Average Path Velocity Research Articles

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Time of day modified the time required for semen collection with electroejaculation and slightly affected the quality of fresh semen in rams.

The aim was to compare some stress responses to electroejaculation (EE), and the quality of fresh semen, when ram semen is collected at dawn (06:00h), noon (12:00h), or evening (18:00h). Twelve Corriedale rams were used, and semen was collected from four rams at each study time on three different days, with a Latin-square design. The time required for EE, the number of vocalizations emitted, heart rate, and rectal temperature were recorded, and fresh semen was evaluated. The time required for EE was shorter at evening than at dawn and noon (399.3s, 480.6s, and 460.2s respectively; pooled SEM = 72.1; P = 0.03). The percentage of sperm with progressive motility was greater at noon than dawn (59.7% and 50.3%; pooled SEM = 5.8; P = 0.05). Curvilinear velocity was greater at dawn than evening (117.0µm/s and 95.5µm/s; pooled SEM = 7.1; P = 0.04), slow linear velocity was greater at evening than at dawn and noon (13.1µm/s, 9.3µm/s, and 8.5µm/s respectively; pooled SEM = 1.7, P = 0.05), and the slow average path velocity was greater at evening than dawn and noon (16.2µm/s, 11.7µm/s, and 10.8µm/s respectively; pooled SEM = 1.9, P = 0.05). In conclusion, the collection time modified the time required for electroejaculation and had only slight effects on the quality of fresh semen. Overall, the time of the day appears to have only slight effects on semen collection and quality.

Exogenous GSH Supplementation to Raw Semen Alters Sperm Kinematic Parameters in Infertile Patients.

Glutathione is an important antioxidant found in all mammalian cells. Sperm motility is positively correlated with seminal reduced glutathione (GSH) levels, and infertile men are known to have lower GSH levels. Studies on GSH supplementation in improving sperm functions in infertility patients are limited. Here, we re-investigate the effect of exogenous GSH supplementation on human sperm motility and kinematic parameters. Residual semen samples from 71 infertility patients who came for routine semen analysis for infertility assessment were studied. Liquefied raw semen was supplemented with GSH (0-10 mM) for 1 h. The untreated sample was the blank control. Only a 5 mM concentration was tested in all 71 samples. After two washes, the sperm was incubated and then analyzed for sperm motility and kinematic parameters by computer-assisted semen analysis (CASA), followed by adenosine triphosphate (ATP), reactive oxygen species (ROS) levels, free thiols, and DNA damage analyses. At 2 hrs post-treatment, GSH supplementation significantly altered many of the kinematics, compared to the control. Straight line velocity (VSL) (p = 0.0459), curvilinear velocity (VCL) (p < 0.0001), average path velocity (VAP) (p < 0.0001), and lateral head amplitude (ALH) (p < 0.0001) were decreased, whereas straightness (STR) (p = 0.0003), linearity (LIN) (p = 0.0008), and beat cross frequency (BCF) (p = 0.0291) were increased in 5 mM group. Wobble (WOB) (p = 0.4917), motility (MOT) (p = 0.9574), and progressive motility (PROG) (p = 0.5657) were unchanged. ATP level was significantly increased in the 5 mM group (p < 0.05). It is concluded that exogenous GSH supplementation does alter sperm kinematics in humans. These altered kinematic parameters together with increased energy (ATP) may have a positive role in influencing the success rates of ART procedures.

Sulforaphane inclusion in a freezing medium augments post-thaw motility, functional and biochemical features, and fertility potential of buffalo (Bubalus bubalis) spermatozoa

Sulforaphane is a natural and highly effective antioxidant safeguarding the reproductive system, and alleviate oxidative stress. This study was designed in order to elaborate L-sulforaphane effect on semen quality, biochemical parameters, and fertility of buffalo (Bubalus bubalis) spermatozoa. Semen was collected from five buffalo bull with artificial vagina (42 °C) three times and evaluated for volume, consistency (color), motility, and sperm concentration. After critical examination, semen was diluted (50 × 106 spermatozoa per ml, 37 °C) in extenders with (2 μM, 5 μM, 10 μM, and, 20 μM) or without (control) sulforaphane, cooled (from 37 to 4 °C), equilibrated (4 °C), filled (straws, 4 °C), and cryopreserved (LN2, −196 °C). Data analysis exhibited that sulforaphane addition in extender augments total motility (%, 10 μM, and 20 μM than control), progressive motility (%), and rapid velocity (%, 20 μM than control), and velocity parameters (average path velocity, μm/s, straight line velocity, μm/s and curved linear velocity, μm/s, 20 μM than control, and 2 μM). Moreover, sulforaphane augments functional features (membrane functionality, mitochondrial potential, and acrosome integrity) of buffalo sperm (20 μM than control). Sulforaphane preserves biochemical features of seminal plasma of buffalo i.e., Calcium (μM), and total antioxidant capacity (μM/L), followed by reduction in lactate dehydrogenase (IU/L), reactive oxygen species (104 RLU/20 min/ 25 million), and lipid peroxidation (μM/ml) in 20 μM than control. Lastly, sulforaphane augments fertility rate of buffalo sperm at 20 μM than control, and 2 μM. Conclusively the existing study revealed that adding L-sulforaphane (20 μM) in a freezing medium augments motilities, kinematics, functional parameters, and fertility rate of buffalo spermatozoa. Correspondingly, sperm favorable biochemical features were also augmented with sulforaphane followed by reduction in oxidative stress parameters. Further studies are highly recommended to define the particular mechanism of action of sulforaphane in augmenting buffalo post-thawed semen quality, and in vitro fertility potential.

Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

Triladyl® improves the cryopreserved quality and in vivo fertilization potential of Beetal buck (Capra hircus) spermatozoa

This study compared differential concentrations of chicken egg yolk (v/v; T1, 10%; T2, 15%; and T3, 20%) in commercial extender Triladyl® with tris-citric acid based extender (v/v; chicken egg yolk: 20%; C, control) for improving the in vitro quality and in vivo fertilization potential of Beetal buck (Capra hircus) spermatozoa at post–thawing. The mean values of progressive motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, μm sec−1), straight line velocity (VSL, μm sec−1), curvilinear velocity (VCL, μm sec−1), supra-vital plasma membrane integrity (SV−PMI, %), mitochondrial transmembrane potential (MMP, %), and viable with intact acrosome (V−IACR, %) were significantly higher(except significantly lower medium velocity; MV, %)in T1 and T2with than control at all stages. Mean values of straightness (STR – VSL: VAP %) and linearity (LIN – VSL: VCL %) were significantly higher in T1 and T2 than control at PD. In vitro longevity, from 0 to 3 h incubation (37 °C), showed significantly low PM (T1) and RV (T1 and T2) percentage loss than control at PT. A significantly positive Pearson’s correlation was noticed between CASA and SV−PMI, MMP, V−IACR, and DNA integrity (%). Significantly higher in vivo fertilization rates (%) were recorded in T1 (73.53) and T2 (74.19) compared to the C (46.88) and T3 (48.28), respectively. In conclusion, addition of even chicken egg yolk at 10% (v/v) in Triladyl® improves the cryopreserved in vitro and in vivo quality of Beetal buck spermatozoa as compared to tris-citric acid extender with 20% (v/v) egg yolk.

A novel approach to sperm selection: Nanoparticle-based purification improves quality of Angora cryopreserved buck’s semen

The Aim: The aim of the study was to investigate the applicability of an up-to-date method, sperm selection with nanoparticles at different temperatures (37ºC, 21ºC, and 4ºC), on Angora buck semen. Material and Methods: Semen samples were collected from 3 Angora bucks with 7 repetitions and each sample was diluted and divided into 4 groups. Nanopurification procedure was applied by using a nanoparticle dose of 10 μg/ml to the three groups at 37ºC, 21ºC, and 4ºC while in the control group samples were cryopreserved without nanopurificiation treatment. Post-thaw motility and morphology parameters of the sperm samples were analyzed by using computer-aided sperm analysis (CASA) and fluorescein microscopy.Results: After nanopurification at 37ºC total motility, linearity (LIN) (P&lt;0.05), curvilinear velocity (VCL), straight-line velocity (VSL), and acrosome integrity parameters (P&lt;0.001) were higher than the control group while the semen samples nanopurified at 21ºC were superior to the control group in terms of LIN, straightness (STR) (P&lt;0.05), VCL, VSL, average path velocity (VAP), wobble and acrosome integrity (P&lt;0.001). There was no significant difference between the control and the 4ºC group.Conclusion: In conclusion, nanopurification procedure on buck semen was useful in terms of parameters that affect fertility and the method can be performed at 21ºC, as well as 37ºC, to adapt the procedure to field conditions;

Development of a diluent storage media and effect of cryoprotectants on semen collected from white spotted bamboo sharks (Chiloscyllium plagiosum) and cooled to 5 ºC

Three experiments were performed to evaluate the effects of osmolarity, temperature, media and cryoprotectants on white spotted bamboo shark (Chiloscyllium plagiosum) spermatozoa. Semen was collected from 7 different males using a massage technique with the animals held in tonic immobilization with gills submerged. A Computer Assisted Sperm Analysis (CASA) system was used to assess sperm motility. Experiment one evaluated the effects of Hank’s balanced salt solution (HBSS) with different osmolarities (200, 400, 800 and 1000 mOsm Kg1) and incubation temperatures (5 °C and 24 °C [room temperature]) over time (6 h, 12 h and 24 h). Membrane integrity and morphology were degraded (P < 0.05) over time; however, sperm extended with HBSS 1000 and HBSS 800 were least affected. Higher total motility (TM) and average path velocity (VAP, P > 0.05) sperm parameters were observed with HBSS 1000 and HBSS 800, respectively. In addition, TM, VAP, and curvilinear velocity (VCL) were all reduced (P < 0.05) compared to sperm held at 5 °C. Experiment two compared the effects of sperm diluted in crystal reef water (CRW), elasmobranch ringers (ER) or raw held at 5 °C. Results indicated that CRW had improved viability, TM, VAP, VSL, VCL, and straightness (STR) when compared to sperm diluted in ER or Raw at 5 °C. The final experiment compared different cryoprotectants (dimethyl sulfoxide [DMSO] 10%, DMSO 20%, Ethylene-glycol [EG] 10%, EG 20%, and Trehalose [Tre] 10%, Tre 20%) during incubation at 5 °C. Across all time points, sperm incubated in Tre (10 or 20%) had increased (P < 0.05) membrane integrity and normal morphology compared to all other media and raw. Across all time points, sperm exposed to Tre (10% and 20%) and 10% DMSO had significantly improved TM (P < 0.05) compared to EG (10% and 20%) and 20% DMSO which were similar to raw. Although sperm collected from WSBS are robust and appear to tolerate a wide range of osmotic conditions and media, media made at 1000 mOsm Kg1 should provide the best in-vitro osmotic pressure, while storing at cooler temperatures should improve storage survival. For future cryopreservation studies, media using TRE and DMSO as cryoprotectants should be evaluated.

Effect of sperm and glycerol concentration on epididymal sperm motility parameters after thawing

The aim of this study was to evaluate the effect of different glycerol concentrations and different sperm concentrations on stallion epididymal sperm motility indicators after thawing. For statistical analysis, 25 stallions were used. Collection of the epidydimal spermatozoa was performed as a retrograde flush of cauda epididymis and ductus deferens within 12 h post castration. After evaluation, the resuspended spermatozoa were centrifuged, the supernatant removed and the spermatozoa resuspended in Gent semen extender to get three different groups with different concentrations of sperm (250 × 106 in ml, 500 × 106 in ml, 1000 × 106 in ml) and different final glycerol concentrations (2%, 4%, and 6%). Therefore, 9 different samples were finally obtained from each stallion. The spermatozoa were packed and placed in a fridge (4 °C) for 2 h, then placed in liquid nitrogen vapour (−80 to −100 °C) and after 25 min plunged into the liquid nitrogen and stored at −196 °C for at least 5 days. The selected straws were individually thawed in a 38 °C water bath for 30 s prior to post-freezing analysis. Motility indicators were assessed at 0, 60, 120, and 180 min after thawing. Parametric test was used for analysis; the measured indicators were total motility, progressive motility, curvilinear velocity, straightness, and average-path velocity. In this study, the best results were reached in samples diluted into a concentration of 1,000 × 106 in ml, regardless of the concentrations of glycerol.

Investigating Visual Monitoring of the Scrotum as a Supplementary Tool for Boar Semen Quality Evaluation.

Farm animals behavior research uses video cameras, mainly for visual observation and recording. The purpose of this feasibility study was to enrich the predictable methods of boar semen production capacity by correlating sperm variables with the scrotal contractions (SC) frequency and intensity. A video camera was used to record the reaction of the scrotum during ejaculation. The respective collected ejaculates were evaluated and semen parameters, such as viability, morphology, membranes functional integrity and kinematics, were determined. The camera recorded the scrotal contractions/relaxations and the video was handled by the Image Processing Toolbox of Matlab (Mathworks Inc., Natick, MA, USA). The SC intensity was verified as a percentage change in the scrotum size among the video frames of maximum contraction and relaxation. The archived data from the frames were analyzed statistically, using a linear mixed effects model that involved sperm assessed parameters. Correlations of the SC intensity with the average path velocity, VAP (R2 = 0.591, p = 0.043) and with the percentage of the cytoplasmic droplets (R2 = 0.509, p = 0.036) were noticed. Previous studies reported the positive correlation of VAP with the number of live-born piglets. In conclusion, video monitoring of the boar scrotal function during ejaculation is useful, but more research is needed to establish its appropriateness as a supplementary method for the prognosis of boar ability to produce high-quality semen.

Alkaline semen diluent combined with R848 for separation and enrichment of dairy goat X-sperm.

A large number of female goats are needed for the dairy goat industry; therefore, the development of a method to ensure the birth of more females than males in a single pregnancy will lead to economic benefits. Increasing the number of X-sperm would be an effective way to increase the proportion of female offspring. In this study, goat semen was incubated at pH 7.4 in alkaline diluent combined with resiquimod (R848) and the number of X-sperm was enriched by the swim-up method. The percentage of X-sperm was determined using the double TaqMan qPCR method. Sperm total motility, progressive motility, average path velocity, straight-line velocity, and curvilinear velocity were measured using a computer-aided sperm analysis system, and the functional parameters of the sperm plasma membrane, the acrosome, mitochondrial activity, ATP content, and reactive oxygen species levels were also measured. Lastly, the ratio of female embryos was determined by in vitro fertilization, and the number of female kids and the pregnancy rate of does was assessed by artificial insemination. The results showed that dilution of semen in an alkaline buffer containing R848 could enrich the number of X-sperm to 85.57% ± 3.27%. The progressive motility, average path velocity, straight-line velocity, curvilinear velocity, mitochondrial activity, and ATP level of the collected X-sperm-enriched semen were significantly reduced, but its total motility, plasma membrane, and acrosome were not affected. The in vitro fertilization experiments showed that the rate of female embryo production using X-sperm-rich seminal fluid could reach 83.25% (174/209), which was significantly higher than the proportion of female embryos in the control group, 47.71% ± 1.80% (104/218). As determined by artificial insemination, the number of female kids in the test group increased by 62.79% (243/387), which was significantly higher than that in the control group (47.65%, 193/405). There was no significant difference in pregnancy rate between the test group and the control group (71.71% vs. 78.48%). Therefore, this study demonstrated that use of a pH 7.4 diluent containing R848 is a simple and effective method of X-sperm enrichment for dairy goat production. Its application would allow does to produce more female offspring for herd expansion and milk production.

Effect of Melatonin and Caffeine Supplementation to Freezing Medium on Cryosurvival of Peruvian Paso Horse Sperm Using a Two-Step Accelerating Cooling Rate.

This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96®, and finally frozen with INRA-Freeze® with either no supplementation (as control), 1 μM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1-4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

Effects of CO₂ Euthanasia of C57BL/6 Mice on Sperm Motility, In Vitro Fertilization, and Embryonic Developmental Competence.

Cryopreservation of epididymal sperm collected after euthanasia is a common method to preserve and distribute valuable mouse models worldwide. However, the euthanasia method used prior to sperm collection must not adversely affect sperm quality. The most common method of euthanasia in mice is CO₂ asphyxiation, but its effect on the quality of sperm collected postmortem is largely unknown. The objective of this study was to determine the effects of CO₂ euthanasia of C57BL/6 mice on both freshly recovered sperm and sperm subjected to freezing and thawing. First, sperm concentration, progressive motility, curvilineal velocity (VCL), average path velocity (VAP), and progressive velocity (VSL) were analyzed for mice euthanized by cervical dislocation (CD), high flow CO₂ (100%), or low flow CO₂ (30%) displacement/minute, respectively. Then, in-vitro fertilization and embryonic development rates were determined using frozen-thawed sperm from each euthanasia method. Neither fresh nor frozen-thawed sperm showed significant differences in sperm concentration, progressive motility, VAP, or VCL when compared to CD and CO₂ groups. However, frozen-thawed sperm collected from CD mice had higher VCL values than did those collected from the low flow mice (P = 0.039). VCL was not different in fresh or frozen-thawed sperm collected after mouse euthanasia by CD as compared with high flow CO₂ or by high flow as compared with low flow CO₂. Frozen-thawed sperm showed no differences among the 3 euthanasia groups for fertilization (P = 0.452) or blastocyst development rates (P = 0.298). The results indicate that CO₂ euthanasia can be used as an alternative to CD to obtain optimal quality mouse sperm for cryopreservation while remaining compliant with welfare requirements.

Interrelation of Kinetic Parameters of Sperm of Servicing Bulls of the Holstein Breed with Its Fertilising Ability

Sperm motility is an essential indicator that characterises the viability and structural integrity of sperm. This parameter is closely related to the fertilisation capacity of male germ cells, so its assessment is an integral part of sperm analysis. The purpose of this study is to investigate the possibility of predicting the fertilisation capacity of sperm of servicing bulls based on kinetic indicators. The study involved laboratory, zootechnical, and statistical methods. A significant variability in the kinetic parameters of sperm of servicing bulls was revealed – from 6.2 to 16.1%. The variation in the percentage of motile sperm in the ejaculate was 78.9-89.8; the percentage of progressive sperm – 50.0-74.5; velocity average path of sperm (VAP) – 132.6-163.7 µm/s; velocity straight line (VSL) – 99.2-138.2 µm/s; curvilinear velocity (VCL) – 223.7-272.3 µm/s; straightness of sperm (STR) – 73.9-85.0%, linearity (LIN) – 45.1-56.1%, wobble (WOB) – 57.2-63.8%. The difference between the minimum and maximum values for all the parameters under study is highly likely (P&lt;0.001). A correlation between various kinetic parameters of sperm is established. Sperm movement rates and their relative values are largely conditioned upon the percentage of progressive sperm in the ejaculate (r=+0.231-0.761). VAP, VSL, and VCL indicators are interrelated (r=+0.550-0.887). The study investigated the average fertilising ability of the sperm of servicing bulls based on the results of insemination of 8,594 cows and 992 heifers in four farms of Zhytomyr and Kyiv oblasts, the variation was within 40.7-61.4%. It was proved that bulls with higher and average fertilising capacity are described by a higher percentage of progressive sperm in ejaculates, and VAP, VSL, and VCL indicators of sperm movement compared to bulls with low fertilising capacity. This pattern is confirmed by correlation analysis (r=+0.538-0.675). Kinetic parameters identified using the CASA system allow predicting the fertilising capacity of sperm and identify servicing bulls with reduced reproductive function

Use of auxiliary techniques for assessing the reproductive capacity of natural service Nellore bulls

The aim of this study was to investigate whether parameters obtained by computer-assisted sperm analysis (CASA), infrared thermography (IRT), and testicular B-mode and Doppler ultrasound can be used as indicators of fertility in natural service Nellore bulls. Twenty-nine Nellore bulls were evaluated on days 66 (-D66), 38 (-D38), and 10 (-D10) before the beginning of natural service mating. In all assessments, semen samples were collected and sperm kinetics were evaluated by CASA, thermographic imaging of the scrotal and ocular region, testicular B-mode ultrasound, and Doppler ultrasound of the spermatic cord. At the end of natural service, the bulls were classified based on the pregnancy rate of individual batches into low fertility (pregnancy rate 66.57 ± 0.62 %), medium fertility (76.47 ± 0.51 %), and high fertility (84.80 ± 0.60 %) groups. No difference in the IRT parameters was observed between groups. High fertility animals exhibited higher average path velocity (VAP = 110.98 µm/s), straight line velocity (VSL = 87.05 µm/s), curvilinear velocity (VCL = 190.78 µm/s), pulsatility index (PI = 0.93), and resistive index (RI = 0.57) than low fertility animals (VAP = 100.02 µm/s; VSL = 79.84 µm/s; VCL = 173.22 µm/s; PI = 0.69; RI = 0.48). Positive correlations were observed between pregnancy rate and VSL (0.21), PI (0.28) and RI (0.32). In conclusion, IRT does not provide fertility indicators in Nellore bulls. The VAP, VSL and VCL obtained by CASA and PI and RI obtained by Doppler ultrasound can be used as indicators of fertility in Nellore bulls.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Simple SummarySperm suffers damage from reactive oxygen species stress (ROS) during cryopreservation. Supplementation of antioxidants is suggested to reduce sperm cryodamage induced by ROS. Proline has been regarded as a powerful antioxidant. However, there is no information on the role proline plays during goat sperm cryopreservation. Thus, this study aims to investigate the effects of proline during goat sperm cryopreservation procedures. In the present study, supplementation of 2 mM proline significantly enhanced post-thaw goat sperm quality by regulating sperm redox homeostasis. Proline supplementation protects goat sperm against ROS stress through PRODH-mediated metabolism.Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Presence of leptin and its receptor in the ram reproductive system and in vitro effect of leptin on sperm quality.

Leptin is a 16 kDa hormone encoded by obese (OB) gene in adipocytes. This molecule not only regulates energy metabolism but also plays a role in the reproduction of mammals. Leptin and its receptor (OBR) have been found in male reproductive systems of human, bovine, equine and pig. The effects of leptin on sperm quality vary widely from different research findings. However, the presence of leptin and its receptor in the ram reproductive system and the in vitro effect of leptin on sperm quality have not reported yet. In the present study, we found that the OB was highly expressed in primary and secondary spermatocytes of the testes, OBR was highly expressed in secondary spermatocytes of the testes. The expressions of OB were in stereocilia of epididymis and in columnar cells of epididymal caput and cauda, the expressions of OBR were in columnar cells of epididymis and in stereocilia of epididymal corpus and cauda. The presence of both OB and OBR in testes, epididymis and sperm were confirmed through RT-PCR, immunolocalization and Western blot analyses. The RT-qPCR results indicated OB and OBR had higher expression levels in epididymal sperm than that of the ejaculated sperm in rams. When sperm were treated with 5 ng/mL leptin, the progressive motility (P < 0.01), straight-line velocity (VSL) (P < 0.05), average path velocity (VAP) (P < 0.05), membrane mitochondrial potential (MMP) (P < 0.01) and viability (P < 0.05) significantly increased, while DNA fragmentation index (DFI) and reactive oxygen species (ROS) significantly decreased compared to the control (P < 0.01), and the other semen parameters such as acrosome integrity and acrosome reaction rate had no significant changes between groups (P > 0.05). In conclusion, this is probably the first report describing localization of leptin and its receptors in the reproductive system of rams and their effects on sperm quality parameters. Our findings suggest that 5 ng/mL leptin treatment enhanced sperm motility, viability and MMP, and decrease DFI and ROS without obvious influence on the acrosome reaction in ram sperm. The potential mechanisms may be related to leptin’s ability to reduce the oxidative stress and apoptosis of sperms and improve their mitochondrial function and energy supply, therefore, to maintain the physiological homeostasis of the sperm.

PSI-1 An Evaluation of Functionality of Cryopreserved Sperm Cells Treated with Heifer-Plus™

Abstract The commercial beef cattle industry produces calves for replacement or slaughter. Female replacements are important to advance herd genetics, therefore, a tool to influence sex ratios may enhance reproductive efficiency. A commercial semen sexing kit, Heifer-Plus™, used in conjunction with artificial insemination, may alter the sex ratio of offspring by increasing motility of the X chromosome-bearing sperm. The aim of this research was to assess the methodology of Heifer-Plus™ through evaluation of motion patterns and kinematic parameters in conventional semen straws. Four bulls representing Angus, Wagyu, and Holstein breeds were utilized. Six straws of cryopreserved semen from each bull were thawed in a water bath (37.5°C for 35 sec). Three straws from each bull were pooled and treated with Heifer-Plus™ and 3 straws were pooled as the no-treatment control. Post treatment, all samples were aliquoted in microcentrifuge tubes and Computer Assisted Semen Analysis (CASA) measurements of motility and kinematic factors were assessed. Semen samples were held at 37.5°C for incubation for three 15-min periods (15-mins, 30-mins, and 45-mins) measuring thermoresistance and longevity of spermatozoa. After incubation, 80 µL of each semen sample were observed on CASA. Data were analyzed using ANOVA method in GraphPad Prism and P-values ≤ 0.05 were considered significant. No effect of treatment on motility parameters or rapid velocity were measured, however there was a decrease in total motility (P=0.0045), progressive motility (P=0.0115) and rapid velocity (P =0.0099) across time intervals. Heifer-Plus™ treated cells had higher incidence of static velocity compared to control (P = .0038). Semen samples were clustered into subpopulation threshold-based on sperm average path velocity (VAP) and linearity (LIN) at each time point. Spermatozoa treated with Heifer-Plus™ consistently exhibited a high velocity and variable linearity therefore were categorized consistently into two categories at each time point.

Sperm selection techniques in cattle: Microfilter device versus conventional methods

Microfluidics and microfilter devices have been developed to mimic the characteristics of the female reproductive tract, minimizing the risk of sperm damage. This study aimed to compare the use of a microfilter device versus conventional methods for sperm selection used in in vitro fertilization (IVF). For selecting spermatozoa, the pooled samples were processed in a microfilter device, swim-up and mini-Percoll gradient. Kinematic and morphometric parameters, vitality and DNA damage were analysed before and after sperm selection. After selection, 10,000 motile spermatozoa per oocyte were used in IVF drops. Embryos were assessed at three (cleavage rate) and seven (blastocyst rate) days post-IVF. Results of sperm kinematic parameters including average path velocity, velocity straight line, curvilinear velocity, linearity, lateral head displacement with the microfilter device were superior to density gradient (p < 0.05), but similar to swim-up method. Likewise, sperm DNA damage was significantly reduced using the microfilter device and swim-up method. Regarding the total sperm recovery rate post selection, results with the microfilter device (17.64%) and mini-Percoll gradient (18.27%) were higher than with swim-up method (6.52%). However, the cleavage and blastocyst rates were the lowest using the microfilter device. In conclusion, sperm selection using the microfilter device and swim-up method can improve kinematic parameters, although the mini Percoll gradient was the most efficient method for embryo production.

Motion characteristics and plasma integrity evaluation of Murrah buffalo semen

Buffalo semen collected from Murrah bull were cryopreserved and evaluated for different motility parameter, kinematics and plasma membrane integrity. Buffalo bulls were maintained uniform standard management and nutritional practices. Semen was collected regularly twice a week semen collection schedule from four (04) Murrah bull. Collected semen was immediately transported to laboratory and evaluated for different macroscopic parameter (color, volume and thickness). Fresh semen was then diluted with saline solution and evaluated for sperm concentration, motility, sperm kinematics and morphology. Semen samples that fill all the standard were selected for freezing and diluted with Tris-egg yolk citrate diluter. Diluted semen was equilibrated, cryopreserved and finally evaluated for post thaw sperm quality. Different motility parameter (total, progressive, static and slow motility) varied significantly (p&lt;0.01) irrespective of different freezing stages. Significantly higher progressive sperm motility and viability of buffalo spermatozoa were observed at fresh semen whereas lower progressive sperm motility and viability was found at post thaw stage. Total and progressive motility reduced by 2.5 and 2.12% following equilibration, whereas following cryopreservation, total and progressive motility reduced by 35.7 and 28.51% and static motility increases accordingly (35.4%). Significantly higher plasma membrane integrity of sperm was observed at fresh semen followed by pre freeze and post thaw semen. Following freezing, integrity of plasma membrane reduces at the rate of 10.81% and 26.7% at pre freezing and post thaw stages. Significantly higher average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were found for fresh semen followed by pre-freeze and post-thaw semen. Frozen buffalo semen with higher progressive motility and motion characteristics may be produced if motility losses can be reduced during freezing stage as this stage results higher motility losses. Asian Australas. J. Biosci. Biotechnol. 2022, 7(2), 75-81