Loss Of Sperm Viability Research Articles

PSXV-13 The activity of thiol cathepsins in seminal plasma of stallions with different percentages of viable sperm after freezing-thawing

Abstract The aim of this work was to study the activity of cathepsins B, L and H in the seminal plasma of stallions with normal (>25%, n = 11) and low (< 25%, n = 13) percentage of viable sperm after freezing-thawing. Sperm of 24 Arabian stallions aged from 5 to 20 years (12.1 ± 4.8 years on average) was collected during the breeding season (February-May). The activity of cathepsins in spermoplasm was studied by the spectrofluorometric method (System 3 Scanning Spectrofluorometry, Optical technology devices, inc. Elmstord, New York, 10523) by Barrett&Kirschke. The viability of the sperm was determined after its freezing-thawing. Sperm smears were eosin stained and live:dead-ratio was examined using an Olympus BX41 phase contrast microscope (Olympus Corporation, Japan). The significance of differences in the studied groups was determined using the Mann-Whitney U-test. There were no significant differences in the activity of catepsins B and L in the spermoplasm of stallions with normal and low percentages of viable spermatozoa after freezing-thawing. It was found that in the group of stallions with a normal percentage of viable spermatozoa after freezing-thawing, the activity of cathepsin H in the spermoplasm was significantly higher than in the group with a low percentage of viable spermatozoa (P = 0.0219). Free radicals formed during freezing-thawing of sperm can damage cell membranes, leading to loss of sperm viability. Thiol cathepsins are involved in the degradation of oxidatively modified proteins and, apparently, it is cathepsin H that is most actively involved in this process in the sperm of stallions. We assume that the low activity of cathepsin H in the seminal plasma of stallions with a low percentage of viable spermatozoa indicates a high involvement of this enzyme in the protection of sperm membranes from oxidative damage. The research was supported by the Russian Science Foundation grant No. 20-16-00101.

Comparative in vitro study on the local tolerance and efficacy of benzalkonium chloride, myristalkonium chloride and nonoxynol-9 as active principles in vaginal contraceptives

Background Spermicides have been identified as a potentially attractive alternative to hormonal contraceptives and/or intrauterine devices. Thus, this study aimed evaluating the efficacy and local tolerance of benzalkonium chloride (BKC) and myristalkonium chloride (MKC) contained in Pharmatex® vaginal formulations and compare them with nonoxynol-9 (N-9), the most common active ingredient in topical vaginal contraceptives. Methods Human normozoospermic samples were assessed for motility, viability, acrosome status and penetration ability after exposure to control, N-9 or different BKC and MKC doses for 0 and 10 minutes. Local tolerance on HeLa cells was evaluated by the Trypan-blue and MTT assays. Results Exposure to BKC and MKC reduced acrosome integrity while promoting total immobilisation and complete loss of sperm viability (p < .001, n = 15). Both compounds also compromised sperm penetration ability upon exposure (p < .001, n = 15). N-9 induced the same outcomes (p < .001, n = 15); nevertheless, it was more toxic to HeLa cells than BKC and MKC (p < .05, n = 14). Conclusions BKC and MKC present strong in vitro spermicidal activity at lower doses than N-9 and were better tolerated after immediate exposure than N-9. Available Pharmatex® galenic formulations were as effective as products based on N-9.

Artemisia vulgaris extract causes precocious acrosome reaction and viability loss but low rate of membrane damage in mouse spermatozoa.

Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 µg/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.

Effect of butylated hydroxytoluene supplementation on ultrastructure of crossbred bovine spermatozoa

The present study was planned to observe the effect of Butylated hydroxytoluene (BHT) on ultra structure changes in bovine crossbred spermatozoa. The sperm plasma membrane is the primary site of damage induced by cryopreservation. There are many harmful conditions such as cold shock, osmotic stress, ice crystal formation or oxidative damage etc which cause sperm cryo-injury and loss of sperm viability and fertility during cryopreservation. At the time of cryopreservation there were considerable ultra structure changes to the acrosomes and middle piece studied by transmission electron microscopy. Semen from two crossbred bulls were collected and extended with egg yolk tris dilutor along with addition of different concentrations of BHT i.e. 0.0mM (Control), 0.5mM (T 1 ) and 1.0mM (T 2 ), respectively. The effect of BHT on ultrastructural alterations was revealed by Transmission electron microscopy during post dilution (Stage I), post equilibration (Stage II) and post thaw (Stage III), respectively. TEM results of spermatozoa at Stage I, II and III showed that major ultrastrucural alterations occur in plasma membrane and outer acrosomal membrane. Group T 1 and T 2 showed significantly lower number of ultrastructural defects compared to control at Stage I, II and III. The study indicated that 1.0mM BHT increases the cryosurvival of sperm as revealed by post thaw lower ultrastructural defects of bovine bull spermatozoa

Ovarian Fluid Mediates the Temporal Decline in Sperm Viability in a Fish with Sperm Storage

A loss of sperm viability and functionality during sperm transfer and storage within the female reproductive tract can have important fitness implications by disrupting fertilization and impairing offspring development and survival. Consequently, mechanisms that mitigate the temporal decline in sperm function are likely to be important targets of selection. In many species, ovarian fluid is known to regulate and maintain sperm quality. In this paper, we use the guppy Poecilia reticulata, a highly polyandrous freshwater fish exhibiting internal fertilization and sperm storage, to determine whether ovarian fluid (OF) influences the decline in sperm viability (the proportion of live sperm in the ejaculate) over time and whether any observed effects depend on male sexual ornamentation. To address these questions we used a paired experimental design in which ejaculates from individual males were tested in vitro both in presence and absence of OF. Our results revealed that the temporal decline in sperm viability was significantly reduced in the presence of OF compared to a saline control. This finding raises the intriguing possibility that OF may play a role in mediating the decline in sperm quality due to the deleterious effects of sperm ageing, although other possible explanations for this observation are discussed. Interestingly, we also show that the age-related decline in sperm viability was contingent on male sexual ornamentation; males with relatively high levels of iridescence (indicating higher sexual attractiveness) exhibited a more pronounced decline in sperm viability over time than their less ornamented counterparts. This latter finding offers possible insights into the functional basis for the previously observed trade-off between these key components of pre- and postcopulatory sexual selection.

Intrasperm Ca2+ modulation and human ejaculated sperm viability: influence of miconazole, clotrimazole and loperamide

Elevation of intrasperm Ca(2+) is reported to influence viability of ejaculated spermatozoa. Human spermatozoa possess inositol triphosphate (IP(3))-sensitive Ca(2+) stores, which can be targeted for increasing intrasperm Ca(2+) level. The influence of agents affecting Ca(2+) homeostasis has been investigated. Miconazole nitrate, clotrimazole and loperamide hydrochloride produced a dose- and time-dependent decrease in viability, each requiring respectively 0.5, 1.0 and 1.0 mM for producing death of all sperm cells immediately upon addition to ejaculated human semen samples. The reduction in sperm viability was accompanied by elevation of intrasperm Ca(2+) and was not affected by presence or absence of extracellular Ca(2+). Significantly (P<0.05) less time was required for producing complete loss of sperm viability and increasing intrasperm Ca(2+) when any of these drugs was added to sperm cells previously treated with selected agents affecting Ca(2+) homeostasis. This enhanced spermicidal activity of miconazole, clotrimazole and loperamide appeared to be due to further mobilization of Ca(2+) from partially depleted intrasperm Ca(2+) stores. Synergism of spermicidal activity and intrasperm Ca(2+) elevation by miconazole or clotrimazole was observed when Ca(2+) efflux from sperm cells was simultaneously inhibited by 2',4'-dichlorobenzamil hydrochloride, a Na(+)-Ca(2+) exchange inhibitor. The spermicidal activity of miconazole, clotrimazole or loperamide due to elevation of intrasperm Ca(2+) and its synergism, therefore, has great potential for exploitation of these drugs as contact spermicides.

Spermicidal activity of sulfonylureas and meglitinide analogues: role of intrasperm Ca2+ elevation

Intrasperm calcium concentration ([Ca2+]is) is known to play a vital role in regulating motility and viability of ejaculated spermatozoa. K ATP channel blockers are reported to block K ATP channels, leading to depolarization of the cell membrane. This activates the voltage-gated calcium channels, resulting in enhanced Ca2+ influx, which eventually elevates the intracellular [Ca2+] level. Hence, it can be hypothesized that drugs acting on K ATP channels could possess the ability to elevate [Ca2+]is. Sulfonylureas such as glibenclamide or gliclazide, as well as meglitinide analogues such as repaglinide, produced a dose- and time-dependent decrease in viability, each requiring 7.5 mM, 10 mM and 6.5 mM, respectively, to produce death of all sperm cells immediately upon addition to ejaculated human semen samples. The reduction in sperm viability was accompanied by an elevation of [Ca2+]is and was affected by removal of extracellular Ca2+. Significantly (P < 0.05) less time was required to elevate [Ca2+](is) and produce complete loss of sperm viability when any of these drugs were added to sperm cells simultaneously with selected agents affecting Ca2+ homeostasis. Thus, the spermicidal activity of these drugs attributed to elevation of [Ca2+]is and their synergism can be potentially exploited for developing contact spermicidal formulations.

Modulation of human sperm function by follicular fluid*

Human follicular fluid (hFF), present in the ampullary environment, can reduce the number of sperm bound to the zona pellucida. The aim of the present study was to investigate the effects of follicular fluid on sperm function. The presence of 50% v/v follicular fluid resulted in a significant reduction in the number of bound spermatozoa with respect to control medium (12.7 +/- 5.5 sp HZ(-1) versus 24.6 +/- 5.7 sp HZ(-1), P = 0.03) as measured by the hemizona binding assay. This reduction in zona binding capacity was not associated with a loss of sperm viability, motility or a premature acrosomal reaction. When capacitated spermatozoa were previously exposed 1 h to follicular fluid, a significant reduction in the number of alpha-d-mannose binding sites on sperm head was detected (23.7 +/- 3.1% versus 15.5 +/- 2.4%, P < 0.05). In addition, sperm fertilizing capacity (assessed as the acrosome reaction to ionophore challenge score) in the presence of follicular fluid was also diminished (38.0 +/- 4.8% versus 22.6 +/- 4.9%, P < 0.01). No modification in the pattern of protein tyrosine phosphorylation which occurs during capacitation was observed in the presence of the fluid. Taken together, the results indicate that the decrease in sperm zona-binding capacity observed in the presence of hFF was related to a lower number of sperm containing alpha-d-mannose receptors.