Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk‐based extender with Trolox supplementation

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml^?1] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post‐thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml^?1 Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml^?1 groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml^?1 ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.     

Anti‐oxidative Status and Semen Quality during Cooled Storage in Stallions

Activity of the anti‐oxidative enzymes glutathione peroxidase (GSH‐Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH‐groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro^® extender either with or without addition of N‐acetyl cysteine or phosphate‐buffered saline (PBS) and stored for 72 h at 5°C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH‐Px, SOD and CAT immediately after semen collections were 10.0 ± 0.6 picokatals, 0.40 ± 0.03 SOD units and 0.70 ± 0.05 nanokatals/10⁶ spermatozoa respectively. TBARS content was 0.06 ± 0.01 nmol and SH‐group content 1.7 ± 0.5 mmol/10⁶ spermatozoa. The loss of motile spermatozoa during storage did not differ between extenders. N‐acetyl cysteine had no effect on semen motility and membrane integrity. The loss in membrane‐intact spermatozoa was highest (P < 0.05) in semen diluted in PBS. Motility and membrane integrity after addition of extender were positively correlated with GSH‐Px and CAT, indicating that anti‐oxidative mechanisms contribute to the initial high percentage of motile and membrane‐intact spermatozoa. However, in these samples the decrease in semen quality was most pronounced. No correlations existed between initial activity of anti‐oxidative enzymes, peroxidation products and semen quality during storage. This indicates that once extender has been added, peroxidative damage to sperm membranes is not the predominant cause of losses in semen quality.     

Addition of pomegranate juice (Punica granatum) in tris‐based extender improves post‐thaw quality,motion dynamics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa

The aim of the present study was to determine the protective effects of pomegranate juice in tris‐based extender on semen parameters, computer‐assisted sperm analysis (CASA) motion characteristics and field fertility of post‐thawed Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Two consecutive ejaculates/collection from each of the five adult Nili Ravi buffalo bulls were collected with artificial vagina at 42°C for a period of 7 weeks, diluted in extender containing different concentrations of pomegranate juice (0.0%, 2.5%, 5%, 7.5% and 10%). Diluted samples were packed and frozen in 0.54 ml French straws. The addition of 10% pomegranate juice in extender significantly improved post‐thaw sperm morphology (%), motilities (CASA total motility, progressive motility (%) as well as VAP, VSL, VCL, STR, DAP, DSL) compared to the control group (p < 0.05). Plasma membrane, acrosome membrane and DNA integrity were significantly higher in extender with 10% pomegranate juice than the control group (p < 0.05). Field fertility rate (60.39% vs. 46.53%) was higher (p < 0.05) in extender with 10% pomegranate juice as compared to the control. It is therefore concluded that the addition of 10% pomegranate juice in tris‐based extender improves post‐thaw semen parameters, CASA motion dynamics and field fertility in Nili Ravi buffaloes.     

Milk,caseinate and lactoferrin addition to equine semen cooling extenders

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk‐ and glucose‐based, C2) 0.6% caseinate, C3) C2 + Lf 200 μg ml^?1, C4) C2 + Lf 500 μg ml^?1 and C5) C2 + Lf 1000 μg ml^?1 extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 μm per ×10⁶ spermatozoa) compared with the fresh semen (8.6 ± 1.9 μm per ×10⁶ spermatozoa). In contrast, the H₂O₂ concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 μm per ×10⁶ spermatozoa) than in the milk extender (430.9 ± 199.8 μm per ×10⁶ spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 μm per ×10⁶ spermatozoa). Caseinate showed to be as efficient as milk to protect equine‐cooled spermatozoon.     

Utilisation of sperm‐binding assay combined with computer‐assisted sperm analysis to evaluate frozen‐thawed bull semen

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm² of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r² = 0.91 and CASA: r² = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.     

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5 mM caffeine-treated groups). Compared to that in the control, the addition of 5 mM caffeine induced an increase in sperm motility (38.9 ± 2.8 versus 32.6 ± 3.4%), and a decrease in nitrite concentration (11.4 ± 2.1 versus 12.8 ± 2.9 µM/µg protein, p < .05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.     

Short‐term storage of salmonids semen in a sodium alginate‐based extender

Short‐term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate‐based extender on sperm function in the short‐term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish^® (Ext‐C) and Storfish^® supplemented with sodium alginate (Ext‐A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O₂^?) level and DNA fragmentation (DNA Frag) were assessed. Ext‐A had positive effect in preservation of sperm motility, viability, ΔΨmit, O₂^? level and DNA integrity in the three species analysed compared to control samples. In Ext‐A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O₂^? level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate‐based extender is effective for protecting sperm quality during 10 days of short‐term storage.     

正常生育力和精子活力低下不育者精子运动计算机辅助分析

用精子活力分析仪对７３名正常生育力男性射出精子进行了精子动力学指标分析；用上述指标分析了２００例精子活力低下不育男性射出精子动力学指标及各项运动参数间相互关系；用己酮可可碱作用精子活力低下不育男性射出精子，观察精子各项运动参数改变。结果：（１）正常生育力组，前向运动精子在总运动精子中比例与精子活动率间不呈相关性；随精子活动率提高，快速相和慢速相精子分布分别逐渐增加和减少，精子ＶＡＰ、ＶＣＬ、ＶＳＬ、ＢＣＦ、ＡＬＨ增加，ＬＩＮ和ＳＴＲ降低。（２）精子活力低下不育组，随精子活动率下降，主要表现在运动速度下降，与正常组比较，精子前向运动能力和运动速度均有明显下降（Ｐ＜０．０５），但ＬＩＮ和ＳＴＲ不管在组内还是与正常组相比均无明显差别（Ｐ＞０．０５），反映出这种前向运动能力的减低是由速度下降引起，速度分布变化以中速相明显。（３）己酮可可碱能明显提高精子活力低下不育者精子的活动率，前向运动率和精子运动快速相的分布；对精子ＬＩＮ，ＳＴＲ影响不明显。     

Gallic and carnosic acids improve quality of frozen‐thawed ram spermatozoa

The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen‐thawed ram spermatozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris‐based extender and divided into five equal portions containing: 0.05 or 2 mM of GA; 0.05 or 0.2 mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5 cm above liquid nitrogen for 12 min then plunged. Computer‐aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2 mM GA had greater total motility than control spermatozoa (39.9 ± 3.01 vs. 29.2 ± 1.31%, mean ± SEM, p < .05). The PMAI was greatest in 0.2 mM CA (13.3 ± 0.68%), whereas HMMP was highest in 0.05 mM CA but lowest in control (22.9 ± 4.95 and 11.4 ± 3.64% respectively; p < .05). In conclusion, for cryopreservation of ram semen in Tris‐based extender, supplementation with 2 mM GA increased post‐thaw motility, whereas supplementation with 0.05 mM CA enhanced mitochondrial function.     

The micronutrient supplements,zinc sulphate and folic acid,did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty‐three OAT men participated in a 16‐week intervention randomised, double‐blind clinical trial with daily treatment of folic acid (5 mg day^?1) and zinc sulphate (220 mg day^?1), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A₃ staining; and semen and blood folate, zinc, B₁₂, total antioxidant capacity ( TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10⁶ ml^?1) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.     

Canine sperm vitrification with sucrose: effect on sperm function

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m ) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.     

l‐Cysteine improves antioxidant enzyme activity,post‐thaw quality and fertility of Nili‐Ravi buffalo (Bubalus bubalis) bull spermatozoa

The effects of l ‐cysteine in extender on antioxidant enzymes profile during cryopreservation, post‐thaw quality parameters and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris–citric acid‐based extender having different concentrations of l ‐cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm ) and frozen in 0.5‐ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre‐freezing and post‐thawing in extender containing 2.0 mm l ‐cysteine as compared to other groups. Post‐thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s^?1), straight line velocity (μm s^?1), curvilinear velocity (μm s^?1), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l ‐cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l ‐cysteine than in the control. In conclusion, the addition of 2.0 mm l ‐cysteine in extender improved the antioxidant enzymes profile, post‐thaw quality and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa.     

Effect of tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on human sperm motility, viability and motion parameters

Male genital tract infections and non-specific inflammatory conditions may be associated with unexplained infertility. Previous studies have shown the presence of cytokines such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the semen of infertile men. However, the mechanism of effect of these cytokines on human sperm function is still controversial. The present study was undertaken to investigate the in-vitro effects of TNF-α and IFN-γ on human sperm motion, viability and the hypoosmotic swelling test (HOST).
Washed spermatozoa from normal volunteers ( n =9) were incubated in the presence/absence of TNF-α (1 μg/mL) plus IFN-γ (0.1 μg/mL). Sperm motility, viability, HOST, and video sequences were recorded at different time intervals (0, 30, 60 and 180 min). Sperm motion parameters were analysed using computer-assisted semen analysis. There was a time-dependent negative effect of TNF-α plus IFN-γ on sperm motility, viability, HOST, and lateral-head displacement (ALH). The maximum decrease was observed between 60 and 180 min for sperm motility (50.8 ± 5.6%), viability (52.8 ± 4.0%), HOST (38 ± 2%) and ALH (4.7 ± 0.1 μm) compared to control samples (62.2 ± 2.8, 62.4 ± 2.9, 58 ± 4, and 5.3 ± 0.4, respectively; All p  < 0.05). There was no significant effect on sperm straight-line velocity and mean linearity when compared to control.
These data suggest that the common inflammatory cytokines TNF-α plus IFN-γ have only partial detrimental effects on sperm motility, viability, membrane integrity and lateral head displacement, which may contribute to the poor fertilizing potential of human spermatozoa during inflammatory conditions.     

Impact of using a fast‐freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast‐freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s^?1 or 75 °C 7 s^?1). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20 min post‐thawing in the fast‐freezing technique than in the conventional one. Plasma membrane integrity was also greater (P < 0.05) in semen frozen with the fast‐freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast‐freezing technique was 76% (19/25), and did not differ (P > 0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep‐horn insemination maximises the use of equine semen. The fast‐freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.     

Incubation of frozen-thawed llama sperm with seminal plasma

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C). A total of 20 ejaculates from five adult llama males (n = 5; r = 4) were evaluated. A significant decrease in sperm motility, membrane function and live sperm was observed in all thawed samples (0%, 10% and 50%) at 0 hr when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > .05), but a significant increase in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples versus raw semen. When evaluating thawed samples over time, a significant decrease of motility and membrane function was observed, while the percentages of total live sperm were preserved over the 3 hr of incubation in all final concentrations evaluated. To conclude, the addition of 10% or 50% of seminal plasma was incapable of preserving motility or membrane function of frozen-thawed llama sperm during 3 hr of incubation.     

Comparison of Tris egg yolk‐based,Triladyl® and Optixell® extender on post‐thaw quality,Kinematics and in vivo fertility of Nili Ravi Buffalo (Bubalus bubalis) bull spermatozoa

Our objectives were to ascertain the comparison of Tris egg yolk‐based, Triladyl ^® and Optixell ^® extender on postthaw quality, CASA parameters and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 35) from five bulls were diluted in Tris egg yolk‐based, Triladyl ^® , Optixell ^® extender and frozen in 0.50 ml French straws. Postthaw sperm CASA motility (%) was higher (p < 0.05) in Optixell extender as compared to Triladyl and Tris egg yolk‐based extender. Although sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross‐frequency (Hz), straightness (%), length of curvilinear path (μm), length of average path (μm), intact plasma and acrosome membrane (%), and DNA integrity (%) were higher (p < 0.05) in spermatozoa cryopreserved in Optixell ^® extender as compared to Tris egg yolk‐based and Triladyl ^® extender. The fertility rates (68.18%, 45.45%, 55.4%) were higher (p < 0.05) in buffaloes inseminated with semen doses frozen in Optixell extender than the Tris egg yolk‐based and Triladyl ^® extender respectively. It is concluded that Optixell ^® extender improves postthaw semen quality and fertility in Nili Ravi buffaloes.     

Effect of cysteine and glutamine added to extender on post‐thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris‐egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post‐thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen–thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen–thawed spermatozoa.     

Tsga10 expression correlates with sperm profiles in the adult formalin‐exposed mice

Testis‐specific gene antigen10 (Tsga10), as a cytoskeletal protein in the sperm tail, impacts the sperm motility. This study investigates the correlation between sperm profile alterations and Tsga10 gene expression in adult mice exposed to formaldehyde (FA) and then treated with antioxidant effect of manganese (Mn²⁺). In this regard, we examined 35 NMRI adult male mice (6–8 weeks age) in 4 groups of control, sham, FA‐exposed and FA+Mn²⁺. The mice in FA+Mn²⁺ group were exposed to FA (10 mg kg^?1 twice a day) for 2 weeks and treated with daily Mn²⁺ administration (5 mg kg^?1) in the second week prior to sacrificing the mice for testis dissection. The right testis was dissected in each group and subjected to RNA extraction and cDNA syntheses for gene expression analysis by real‐time PCR. The findings revealed that FA decreased sperm parameters and Tsga10 expression (52.6 ± 24.37%). However, the injected powerful manganese antioxidant improved sperm profile through overexpression of Tsga10 (121.6 ± 27.13%) under FA‐induced stressful condition which proves the correlation between sperm profile and Tsga10 expression (P ≤ 0.05). This study also shows that Tsga10 expression protects sperm dysfunction in FA+Mn²⁺ group and resulting in better preservation of spermatozoa and improvement of male fertility.     

Cystic fibrosis transmembrane conductance regulator is correlated closely with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters

Cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated to be expressed in mature spermatozoa and correlated with sperm quality. Sperm CFTR expression in fertile men is higher than that in infertile men suffering from teratospermia, asthenoteratospermia, asthenospermia and oligospermia, but it is unknown whether CFTR is correlated with sperm parameters when sperm parameters are normal. In this study, 282 healthy and fertile men with normal semen parameters were classified into three age groups, group (I): age group of 20–29 years (98 cases, 27.1 ± 6.2), group (II): age group of 30–39 years (142 cases, 33.7 ± 2.6) and group (III): age group of more than or equal to 40 years (42 cases, 44.1 ± 4.6). Sperm concentration, total count and progressive motility were analysed by computer‐assisted sperm analysis. Sperm morphology was analysed by modified Papanicolaou staining. Sperm CFTR expression was conducted by indirect immunofluorescence staining. There was a significant positive correlation (P < 0.001) between CFTR expression and sperm progressive motility (r = 0.221) and normal morphology (r = 0.202), but there were no correlations between sperm CFTR expression and semen volume, sperm concentration, sperm total count as well as male age (P > 0.05). Our findings show that CFTR expression is associated with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters, but not associated with the number of spermatozoa and male age.     

Effect of Seminal Plasma Fractions on Stallion Sperm Survival after Cooled Storage

This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm‐rich fractions (SRF) or sperm‐poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 × 10⁶ sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions’ second (SP‐SRF) or last cup (SP‐SPF). Skim milk extender (K) and skim milk extender supplemented with modified Tyrode's medium (KMT) were used as control treatments. After a 24‐h storage period in a transport container, spermatozoa were evaluated for motion characteristics and plasma membrane integrity by calcein acetoxymethyl (AM)/propidium iodide staining. The percentage of spermatozoa with intact plasma membranes after storage was lower in SP‐SRF than in SP‐SPF, and the highest in K (P < 0.05). Progressive motility (PMOT) was lower for sperm stored in SP‐SRF than for sperm stored in SP‐SPF (P < 0.05), but there was no significant difference in total motility (TMOT). Sperm stored in KMT (P < 0.05) registered the highest TMOT and PMOT percentages. Osmolarity was significantly higher and pH lower in K than in KMT or SP. Treatment with SP‐SPF from three stallions benefited the PMOT of sperm from one stallion. These preliminary findings suggest that SP from SRFs may be more harmful during storage than SP from SPFs. Removal of SP improves sperm survival in KMT extender, and exchanging SP between stallions seems to influence sperm survival.