beneficial in obtaining better results in treatment of male idiopathic infertility.

scholarly journals The use of insulin-like growth factor 1 improved the parameters of the seminogram in a patient with severe oligoasthenoteratozoospermia

2019 ◽  
Vol 7 ◽  
pp. 2050313X1983415
Author(s):  
José Arturo Mora Rodríguez ◽  
Leonardo M Porchia ◽  
Felipe Camargo ◽  
Esther López-Bayghen
Keyword(s):  
Growth Factor ◽  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Quality Parameters ◽  
Intradermal Injection ◽  
Sperm Parameters ◽  
Insulin Like Growth Factor ◽  
Progressive Motility

Male patients suffering from oligoasthenoteratozoospermia typically failed to achieve pregnancy, even with assisted reproductive technologies. Growth hormone and insulin-like growth factor 1 have been shown to regulate sperm quality parameters; therefore, the insulin-like growth factor 1 supplement could improve sperm parameters. Here, we determine the effect insulin-like growth factor 1 has on sperm parameters in a patient suffering from oligoasthenoteratozoospermia. A 47-year-old male was administered once a day 1.5 IU of insulin-like growth factor 1 by intradermal injection for 2 months. Seminogram analysis was performed before and after. Treatment with insulin-like growth factor 1 resulted in a 15.5-fold improvement in sperm concentration (1.1 × 106 vs 18.3 × 106 per mL), 71.4% change in volume (0.7 vs 1.2 mL), increased progressive motility (2% vs 43%), and the total volume of sperm with progressive motility (0% vs 23.6%). Here, we show that administering a daily dose of insulin-like growth factor 1 can improve sperm quality parameters.

scholarly journals Sperm quality assessments for endangered razorback suckers Xyrauchen texanus

Reproduction ◽  
2011 ◽  
Vol 141 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jill A Jenkins ◽  
Bruce E Eilts ◽  
Amy M Guitreau ◽  
Chester R Figiel ◽  
Rassa O Draugelis-Dale ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Natural Populations ◽  
Reproductive Capacity ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Isotonic Buffer ◽  
Quality Assessments ◽  
Sperm Motion

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckersXyrauchen texanuscollected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2;P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2;P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985;P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=−0.83;P=0.0116) and mitochondrial function (r=−0.91;P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=−0.77;P<0.0001) and pre-freeze viabilities (r=−0.66;P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.

scholarly journals Effects of Sperm Parameters on Pregnancy Rate in Patients Undergoing Intrauterine Insemination

2020 ◽  
pp. 1
Author(s):  
Mehmet Solakhan ◽  
Mustafa Demir
Keyword(s):  
Intrauterine Insemination ◽  
Sperm Count ◽  
Sperm Concentration ◽  
Sperm Parameters ◽  
Motile Sperm ◽  
Progressive Motility ◽  
Age Related ◽  
Significant Difference ◽  
Sperm Volume ◽  
Infertile Couples

<p><strong>OBJECTIVE:</strong> In this study, the effects of sperm parameters on the success of intrauterine insemination were investigated. </p><p><strong>STUDY DESIGN:</strong> The data from 309 infertile couples who were admitted between 2012-2018 without a female factor were analyzed retrospectively and included in the study. After the administration of gonadotropin and hCG (5000-10000 IU), single insemination was performed in 36-40 hours in all cycles. All couples underwent routine infertility screening. The relationship between sperm parameters (motility, morphology, sperm count), patient age, duration of infertility with intrauterine insemination success was evaluated.</p><p><strong>RESULTS:</strong> There was no statistically significant difference between the two groups in terms of mean age and age related-parity. There was no statistically significant difference between male ages, liquefaction, and sperm volumes between the two groups (p=0.898, p=0.448, p=0.651). Before washing; There was a statistically significant difference between the sperm concentration, percentage of total motile sperm, percentage of progressive motility sperm, percentage of normal sperm morphology, and total sperm count between the two groups (p=0.0001, p=0.0001, p=0.0001, p=0.0001, p=0.0001). After sperm washing; the results were similar to those obtained before washing. While statistically significant difference was observed between sperm volume and sperm concentrations (p=0.023, p=0.018), no significant difference was observed between the two groups in total sperm count (p=0.612).</p><p><strong>CONCLUSION:</strong> As a result, during the application of intrauterine insemination to infertile couples, total motile sperm count, progressive motility sperm count ratio and high sperm ratio with normal morphology used in order to increase pregnancy success can be considered as criteria that increase the chances of success.</p>

72 EFFECT OF SINGLE-LAYER CENTRIFUGATION WITH EQUIPURE™ ON MOTILITY KINEMATICS OF FROZEN - THAWED DONKEY SPERM

2013 ◽  
Vol 25 (1) ◽  
pp. 183 ◽  
Author(s):  
I. Ortiz ◽  
J. Dorado ◽  
D. Acha ◽  
L. Ramirez ◽  
M. Urbano ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Single Layer ◽  
Bottom Layer ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Sperm Analysis ◽  
Progressive Motility ◽  
Head Displacement ◽  
Straight Line ◽  
Computer Assisted Sperm Analysis

Single-layer centrifugation (SLC) with EquipureTM Bottom Layer has been used to enhance the quality of stallion semen samples; however, no studies have been performed on donkeys. The aim of this study was to determine if SLC with EquipureTM Bottom Layer improves kinematic parameters on frozen–thawed donkey sperm. Semen was collected from 4 Andalusian donkeys by artificial vagina. Three ejaculates from each donkey were centrifuged with EquiproTM, supernatant was removed, and pellet was re-extended in the freezing medium GentTM to a final concentration of 200 × 106 spermatozoa per milliliter. Sperm were slowly cooled to 5°C for 2 h, loaded in 0.5-mL plastic straws, and frozen in liquid-nitrogen vapors. After at least one week of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: aliquot 1 was used as such (control) and aliquot 2 was processed by SLC using EquipureTM Bottom Layer. Computer-assisted sperm analysis was performed, and sperm kinematics total motility (%), progressive motility (%), curvilinear velocity (VCL; µm s–1), velocity straight line (VSL; µm s–1), velocity average path (VAP; µm s–1), linearity (LIN; %), straightness (STR; %), wobble (WOB; %), lateral head displacement (ALH; µm), and beat cross frequency (BCF; Hz) were statistically compared using GLM model between frozen–thawed semen samples processed or not with EquipureTM. Results were expressed as mean ± standard error. Significant differences (P < 0.05) were found between SLC-selected and unselected semen for total motility (77.44 ± 5.83 v. 58.89 ± 6.07), progressive motility (76.88 ± 4.52 v. 56.59 ± 5.44), VCL (137.50 ± 0.75 v. 133.0 ± 0.99), LIN (69.43 ± 0.31 v. 68.23 ± 0.41), STR (78.45 ± 0.29 v. 76.90 ± 0.37), WOB (85.06 ± 0.18 v. 83.91 ± 0.26), ALH (2.76 ± 0.01 v. 2.44 ± 0.01), and BCF (9.13 ± 0.05 v. 8.53 ± 0.06), respectively. No significant differences were observed for VSL (102.89 ± 0.70 v. 104.32 ± 0.95) and VAP (123.21 ± 0.71 v. 121.50 ± 0.98). Most of the computer-assisted sperm analysis parameters used in the present study have been previously identified as reliable markers of sperm motility in relation to sperm quality and fertility. It has also been reported that VCL appears to be critical for the formation of the sperm reservoir and penetration of the zona pellucida. In addition, other variables improved in the SLC-selected samples have been described as measure of progressivity (LIN, STR) and spermatozoa vigor (BCF, ALH). These preliminary results suggest an additional option for improving sperm quality in donkey semen doses. In conclusion, SLC with EquipureTM can be used to enhance kinematic parameters on frozen–thawed donkey sperm.

121 Effect of pentoxifylline on motility of good- and poor-quality frozen-thawed equine sperm

2020 ◽  
Vol 32 (2) ◽  
pp. 187
Author(s):  
M. Felix ◽  
I. Ortiz ◽  
H. Resende ◽  
J. Brom-de-Luna ◽  
C. Love ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Phosphodiesterase Inhibitor ◽  
Petri Dish ◽  
Poor Quality ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Significant Difference ◽  
The Right

Equine semen used for intracytoplasmic sperm injection (ICSI) is typically frozen-thawed and may be of poor quality. To prepare sperm for ICSI, semen is typically centrifuged to remove freezing extender. However, centrifugation can cause damage to sperm, which is especially meaningful if sperm quality is already poor. We evaluated a method for selection of sperm without centrifugation, using a “swim-over” technique, and assessed the effect of pentoxifylline, a phosphodiesterase inhibitor that increases sperm motility in other species. To mimic poor-quality semen, we thawed frozen semen (1×) and re-froze it three additional times (4×). Aliquots (0.25 µL; 50,000 sperm) of 1× or 4× semen were placed at the bottom of the right leg of an “H,” made using 15µL of medium by tracing a template placed below a Petri dish. The medium used (Hanks’ balanced salt solution with 40mg mL BSA and added lactate and pyruvate) contained different concentrations of pentoxifylline (0, 0.5, 1, 2 or 4mgmL−1). One µL of medium was removed from the tip of the left arm of the H after 15 and 30min incubation, and the number of sperm were counted. In a second study, we evaluated the effect of pentoxifylline on sperm motility parameters using computer-assisted sperm motility analysis. After thawing, 1× and 4× semen was washed to remove freezing extender and resuspended in the same medium but with 7mgmL−1 bovine serum albumin (BSA), containing the different pentoxifylline concentrations. In Study 1, the number of collected sperm did not differ significantly for 1× sperm exposed to 0 to 4mgmL−1 pentoxifylline (means of 15 to 23 sperm at 15min, and 18 to 25 sperm at 30min). Similarly, in 4× frozen semen, there was no significant difference in number of collected sperm between 0mgmL−1 and 2 or 4mgmL−1 pentoxifylline concentrations (&lt;1 to 6 at 15 min; 5 to 6 at 30min). In Study 2, at 0min,% total motility was significantly higher in 1 and 2mgmL−1 pentoxifylline than in 0mgmL−1 for 1× sperm (47.8±1.7 and 49.3±1.9, vs. 32.1±3.9, respectively; P=0.018) and significantly higher for 1, 2, and 4mgmL−1 pentoxifylline than for 0mgmL−1 for 4× sperm (3.9±0.9, 5.7±0.4, and 8.2±0.5, vs. 1.2±0.4; P=0.0001). Similar results were found at 15 and 30min for 1×, and at 15min for 4×. Pentoxifylline at 1 to 4mgmL−1 significantly increased the percentage of progressive motility in 1× sperm at 30min (17.8±1.3, 21.8±2.7, and 20.3±1.2, vs. 10.0±0.4; P=0.002) and, at 4mgmL−1, increased the percentage of progressive motility in 4× sperm at 0min (1.43±0.1 vs. 0.2±0.1; P=0.005) and 15min (1.4±0.2 vs. 0.1±0.0; P=0.0001). Exposure of poor-quality semen to pentoxifylline at 4mgmL−1 improved total and progressive motility but did not increase the recovery of motile sperm in a swim-over collection preparation.

scholarly journals Tetrazolium Salt WST-8 as a Novel and Reliable Chromogenic Indicator for the Assessment of Boar Semen Quality

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2293
Author(s):  
Yu-Hsin Chen ◽  
Chean-Ping Wu ◽  
Hsiu-Lien Lin ◽  
Ren-Bao Liaw ◽  
Yung-Yu Lai ◽  
...  
Keyword(s):  
Cell Viability ◽  
Semen Quality ◽  
Sperm Quality ◽  
Reduction Rate ◽  
Sperm Parameters ◽  
Cell Counting ◽  
Computer Assisted ◽  
Tetrazolium Salt ◽  
Reliable Prediction ◽  
Boar Semen

A tetrazolium salt, 2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium (WST-8), has been used widely to determine cell viability; however, its application in the field of reproduction is still limited due to this assay merely providing information regarding cell viability. The aim of this study was to correlate the WST-8 reduction rate with various sperm quality-related parameters (i.e., sperm viability, motility, progressive motility, acrosome integrity and mitochondria integrity) in order to provide a rapid, reliable and affordable assessment for boar semen quality evaluation. Using different ratios of active/damaged sperm cells, we first validated our sample preparations by standard flow cytometry and computer-assisted sperm analysis. Further analyses demonstrated that the most efficient experimental condition for obtaining a reliable prediction model was when sperm concentration reached 300 × 106 cells/mL with the semen/cell-counting kit-8 (CCK-8®) ratio of 200/10 and incubated time of 20 min. Under this set up, the WST-8 reduction rate (differences on optic density reading value, ΔOD at 450 nm) and sperm parameters were highly correlated (p < 0.01) for all sperm parameters evaluated. In the case of limited semen samples, a minimal semen concentration at 150 × 106 cells/mL with the semen/CCK-8® ratio of 200/20 and incubation time for 30 min could still provide reliable prediction of sperm parameters using the WST-8 assay. Our data provide strong evidence for the first time that the WST-8 assay could be used to evaluate boar semen quality with great potential to be applied to different mammalian species.

Colloid single-layer centrifugation improves post-thaw donkey (Equus asinus) sperm quality and is related to ejaculate freezability

2015 ◽  
Vol 27 (2) ◽  
pp. 332 ◽  
Author(s):  
I. Ortiz ◽  
J. Dorado ◽  
D. Acha ◽  
M. J. Gálvez ◽  
M. Urbano ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Single Layer ◽  
Quality Parameters ◽  
Sperm Parameters ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Equus Asinus ◽  
The Relationship ◽  
Fresh Semen

The aim of this study was to determine whether colloid single-layer centrifugation (SLC) improves post-thaw donkey sperm quality and if this potential enhancement is related to ejaculate freezability. Semen from Andalusian donkeys was frozen following a standard protocol. SLC was performed on frozen–thawed semen and post-thaw sperm parameters were compared with uncentrifuged samples. Sperm quality was estimated by integrating in a single value sperm motility (assessed by computer-assisted sperm analysis), morphology and viability (evaluated under brightfield or fluorescence microscopy). Sperm freezability was calculated as the relationship between sperm quality obtained before freezing and after thawing. Ejaculates were classified into low, medium and high freezability groups using the 25th and 75th percentiles as thresholds. All sperm parameters were significantly (P < 0.01) higher in SLC-selected samples in comparison to uncentrifuged frozen–thawed semen and several kinematic parameters were even higher than those obtained in fresh semen. The increment of sperm parameters after SLC selection was correlated with ejaculate freezability, obtaining the highest values after SLC in semen samples with low freezability. We concluded that, based on the sperm-quality parameters evaluated, SLC can be a suitable procedure to improve post-thaw sperm quality of cryopreserved donkey semen, in particular for those ejaculates with low freezability.

scholarly journals The effect of lipopolysaccharide from uropathogenic Escherichia coli on the immune system, testis tissue, and spermatozoa of BALB/c mice

2021 ◽  
Author(s):  
Khadije Rezai Khanmohammad ◽  
Mohammad Bagher Khalili ◽  
Maryam Sadeh ◽  
Ali Reza Talebi ◽  
Akram Astani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune System ◽  
Sperm Quality ◽  
Sodium Dodecyl ◽  
Uropathogenic Escherichia Coli ◽  
Sperm Parameters ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tract Infections ◽  
Testis Tissue

Objective: Uropathogenic Escherichia coli is known to cause urinary tract infections, and the endotoxin (lipopolysaccharide [LPS]) of this bacterium may cause deficiencies of sperm quality and morphology. In the present study, the effects of LPS on mouse sperm were studied, and the levels of interleukin (IL)-17A and possible changes in testis tissue were evaluated.Methods: LPS of uropathogenic E. coli was extracted using the methanol-chloroform method, followed confirmation using sodium dodecyl sulfate-polyacrylamide electrophoresis. Purified LPS (100 µg/kg) or phosphate-buffered saline was injected intraperitoneally into BALB/c mice for 7 days consecutively in the test and control groups, Mice were sacrificed on days 3, 7, and 42 after the first injection. Blood was tested for levels of IL-17A using the enzyme-linked immunosorbent assay method. Testis tissue and sperm were collected from each mouse and were studied according to standard protocols. Results: The mean sperm count and motility significantly decreased (p=0.03) at 3, 7, and 42 days after the injections. The level of IL-17A in the test groups increased, but not significantly (p=0.8, p=0.11, and p=0.15, respectively). Microscopic studies showed no obvious changes in the morphology of the testis tissue; however, significant changes were observed in the cellular parenchyma on day 42. Conclusion: LPS can stimulate the immune system to produce pro-inflammatory cytokines, resulting in an immune response in the testis and ultimately leading to deficiency in sperm parameters and testis tissue damage. In addition, the presence of LPS could significantly impair sperm parameters, as shown by the finding of decreased motility.

30 Bull sperm kinetics after semen cryopreservation in extender containing propagermanium

2019 ◽  
Vol 31 (1) ◽  
pp. 141
Author(s):  
T. E. Cruz ◽  
A. Martins Jr ◽  
F. N. Marqui ◽  
D. G. Souza ◽  
T. I. H. Berton ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Lipid ◽  
Free Radical Scavenger ◽  
Sperm Parameters ◽  
Radical Scavenger ◽  
Computer Assisted ◽  
Kinetics Parameters ◽  
Significance Level ◽  
Freezing Medium ◽  
Bull Sperm

There is a negative association between a plentiful production of oxygen reactive species and spermatozoa kinetics parameters. Thus, antioxidants have been added to the freezing medium to improve sperm quality due to their protective effect against membrane lipid peroxidation. Propagermanium (GE132) is an organometallic compound that has never been used in freezing medium despite its known antioxidant effect as a free radical scavenger. This study aimed to investigate the effects of different concentrations of GE132 added in a commercial freezing medium (CFM) on frozen-thawed sperm motion. Nine ejaculates of 3 Nellore bulls (3 replicates), collected by an artificial vagina, were evaluated, pooled, and divided into groups D (semen was diluted and kept at 33°C for 30min before cooling) and C (semen was cooled immediately after dilution). Both groups were submitted to the same experimental treatment, as follows: addition of 0, 500, and 1000µg mL−1 of GE132 in a CFM resulting in subgroups D0, D500, D1000, C0, C500, and C1000. The sperm samples were diluted to a final concentration of 30×106 spermatozoa per straw (0.25mL) and then cooled at 4°C for 5h before freezing. Sperm samples were assessed using a computer assisted sperm analyser at 5 and 60min post-thawing for total motility (TM;%), progressive motility (%), curvilinear velocity (μm s−1), velocity straight line (μm s−1), velocity average path (μm s−1), amplitude of the lateral head displacement (ALH; μm), beat cross frequency (Hz), linearity (LIN;%), and straightness (STR;%). Data were analysed using the R software package version 3.4.4 (2018; https://www.r-project.org/). An ANOVA was applied to assess statistical differences, and Tukey’s test was used to determine differences among subgroups. A significance level of P&lt;0.05 was adopted. No significant differences (P&gt;0.05) were observed among subgroups for all sperm parameters except for TM, in which C0 presented higher (P&lt;0.05) value (68.72±3.36) than D0 (54.67±5.59), D5 (57.10±2.34), and C10 (54.20±2.73), with similar results between D10 (59.5±4.22) and C5 (59.52±4.64). There were significant differences within subgroups when comparing 5 and 60min post-thawing for TM, ALH, LIN, and STR. Total motility decreased 17.2 and 9.9% in C5 and C10, respectively. Similarly, values of ALH decreased 0.2, 0.4, and 0.2µm in D0, D5, and C5, respectively. However, the increase in LIN was 11% in D10, whereas the values for STR increased in D10 (10%), C5 (6.3%), and C10 (7.3%). The addition of GE132 to the CFM did not enhance all the sperm parameters after cryopreservation except for a slight improvement in ALH, LIN, and STR over time and TM among groups. The lack of additive effect could be due to the presence of antioxidants in the CFM; therefore, further investigation with fluorescent probes using flow cytometry and free-antioxidants freezing medium could lead to a new approach for bull sperm freezing. We acknowledge Tairana AI Station, Master Fertility, and Botupharma, Brazil.

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