Sperm Kinetic Parameters Research Articles

Localization and expression analysis of sperm-specific glyceraldehyde 3-phosphate dehydrogenase in bull spermatozoa with contrasting sperm motility.

Poor sperm motility leading to male infertility has become a profound crisis to be addressed in this contemporary era. In many cases, the origin of poor sperm motility remains unexplained. Few studies reported the indispensable role of sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDHS) in sperm motility, however, studies on GAPDHS are severely confined. The present study aimed to assess the localization patterns, expression levels, and enzyme activity of GAPDHS in normal and asthenozoospermic bulls and to examine their association with sperm functional parameters. The bull semen samples were classified into high-motile and low-motile groups (n=7 per each group) based on the ejaculate rejection rate. Sperm kinetic parameters were assessed using computer-assisted sperm analysis (CASA). Sperm viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and intracellular calcium levels were measured through flow cytometry. Subsequently, GAPDHS localization was observed via immunocytochemistry. The expression levels and enzyme activity of GAPDHS were estimated using western blotting and a GAPDHS activity assay kit. Sperm viability, MMP, ROS, and live sperm intracellular calcium levels did not differ significantly between high and low motile groups. A significant positive correlation was found between MMP and sperm viability, whereas no significant association was found between MMP and sperm progressive motility. The GAPDHS was localized in the principal piece, head-midpiece junction, and at the acrosome region of bull sperm. GAPDHS localization intensity, expression levels, and enzyme activity were found significantly (p<0.05) higher in the high motile group than in low motile group. Furthermore, we noticed a significant positive correlation between GAPDHS activity and sperm kinetic parameters. The analysis of GAPDHS localization patterns, expression levels, and enzyme activity indicated its potential role in sperm motility, suggesting that GAPDHS could serve as a candidate biomarker for sperm motility and male fertility.

Association Between Zinc Ion Concentrations in Seminal Plasma and Sperm Quality: A Chinese Cross-Sectional Study.

This study investigates the impacts of zinc ion concentration in in seminal plasma and the total amount of Zn2+ per ejaculation on sperm quality evaluation parameters. In addition, we assessed the reliability of using zinc content in seminal plasma to evaluate sperm quality. We analyzed semen from 964 men and found that men over 40years old had significantly lower concentrations of Zn2+ in ejaculated semen compared to other age groups (p < 0.05), with no significant difference in Zn2+ concentrations among other age groups (p > 0.05). The total amount of Zn2+ in one ejaculation did not show a statistical difference between the normal semen and the abnormal semen groups (p > 0.05). Statistical differences were observed in sperm kinetic parameters and DNA fragmentation index between the normal Zn2+ total amount group and the abnormal group (p < 0.05). However, there was no statistical difference in morphological parameters (p > 0.05). Spearman correlation analysis showed a negative correlation between Zn2+ concentrations in seminal plasma, age, and sperm fructose and a positive correlation between semen volume, abstinence time, sperm concentration, neutral α-glycosidase, and citric acid content (p < 0.05). The receiver operating characteristic curve analysis demonstrated that Zn2+ concentrations had poor accuracy and specificity in assessing sperm quality (p > 0.05). Although there is a partial correlation between Zn2+ concentrations in seminal plasma and certain semen quality parameters, relying solely on Zn2+ concentrations to evaluate sperm quality lacks accuracy and specificity.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C – PKC; phospholipases A2 and Cζ – PLA2, PLCζ; soluble adenylyl cyclase 10 – sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 – Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

First dose optimization study on freezing Anatolian buffalo semen

The main objective of sperm production centers is to produce as many straws as possible from the obtained ejaculates using the optimal dilution rate. To this end, this study is the first to evaluate the effect of different semen extender rates on Anatolian buffalo semen quality. Ejaculates were collected by artificial vagina from three Anatolian buffalo bulls. These ejaculates were divided into three aliquots and filled into 0.25 ml straws with soy-based extenders at concentrations of 35, 25, and 15 million sperm/straw (n=105). The straw samples of different sperm concentrations were frozen. The quality of sperm was evaluated after thawing (37 °C, 30 sec) and following the thermoresistance test (37 °C, 3 h). The post-thaw total motility and progressive motility values were similar between the groups. However, following the thermoresistance test, there was a significant decrease in total motility in the 35 million sperm/straw group, and the progressive motility was significantly higher in the 25 million sperm/straw group. There was no statistically significant difference between the groups in terms of sperm kinetic parameters, except for VSL after thawing, as well as VAP and LIN values following the thermoresistance test. The overall mean PMAI and STR values were the highest in the 25 million sperm/straw group. In conclusion, it is recommended to dilute the Anatolian buffalo semen at a concentration of 25 million/0.25 ml when freezing it with a soy-based semen extender. In addition, it is considered that soy-based extenders compensate for cryo-damage to sperm motility for a short time, and the thermoresistance test should be applied for objective evaluation in dose optimization studies.

The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on invitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL−1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 μm s−1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 μm s−1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P &lt; 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.

Inclusion of 1,3-dimethylxanthine improved quality parameters of bull semen

Methylxanthines are a unique class of drug, derived from the purine base xanthine and 1,3-dimethylxanthine (Theophylline) is the most commonly used methyl xanthine. The present study was conducted to study the effect of supplementation of 1,3-dimethylxanthine in semen extender on sperm motility and morphological parameters of semen. A total of 15 semen ejaculates were collected from three adult cattle bulls. Each ejaculate was divided into five groups. First group was kept as control (C) in which samples were diluted with Tris-egg yolk-glycerol extender and samples of other 4 groups (T-5, T-10, T-20 and T-40) were supplemented with 1,3-dimethylxanthine at a dose rate of 5, 10, 20 and 40 mM respectively. The diluted semen samples were incubated at 37°C in water bath and evaluated for various seminal parameters at 15 and 30 min of incubations. Sperm motility, sperm kinetic parameters and sperm abnormalities of semen of all groups were assessed using computer assisted sperm analyser (CASA). Morphological parameters of spermatozoa of control and supplemented groups were also evaluated. The results revealed that 1,3-dimethylxanthine supplementation resulted in significant increase in motile sperm concentration, kinematic parameters, sperm viability, sperms with intact plasma membrane as compared to other groups. However, sperm abnormalities viz., bent tail, coiled tail, distal droplet and distal midpiece reflex varied non-significantly in all groups. Thus, it can be concluded that 1,3-dimethylxanthine (Theophylline) inclusion at 10 mM concentration in semen extender improved the quality of semen.

Bioclimatic thermal stress indices and their relationships with andrological characteristics in hair rams.

This study evaluated relationships among reproductive parameters and the bioclimatic indices: temperature and humidity index (THI), equivalent temperature index (ETI), black globe temperature and humidity index (BGTHI), and thermal comfort index (TCI), during the first 45 days of spermatogenesis (SP-45) and during the 15 days of sperm transit through the epididymis (STP-15) that preceded the reproductive assessments (ReA). Such information is useful in determining the optimal breeding season in Northeast Brazil. Santa Inês rams (n = 25) underwent two ReA in three periods of the year (D-P = dry; R-P = rainy and RD-P = rainy/dry transition), and the bioclimatic indices were calculated at the corresponding SP-45 and STP-15 timepoints prior to each ReA. Sperm kinetic parameters in D-P were depressed compared to R-P and RD-P (P < 0.05). The index values had an antagonistic relationship with most parameters and regression analysis demonstrated that the BGTHI and the TCI had a negative association with the progressive motility, curvilinear, straight line, and average path velocities, and a positive association with slow sperm in the ejaculate in SP-45 and STP-15 phases (P < 0.01). Semen quality kinetics is affected throughout the year by the environment and it is apparent that it is impaired in D-P and better in R-P and RD-P seasons. The BGTHI and TCI measured in the sperm production phase classified the environment most coherently and presented better association with the behavior of sperm kinetics.

Improving the quality of chilled semen from Thai native chicken using phosphorus and vitamin B12 supplementation in semen extender

This study aimed to determine phosphorus and vitamin B12 supplementation effect in semen extender on the quality and fertility ability of chilled Thai native rooster semen. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese x Vietnam crossbreed) were included. Semen was collected by applying dorsal–abdominal massage once a week, pooled, diluted to 500 million sperms per dose, and divided into six groups. The semen samples used for control group were diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2–6: semen samples were diluted with modified BPSE and enriched with phosphorus and vitamin B12 (Octafos® Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%. Semen fertility ability was tested in six replications by inseminating layer hens. Thirty-six Thai native hens were randomly assigned to three groups (control, 0.04%, and 0.08%) of 12 hens and were inseminated with a dose of 0.2 mL on collecting day. Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using a computer-assisted sperm analysis system (SCA®, Proiser S.L., Valencia, Spain). Sperm viability, mitochondrial activity, acrosome integrity, plasma membrane integrity, and malondialdehyde (MDA) concentration were also evaluated. The sperm motion characteristics were the highest in the 0.04% supplementation group on all days of collection, especially the VCL and VAP (P < 0.05). The viability, mitochondrial activity, plasma membrane and acrosome integrity of spermatozoa were greater in the 0.04% supplementation group than in the control groups (P < 0.05). The 0.04% supplementation group had the lowest MDA concentration in all days of collection. The 0.04% supplementation group were higher both fertility (66.59% vs. 48.50%: P < 0.05) and hatching rates (58.80% vs. 43.18%: P < 0.05) than in the control group. In conclusion, 0.04% phosphorus and vitamin B12 concentrations supplementation in semen extender improved rooster semen quality and fertility in chilled rooster semen.

Effect of In Vitro Addition of Melatonin and Glutathione on Seminal Parameters of Rams in Diluted Semen and after Thawing.

The present study aimed to evaluate the effects of melatonin (MLT), glutathione (GSH), and their combination on ram semen quality after thawing. During eight weekly sessions, semen from three Merino rams was pooled, diluted with an egg-yolk-based semen extender, and divided into four groups: control, 1 mM MLT, 5 mM GSH, and 1 mM MLT + 5 mM GSH. Diluted semen was evaluated before and after the freezing process. The supplementation of diluted semen with GSH at 5 mM had a deleterious effect on total motility progressive (TPM), curvilinear velocity (VCL), straight-line velocity (VSL), average-path velocity (VAP), linearity (LIN), and straightness (STR) and increased slow spermatozoa (%). MLT at 1 mM also had a negative effect on TPM, VSL, and STR in diluted semen. In thawed semen, 1 mM MLT increased the TPM compared with the control group. VSL was lower in the 5 mM GSH group than in the 1 mM MLT group. Additionally, the combination of both antioxidants attenuated the negative effect of 5 mM GSH on TPM, VSL, and BCF. These results indicate that 5 mM GSH impairs or does not improve sperm kinetic parameters in either diluted or thawed semen. They also suggest that MLT combined with GSH plays a protective role against these effects.

Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen

Abstract This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility &gt; 80%. After thawing, all treatments provided semen with total sperm motility &gt; 40%, with no significant difference (P &lt; 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.

Inclusion of catalase in semen extender modulates the post thaw semen quality and oxidative stress profiles in mithun spermatozoa

Present study was designed to assess the effect of catalase on post-thaw semen quality parameters (SQPs), sperm kinematics, antioxidant and oxidative stress profiles and sperm cholesterol efflux in mithun (Bos frontalis). A total of 25 ejaculates were selected based on biophysical parameters and each sample was split into four equal aliquots and diluted (to get final concentration of 60 million spermatozoa per mL) with the TCG extender containing either 0 U/mL, 50 U/mL, 100 U/mL, and 150 U/mL catalase in the Group I, II, III and IV, respectively. Frozen-thawed samples were analysed for motility parameters (progressive forward and in bovine cervical mucus [BCMPT]), kinetic and velocity parameters by computer-assisted sperm analyser (CASA), viability, sperm morphological and nuclear abnormalities, acrosomal integrity, plasma membrane and nuclear integrities, sperm intra-cellular enzymatic leakage and seminal plasma biochemical (sperm cholesterol and oxidative stress markers) profiles. Study revealed an enhancement in viability, acrosomal integrity, plasma membrane integrity, motility (progressive and in cervical mucus), sperm cholesterol content and reduction in sperm morphological and nuclear abnormalities, leakage of intracellular enzymes in Group III. Moreover, intactness of acrosome and biochemical membranes were protected significantly in addition to significant improvement in kinetic and velocity profiles in extender containing 100 U/ml catalase. Correlation analysis revealed that sperm kinetic parameters, SQPs and antioxidant parameters had significant positive correlation with each other whereas these profiles were negatively correlated with sperm morphological abnormalities, enzymatic leakage and lipid peroxidation in catalase treated sperm. The results clearly indicated that inclusion of 100 U/ml catalase holds a clear advantage over control or 50 U/ml or 150 U/ml catalase in cryopreservation of mithun semen. It can be concluded from the present study that catalase supplementation in semen extender can be effectively utilized to reduce the oxidative stress and to improve the post-thaw semen quality in mithun.

Penicillin and amikacin mixture has bactericidal activity equivalent to gentamicin, tylosin, lincomycin and spectinomycin mixture in equine frozen semen.

Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, a bioequivalence study was performed to assess the bactericidal activity of GTLS and PA in equine frozen semen. Nine mature, healthy stallions were used in the study. Split ejaculates were processed using media without antibiotics (Control) or with different antibiotics. For the GTLS group, centrifugation medium and freezing extender were prepared with gentamicin 250 μg/ml, tylosin 50 μg/ml, lincomycin 150 μg/ml and spectinomycin 300 μg/ml. For the PA group, the centrifugation medium was prepared with potassium penicillin G (PPG) 1200 units/ml and the freezing extender was prepared with PPG 1200 units/ml and amikacin 500 μg/ml. Semen processed in extenders without antibiotics had higher (p< .005) bacterial loads throughout all cryopreservation processing steps than semen samples processed using antibiotics. There were no differences in semen bacterial load after centrifugation, 15 and 30min after final extension, and after thawing between GTLS and PA groups, but PA had faster (p< .05) kill-time kinetics than GTLS. Only minor differences in sperm kinetic parameters were observed among groups. In conclusion, this study demonstrated bioequivalence between GTLS and PA in mitigating end-point bacterial loads. Prudent concentrations of the antibiotic mixtures evaluated in this study can be considered both effective and sperm-safe for equine frozen semen.

Effects of leukocyte elastase in semen on sperm quality.

The study analyzed the effect of leukocyte elastase (LE) in 460 semen on sperm quality, and explore the reference interval of normal level of LE in semen. The differences of LE levels between normal semen and few, weak and abnormal semen were analyzed. Referring to domestic standards, the samples were divided into normal group (LE ≤ 250 ng/mL), occult infection (250 < LE ≤ 1000 ng/mL), and infection group (LE > 1000 ng/mL), and the differences in semen quality among the groups were compared. According to European standards, the samples were divided into normal group (≤600 ng/mL) and abnormal group (>600 ng/mL), and the differences in semen quality between the 2 groups were compared. The correlation between LE levels in semen and semen quality were analyzed. The positive rates of LE in the normal semen group and abnormal semen groups were 30.7% versus 34.7%, and there was no significant difference between the two groups (P > .05). When the semen divided into 3 groups, there was no significant difference between the physicochemical parameters, kinetic parameters, movement trajectory parameters, morphological parameters, and sperm DNA fragmentation index (DFI) (P > .05). There were significant differences in sperm morphology and sperm DFI between the two groups at 600 ng/mL (P < .05). Spearman correlation analysis showed that there was no significant difference between the level of LE in semen and physicochemical parameters, sperm kinetic parameters, sperm movement trajectory parameters, sperm morphological parameters, and sperm DFI (P > .05). It is appropriate to use 600 ng/mL as the threshold for the concentration of LE in semen; the correlation between the level of LE and sperm quality is not significant.

Linseed oil in boar's diet improved in vivo fertility and antioxidant status.

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography-mass spectrometry (GC-MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.

Investigating the need for antibiotic supplementation to the extender used for semen cryopreservation in collared peccaries

The objective was to investigate the effects of semen freezing extender supplementation with antibiotics on bacterial load of semen samples, sperm functional and morphological metrics in the collared peccary. Fresh ejaculates from 10 males were extended in Tris-egg yolk-glycerol supplemented or not (control) with gentamicin (70 μg/mL) streptomycin-penicillin (SP; 1 mg/mL−1000 IU/mL) or and cryopreserved in liquid nitrogen. Bacterial load, sperm motility patterns, morphology, membrane functionality and integrity, mitochondrial activity, chromatin integrity and sperm-binding ability were evaluated in fresh and frozen-thawed samples. Regardless of the use of antibiotics, the sole cryopreservation provoked a significant decrease (P < 0.05) in bacterial load compared to fresh samples (from average values > 1 x 106 CFU/mL to <0.4 × 106 CFU/mL). Post-thawing sperm kinetic parameters were not affected by the absence or presence of different antibiotics, except for beat cross frequency that was significantly (P < 0.05) impaired by SP supplementation compared to the group without antibiotics. After thawing, sperm morphology, membrane functionality and integrity, and mitochondrial activity were also not affected by the presence or absence of antibiotics; however, a significant decrease was observed in the group without antibiotics (P < 0.05) in comparison to fresh samples. Regarding sperm-binding ability, there were no differences among the different groups. While collared peccary semen could be efficiently cryopreserved in the absence of antibiotics in the extender, the use of both gentamicin or the streptomycin-penicillin combination is recommended as effective antibiotic supplementation for a further control of bacterial loads without affecting sperm parameters.

High sperm concentration during cryopreservation decreases post-thaw motility percentage without compromising in vitro fertilization outcomes in common carp

To avoid mechanical compression of spermatozoa during the cryopreservation, a high dilution ratio with the cryoprotective medium is applied. Due to this, the sample volume increases, which entails increased cryobank capacity. Moreover, the high volume of cryomedias does not allow to fertilize of large volumes of eggs in practical artificial reproduction of common carp aquaculture. The current study demonstrated that reasonable lowering of dilution rates might still be effective for carp sperm cryobanking —elevation of spermatozoa concentration up to 13 × 109 spz mL−1 resulted in a significant decrease in post-thaw sperm motility percentage to 20% compared with 39% in 0.5 × 109 spz mL−1. Nevertheless, no significant differences in sperm kinetic parameters (VCL and LIN) were found in this case. The fertilization outcome (embryo development and hatching rates) was similar after applying thawed sperm samples with “optimal” (2.2–2.4 × 109 spz mL−1) and “sub-optimal” (11.0–13.0 × 109 spz mL−1) concentrations (sperm/egg ratio at fertilization was in the range of 0.3–4.5 × 105 spz/egg). Thus, applying a low dilution rate such as one part of sperm to one of the cryoprotective mediums is favorable for decreasing cryo-storage space and the sperm volume needed to fertilize big egg numbers. The experiment also shows that the 4.5 × 105 spz/egg ratio is not sufficient for good fertilization, and it is necessary to use higher sperm concentrations per egg or improve the method of fertilization.

High concentration of growth differentiation factor-9 (GDF-9) to the ram semen had a negative effect on the sperm positive rheotaxis.

Herein, the microfluidic device technique was used to investigate the effects of GDF-9 concentrations and exposure time on the ram sperm positive rheotaxis (PR). Semen was collected from six rams and utilized for PR, motility and sperm kinetic parameter analysis using a computer-assisted sperm analysis program with controlled flow velocity following 0, 10, 20 or 30 min of incubation at 37°C with GDF-9 (200, 400or 600 ng/ml; semen sample without GDF-9 was used as a control). Results revealed that there was not an interaction between effects of GDF-9 concentrations and incubation duration on PR% (p= .457) and TM% (p= .099). A simple main effects analysis showed that GDF-9 concentrations had an effect on PR% (p= .003). However, the incubation duration did not have an effect on PR% (p= .101). GDF-9 concentrations had not an effect on TM% (p= .817). By contrast, the incubation duration affected on TM% (p= .026). A higher PR% was found (p< .05) at 200 ng GDF-9 after 10min (46.7± 10.3) and 20 min (45.5± 11.5) of incubation. After 30 min of incubation, the PR% was found lowest (p< .05) at 400 ng of GDF-9 (30.6± 14.1) and 600 ng of GDF-9 (32.2± 9.6). There was no difference (p> .05) in the sperm kinetic parameters between the four treatment groups. In conclusion, the ram sperms had the best rheotaxis properties after 10 and 20 min of incubation with 200 ng of GDF-9 and were sensitive to high concretions.

Evaluation of sperm membrane functionality during epididymal transit in red-rumped agouti (Dasyprocta leporina).

We studied the sperm membrane functionality through the epididymal transit by comparing different hypoosmotic solutions and verifying possible associations among osmotic response and functional parameters of sperm in red-rumped agouti (Dasyprocta leporina). For this purpose, epididymal sperm from six sexually mature male agoutis were collected via flotation. Then, analyses of sperm parameters and hypoosmotic swelling test using different hypoosmotic solutions (0, 50 and 200mOsm/L) in different regions of the epididymis (caput, corpus and cauda) were performed. There was an increase (p<.05) in the values for sperm concentration, the total number of sperm recovered, total and progressive motility, average path velocity, straight-line velocity, curvilinear velocity, and rapid and medium subpopulations following the caput-corpus-cauda direction. Regardless of the hypoosmotic solution, the agouti sperm membrane presented similar functional integrity in all the epididymal regions. Moreover, the highest (p<.05) osmotic responses were reached with the use of 50mOsm/L solution in comparison to 0 and 200mOsm/L for all the regions. Significant correlations among osmotic response and some sperm kinetic parameters were observed, especially in epididymal caput, while no correlations were found in the region of the cauda. In summary, red-rumped agouti sperm present similar membrane functionality during epididymal transit, but there are evident correlations among such functionality and sperm kinetic parameters, especially in the caput region. Moreover, we indicate the use of a 50mOsm/L hypoosmotic solution for the analysis of this parameter through the hypoosmotic swelling test.