Effect of cooling and freezing on the kinematic parameters of ram spermatozoa sexed by modified protocol with TLR7/8 ligand R848

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The addition of prostaglandin F2α (PGF2α) to boar semen prior to insemination improves the conception and farrowing rates in sows. It is accepted that this is due to increased myometrial contractility, which improves the spermatozoa movement. However, there are limited data about the effect of the exogenous PGF2α analogs on sperm motility parameters and morphology. The aim of the current study was to define if there are changes in motility, morphology and kinematic parameters of spermatozoa on 1stand 24thhour after addition of PGF2α analogue to extended boar semen. A total of 18 ejaculates, obtained from clinically healthy boars were diluted 1:3 in semen extender, and each of them was separate into four aliquots, 50 ml each. PGF2α was added to 3 of them in concentrations of 6, 12 and 25 μg/ml, and the fourth served as untreated control. The motility, kinematic parameters and morphology of spermatozoa were evaluated on 1stand 24thhours after addition of PGF2α. There was no significant difference in sperm morphology, total and progressive motility between the untreated and treated groups. There was however a significant decrease in the rapid velocity and some of the kinematic parameters (VCL, VSL and VAP) in the group treated with 25 μg/ml compared to the control at the 1sthour after PGF2α treatment, which (except for the rapid velocity) persisted to the 24thhour. The results indicate that addition of Oestrophan (Bioveta, CZ) to the extended boar semen did not improve the sperm motility, morphology and kinematic parameters of the spermatozoa.

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 °C) on sperm quality

IntroductionSignificant improvement of sperm motility within one month effected by oral supplementation of selenium and vitamin E was described in four infertile male dogs which failed to conceive in their last three matings with different bitches.Material and MethodsThe dogs (a Golden Retriever, an English Cocker Spaniel, and two Tibetan Mastiffs) were supplemented daily with selenium (Se) (0.6 mg/kg organic Se yeast) and vitamin E (vit. E) (5 mg/kg) per os for 60 days. Semen was collected on days 0, 30, 60, and 90. The sperm concentration and motility parameters were evaluated by the CASA system, sperm morphology was explored by Diff-Quick staining, and live and dead spermatozoa were differentiated by eosin/nigrosin staining. The concentrations of Se and vit. E were measured in peripheral blood serum on semen collection days.ResultsBefore administration, the concentrations of Se in blood plasma were low (86.0–165.0 μg/L). After 30 days of treatment there was an observable improvement in total and progressive sperm motility and kinematic parameters (VAP, VSK, VCL, ALH, BCF, and RAPID). The percentages of live and normal morphology sperm cells were also higher. There was also an observable increase in Se and vitamin E concentrations in blood serum. Bitches were successfully mated and delivered four to six puppies.ConclusionSupplementation with Se and vit. E improved rapid sperm motility and restored fertility in infertile dogs with low Se status.

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 °C

Polychlorinated biphenyls (PCBs) are common environmental contaminants that represent a considerable risk to reproductive toxicity in exposed human populations. Although some experimental studies have suggested an association between the levels of PCBs and semen quality, the direct effects of PCBs on human sperm parameters remain largely unexplored. To this aim, a short-term in vitro incubation experiment that better imitated the putative exposure of sperm to Aroclor 1254 (a commercial PCB mixture) in male reproduction tissue was conducted. Human sperm were incubated with various concentrations (0, 1, 5, or 25 mg l−1) of Aroclor 1254 for different amounts of time (3 and 6 h) in vitro. Sperm motility parameters were analyzed with computer-assisted sperm analysis (CASA). The proportion of sperm with high mitochondrial membrane potential (ΔΨm) and the levels of intracellular reactive oxygen species (ROS) were detected to explore the probable cause of sperm impairment. Human sperm exposed to continuous Aroclor 1254 exhibited: (i) reduced sperm motility and kinematic parameters, (ii) a proportion of sperm with high ΔΨm that decreased in a dose-dependent manner (P < 0.05), and (iii) increased levels of ROS compared with controls (P < 0.05). In conclusion, Aroclor 1254 can decrease sperm motility, which may culminate in increased ROS and general mitochondrial dysfunction, thus affecting the fertilization potential of sperm. Our findings suggest a broader understanding of the effect of Aroclor 1254 on human sperm.

In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848

BACKGROUND: Some antimicrobials could adversely affect sperm quality during sperm cryopreservation and antibiotic treatment with subsequent effects on fertility outputs. To our knowledge, no similar studies have been conducted on breeding roosters, especially for oxytetracycline (OTC). OBJECTIVE: To investigate both in vitro and in vivo impact of oxytetracycline on sperm parameters in breeding roosters. METHODS: Sperm motility parameters were objectively analyzed using the CASA system including total motility (TM %), progressive motility (PM %), all sperm velocities, the sperm count, and cell viability during 9 days of in vivo treatment. In the in vitro investigation, the pooled sperm was diluted and divided into a control aliquot (diluted in 0.9% NaCl) and treated samples. Motility parameters were assessed after 0, 1, 2, 3, 4, 5, and 6 hours of storage at 37ºC. In the in vivo study, 1 g/L of OTC was administrated to five individuals for nine consecutive days. Fresh semen samples were analyzed at T0 (before treatment) and after 6 (T6) and 9 days (T9) of treatment. RESULTS: OTC caused significant impairment of sperm quality in vivo. A drastic reduction in sperm concentration, viability, TM, PM, and all kinematic parameters was observed after 6 days of treatment. However, at day 9 sperm quality had improved to be nearly similar to T0. In vitro, OTC induced similar sperm impairment on all sperm motility parameters. CONCLUSION: Oxytetracycline exhibited negative effects on rooster sperm both in vivo and in vitro and appears consequently not suitable in cryopreservation extenders.

BackgroundSperm cryopreservation is a consolidate option for long‐term male fertility preservation. The freezing/thawing procedure causes detrimental effects to spermatozoa, including damage to viability, motility, membrane composition, and DNA, whereas the effect on sperm chromatin compaction is less studied.ObjectivesThe primary aim of this study was to investigate the impact of cryopreservation on sperm chromatin compaction. Furthermore, the effect of cryopreservation on sperm parameters (motility, viability, chromatin compaction, and DNA fragmentation) was also assessed in relation to the storage time in liquid nitrogen.Materials and MethodsSemen samples, collected from 126 (92 normozoospermic and 34 oligozoospermic) patients undergoing routine semen analysis in the Andrology Laboratory of Careggi University Hospital of Florence, were frozen by conventional fast vapor freezing method. Sperm motility, viability, kinematic parameters (by computer‐aided sperm analysis [CASA]), chromatin compaction (by staining with both aniline blue [AB] and Chromomycin A3 [CMA3]), and sperm DNA fragmentation (sDF, by TUNEL/Propidium Iodide [PI]) were evaluated before freezing and after thawing at different timepoints.ResultsAfter 7 days of storage, a significant decline in sperm motility, viability, and kinematics parameters, as well as a significant increase in the percentage of sperm positivity to CMA3, AB, and sDF, were observed. It is noteworthy that while motility and viability decreased in almost all subjects, the increase in CMA3 and AB positivity was observed in 68.0% and 79.2% of samples, respectively. A progressive deterioration of sperm motility and viability, less evident for chromatin structure, was observed at longer times of storage (28 and 180 days).DiscussionOur results indicate that freezing/thawing procedures can alter chromatin structure. A reduction in protamine content and/or a modification in chromatin assembly can be hypothesized. Furthermore, the length of storage in liquid nitrogen appears to progressively affect sperm parameters, although it should be confirmed in larger cohort of subjects.ConclusionCurrent sperm cryopreservation protocols need to be improved with new strategies and personalized procedures aimed to minimize the damage.

The present work was designed to study the effect of bovine serum albumin (BSA) on the motility characteristics of fresh ram spermatozoa collected at different periods of the year and on their motility status under stress conditions. Moreover, the ability of BSA to replace egg yolk in semen medium was assessed using chilled spermatozoa. Fresh Awassi ram semen samples were collected in April and in June and incubated with two BSA levels (5 mg/mL and 10 mg/mL). Motility parameters of fresh spermatozoa samples treated or not with hydrogen peroxide (H2O2), with 5 mg/mL or 10 mg/mLof BSA were compared. The effects of partial and total replacement of egg yolk by 5 mg/mL of BSA on motility characteristics of chilled spermatozoa were assessed by computer-aided sperm analyser (CASA). The addition of BSA significantly increased (P&lt;0.05) the values of CASA parameters in April, while the same values ​​did not significantly changed during June. BSA improved the motility parameters (P&lt;0.05) in the samples treated with H2O2. Replacing a part of egg yolk by BSA enhanced the values of velocity parameters, while the total substitution resulted in a significant decrease (P&lt;0.05) in all CASA motility parameters. It was concluded that BSA had the ability to improve the motility of fresh spermatozoa at certain periods of the year and the motility of spermatozoa under stress conditions. BSA was capable to replace an important part of egg yolk in semen preservation media for the chilled ram spermatozoa.

The present study investigated effects of NOC-18 and TRIM on kinematic parameters of epididymal ram sperm in vitro. After incubation of epididymal sperm samples for 45 and 90 min in the presence of NOC-18 (0.0005, 0.5, and 500 µM) and TRIM (0.1, 10, and 1000 µM), motion parameters were evaluated by computer-aided sperm analysis. There was a significant increase in the fast progressive motility of sperm in the NOC-treated group at a 0.0005 μM. A significant decrease in immotility (at 45 min), BCF (at 45 min), and nonprogressive motility (at 90 min) in the NOC-treated sperm was observed when compared to controls. There was significant increase of the slow progressive (at 45 min) and nonprogressive motility and decrease of the fast progressive motility, VCL, VSL, VAP, ALH, MAD (at 90 min), and STR (at 90 min) of sperm in NOC-treated groups at a concentration of 500 μM as compared to controls. Different concentrations of TRIM appeared significant (p < 0.05) in reducing in the fast progressive motility, VSL, VCL, VAP, ALH, LIN, and STR of sperm compared to controls. There was also a significant (p < 0.05) decrease in WOB (in 1,000 µM of TRIM) and MAD (at 45 min, all concentrations of TRIM; 90 min, only in 0.1 and 10 µM of TRIM). Nonprogressive motility (45 min, all concentrations of TRIM; 90 min, only in 1,000 µM of TRIM), immotility (45 min, 10 µM of TRIM; 90 min, all concentrations of TRIM), and BCF parameters of sperm (all concentrations of TRIM) were significantly (p < 0.05) increased as compared to their controls. In conclusion, physiology of sperm motility in rams is affected by nitric oxide, although, exogenous NO does not increase sperm motility. Excess NO may initiate toxic conditions resulting in decreased sperm motility.

Epidermal growth factor (EGF) is one of the important cytokines that play a role in fertility. It is known that EGF affects both male and female reproduction, but its effect on sperm parameters is not fully understood. Up to the present, the effect of EGF on ram sperm motility parameters has not been published. We analyzed motility parameters of ejaculates after 24, 48, and 72 hours from the EGF addition. EGF was added to chilled ram sperm at concentrations of 0, 100, 200, and 400 ng·ml−1. Analyses were realized using computer, assisted semen analyzer (CASA)—Hamilton Thorn motility analyzer (version 7). The effect of EGF was already visible after 30 min of incubation. Significant effect on ram sperm total motility and progressive movement was observed at higher EGF concentrations after 48 h of incubation. Our results show that EGF affects sperm motility parameters depending on concentration and time of exposure.

In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome

Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.