Acrosomal Status Research Articles

Comparison between Tris-buffer and INRA-82 extenders on the quality of chilled rabbit spermatozoa

<p>The aim of the present study was to compare the effects of tris-buffer and INRA-82 extenders on the quality of cooled rabbit spermatozoa. Pooled semen samples were collected from eight New Zealand White rabbit bucks, divided into three groups and diluted 1:5 with three different semen extenders: INRA-82, tris-citrate glucose (TCG) and tris-citrate trehalose (TCT). Following dilution, the samples were stored at 5°C for up to 48 h. Sperm motility was recorded at 24, 36 and 48 h post-cooling. Furthermore, sperm viability, morphology, membrane integrity, acrosome status and DNA integrity were assessed at 24 h post-cooling. We found that progressive motility percentages were significantly higher (<em>P</em><0.05) in samples diluted in INRA-82 (61.00, 53.50, 44.00% at 24, 36 and 48 h post-cooling, respectively) than those diluted in either TCG (39.25, 32.00, 19.75%) or in TCT (47.25, 40.50, 29.00%). We also reported that sperm viability, percentage of normal spermatozoa, percentage of spermatozoa with intact acrosome and DNA integrity after 24 h cooling were significantly higher (<em>P</em><0.05) in INRA-82 diluted samples compared to TCG and TCT diluted ones. In summary, our results show that dilution of rabbit semen in INRA-82 improved sperm physiological parameters post-cooling compared to those diluted in TCG or TCT. Our findings also suggest that INRA-82 is a promising diluent that can be used effectively to maintain the viability of chilled rabbit semen.</p>

The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = −0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility.

Comparative efficacy of conventional stains for evaluation of plasmalemma and acrosome integrity of buffalo spermatozoa

The aim of the present study was to compare the efficiency of various conventional single and dual staining protocols for assessment of spermatozoa attributes. The efficiency of single stains such as Eosin-Nigrosin (EN) and Trypan blue (TB) for viability and Giemsa (G) for acrosome integrity vis-a-vis dual stains such as Trypan blue-Giemsa (TB-G) and Eosin Nigrosin-Giemsa (EN-G) were compared. Ejaculates (12) from 3 buffalo bulls were used for evaluation of livability and acrosome integrity using said stains. The smears stained with EN-G were difficult to visualize distinctly and provided obscure results. A nonsignificant difference was found between EN, TB and TB-G for spermatozoa viability. However, at recommended concentration, TB yielded faint images, thus making the observation unnecessarily tedious. However, staining with TB-G was found advantageous for simultaneous determination of viability and acrosome status. Moreover, the results between G and TB-G deferred nonsignificantly indicating similar effectiveness of both the stains. Overall, TB-G can be a reliable, quick and convenient method for determining viability and acrosome integrity simultaneously for routine semen evaluation.

Viability longevity and quality of epididymal sperm stored in the liquid state of European red deer (Cervus elaphus elaphus)

The aim of the study was to evaluate viability longevity and quality of liquid-stored epididymal sperm of European red deer (Cervus elaphus elaphus). Sperm samples were recovered from epididymides of 15 mature stags. Samples were diluted to 100 × 106 sperm⁄mL in modified Salomon’s extender and stored at 5 °C for 25 days. Sperm were analyzed to determine total motility (TMOT), progressive motility (PMOT) and motion variables [CASA system], acrosomes with normal apical ridges (NAR), viability and acrosomal status (FITC-PNA/PI), plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) on the first day (D1) and every other day of storage (D3-D25). Data were analyzed using a one-way analysis of variance (ANOVA). Spermatozoa remained motile until D25, whereas TMOT exceeded 60%, NAR and PMI exceeded 80%, FITC-PNA/PI exceeded 75%, and MMP approximated 70% until D11. There was a lesser (P < 0.05) value for these variables on D5 (relative to D1). Furthermore, there was a decrease in values for motility variables as duration of storage increased, excluding amplitude of lateral head displacement, on D3. The results indicate that liquid-stored epididymal sperm of European red deer are characterized as having a long viability (25 days) with a retention of sperm quality for as long as 11 days in the liquid storage state. In vitro or in vivo studies, however, are required to confirm the findings in the present study.

The in vitro effect of kanamycin on the behaviour of bovine spermatozoa

The use of antibiotics is a common part of animal biotechnologies. Especially, the use of antibiotics in semen extenders is necessary. However, the effect of antibiotics on the spermatozoa structure and function is still not completely examined. Therefore, the aim of our study was to investigate the effect of kanamycin on bovine spermatozoa at concentrations of 80 and 160 µg/mL during the 24 h in vitro cultivation. Semen samples were collected from clinically healthy Holstein-Friesian bulls. At times of 0, 2 and 24 h the motility assessment, mitochondrial activity, acrosomal and membrane integrity evaluation were performed. The sperm motility was measured using the Computer-assisted sperm analysis (CASA). Mitochondrial activity was evaluated through the Mitochondrial Toxicity Test (MTT). The acrosomal status was determined using the fast green/rose bengal staining on slides. Similarly, the membrane integrity was analysed using the eosin-nigrosin staining. Our results revealed the dose- and time-dependent effect of kanamycin under the in vitro conditions. In conclusion, the selected concentrations of kanamycin may have adverse effects on the motility, mitochondrial activity, acrosomal and membrane integrity during semen processing. Considering the relatively low concentrations used, we do not recommend to use kanamycin as a supplement in bovine semen extenders.

120 Use of fixable dyes to analyze equine sperm membrane integrity and acrosome reaction after A23187 treatment

Conventional IVF is not successful in the horse, and current work is focused on factors affecting sperm capacitation in this species. Challenges arise in assessing equine sperm incubated in media containing capacitation promotors, as some of these factors cause sperm head-to-head binding (aggregation). Our preliminary microscopic findings showed that sperm aggregates are largely of viable sperm, whereas nonviable sperm individualize. Thus, data obtained using technologies that analyse only individual cells and gate out aggregates, such as flow cytometry, may not accurately represent the study population. We developed a fixable live/dead/acrosome staining protocol (LD-PSA) that minimizes sperm aggregation. The aim of this study was to evaluate the percentage of viable and acrosome-reacted equine sperm after A23187 treatment, using either LD-PSA or a standard staining protocol (PI-PSA). Sperm from 9 ejaculates were suspended in Hanks’ balanced salt solution (HBSS) and exposed for 10min at 37°C to vehicle (V) or to 10 µM A23187 (C10). The sperm were washed, resuspended in HBSS medium with added lactate and pyruvate and containing 7mgmL−1 of bovine serum albumin (BSA), and assessed immediately (0h) or incubated at 37°C for 2h (the period needed for equine sperm to respond to A23187). Motility was analysed using computer-assisted semen analysis. Each treatment was stained by PI-PSA: propidium iodide and fluorescein isothiocyanate-Pisum sativum agglutinin (PSA) in DPBS; and by LD-PSA: Live/Dead Fixable Red, paraformaldehyde 2%, Triton×1%, and FITC-PSA in Dulbecco's phosphate-buffered saline with Accumax (Stem Cell Technologies), a commercial proprietary cell agglutination inhibitor, before flow cytometric analysis. Differences were analysed using repeated-measures two-way ANOVA. The% total motile sperm (TMOT) for V and C10 treatments were 76.3±3.0 and 71.2±4.7 at 0h (P&amp;gt;0.05), and 70.5±14.8 and 2.4±0.8 at 2h (P&amp;lt;0.05). On flow cytometry, the percentage of events outside the gate for V sperm (0h and 2h combined) was 31.9% in PI-PSA and 21.9% in LD-PSA samples (P&amp;lt;0.01). Measured viability in V samples was significantly lower when stained with PI-PSA than with LD-PSA at 0h (49.2±4.6 vs. 67.1±4.9) and tended to be lower (P=0.07) at 2h (44.0±4.9 vs. 55.1±2.8). Notably, the viability recorded in PI-PSA was 26 percentage points lower than was the TMOT at both 0h and 2h, indicating nonrepresentative results, as nonviable sperm should not be motile. By LD-PSA, this difference was 9 points at 0h and 15 points at 2h. Vehicle sperm showed significantly higher AR values in PI-PSA than in LD-PSA at 0h (30.4±4.0 vs. 17.7±2.4) and 2h (41.9±4.5 vs. 24.0±1.8), as did C10 sperm at 0h (28.9±2.7 vs. 18.0±2.5). The lower values for viability than total motility likely reflect agglutination of viable sperm and thus their exclusion from analysis on flow cytometry. The anti-clumping measures employed in the LD-PSA protocol were associated with increased correspondence of measured viability with TMOT. Thus, LD-PSA may offer a more accurate technique to assess viability and acrosome status of equine sperm incubated in capacitating conditions.

Stage-specific metabolomic changes in equine oviductal fluid: New insights into the equine fertilization environment

A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (1H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF samples were lactate, myoinositol, creatine, alanine and carnitine. Only fumarate and glycine showed significant differences in their concentrations between PRE and PST OF samples, with higher concentrations in the PST samples. In a preliminary study, stallion spermatozoa (n = 3 ejaculates) were incubated with different concentrations of PST OF from one mare (0, 0.0625, 0.125, 0.25, 0.5 or 1%; v:v). After 4 h of sperm incubation, protein tyrosine phosphorylation (PY) by western blotting, sperm motility, and acrosomal status were evaluated. An increase of PY was observed in sperm from two stallions when treated with 0.0625% and 0.125% of OF; however no change in PY was noted in the other stallion. There were no effects of OF on spermatozoa motility or acrosome status. These results provide the first information on the metabolomics of equine OF at different stages of the estrus cycle, and present the possibility that OF may affect PY in stallion spermatozoa.

Contraceptive efficacy of sperm agglutinating factor from Staphylococcus warneri, isolated from the cervix of a woman with inexplicable infertility

BackgroundVoluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility.MethodsGenomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-α sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg++-dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice.ResultsThe 80 kDa rSAF showed complete sperm agglutination, inhibited its Mg2+-ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests.ConclusionThe Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.

Sperm cellular and nuclear dynamics associated with bull fertility

The objective of this study was to ascertain cellular characteristics and the dynamics of the sperm chromatin proteins protamine 1 (PRM1) and protamine 2 (PRM2) in the sperm of Holstein bulls having a different fertility status. Important sperm variables were analyzed using computer-assisted sperm analysis (CASA). Sperm membrane, acrosome status, DNA integrity were also assessed using propidium iodide (PI), fluorescein isothiocyanate conjugated to Arachis hypogaea (FITC-PNA), and acridine orange (AO) followed by flow cytometry. In addition, abundances of PRM1 and PRM2 were analyzed using flow cytometry experiments. Differences in sperm decondensation capacity were assessed in bulls of varying fertility using a decondensation assay. As determined using CASA, average pathway velocity, amplitude of lateral head displacement and straightness were different (P < 0.05) for sperm from high and low fertility bulls. There, however, were no differences between the high and low fertility bulls for characteristics of sperm plasma membrane, acrosome, and DNA integrity (P > 0.05). Relative abundances of PRM1 and PRM2 in sperm from the high and low fertility bulls were inversely related (P < 0.0001). Percentages of decondensed sperm were different between high and low fertility bulls (P < 0.0001) and total numbers of decondensed sperm were greater in low fertility bulls than high fertility bulls (R2 = 0.72). Results of the present study are significant because molecular and morphological phenotypes of sperm that were detected affect fertility in livestock species.

Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; nHF = 10 bulls) or low fertility bulls (LF; nLF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC1(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (Cpos), intact plasma membrane (PIneg), unstained acrosome (PNAneg), low intracellular Ca2+ levels (Fneg), and high mitochondrial membrane potential (Mpos). In both fertility groups, Mpos cells comprised more than 90 and 84% of PInegPNAneg sperm at 0 and 3 h, respectively. The percentage of CposPInegPNAnegFnegMpos sperm did not differ between HF and LF ejaculates; however, the percentage of Fneg cells within the PInegPNAneg and PInegMpos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of Fneg sperm within the PInegMpos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI.

Assessment and preservation of liquid and frozen-thawed Black crested mangabey (Lophocebus aterrimus) spermatozoa obtained by transrectal ultrasonic-guided massage of the accessory sex glands and electroejaculation

The Black Crested Mangabey (Lophocebus aterrimus) is an African monkey listed as Near Threatened by the IUCN and in captivity the population is limited to 34 males. The aim of this study was to evaluate two Black Crested Mangabey males, maintained in captivity in a zoological garden and suspected of infertility, with a complete examination of their genital tract using ultrasonography, followed by recovery of semen using transrectal ultrasonic massage of the accessory sexual glands (TUMASG) and electroejaculation. One male had small testicular and accessory sex gland sizes indicative of senile hypoplasia. The other male was suspected of infertility. Four semen samples were obtained. Fresh semen was initially evaluated, diluted in Refrigeration Medium Test Yolk buffer, cooled at 15 °C and cryopreserved. Endocrine profiles (testosterone, oestradiol, FSH, LH, cortisol), prostatic specific antigen and semen variables (volume, concentration, motility by CASA, viability and acrosome status using flow cytometry, morphology, morphometry utilising TEM) were evaluated in raw, cooled and cryopreserved samples. There was no detrimental effect of cooling or cryopreservation on sperm viability and acrosomal integrity. Similar percentages of viable and acrosome-intact spermatozoa were present in cooled (for 6 h) and frozen-thawed semen samples (75.1% compared with 69.0%, P > 0.05), while progressive motility was greater in cooled, compared with frozen-thawed samples (81.5% compared with 67.3%). This study was the first in which there was evaluation of sperm variables in this species and, although this study is limited by the number of animals it provides background information for further studies using assisted reproductive technologies.

Influence of adding different energy sources to the bull and ram spermatozoa exposed to different refrigerating times

Semen storage changes sperm synthesis and biochemical characteristics and hence hinders ferti-lizing ability. Mixture of two various sugar molecules appeared an improved positive impact on stored spermatozoa of different species. The current study aimed to investigate the effect of fructose, sucrose and raffinose supplementation to Tris-glucose-egg yolk extender (TGY) beside comparing their effect with the effect of commercial Triladyl® extender on bull’ and ram’ semen stored at 5 °C for 96 hours. The semen samples were pooled, extended and divided into five portions. TGY or Triladyl® extenders were diluted at rate 1 part of raw semen: 5 parts of the extender. The TGY diluent was split into four diluent parts in sterilized test tubes. TGY and Triladyl® extenders contained one type of sugar, while the other three extended semen samples contained two types of sugar including TGY plus 0.990 g fructose (TGYF), 0.700 g sucrose (TGYS) and 0.700 g raffinose (TGYR) /100ml extender. The ex-tended semen was stored at 5°C for 0, 24, 48, 72 and 96 hours. The obtained data show that spermato-zoa parameters significantly (P < 0.01) decreased with increasing preservation period of all diluent samples. Meantime, supplementation with fructose, sucrose or raffinose with TGY extended samples improved significantly (P < 0.01) sperm properties (motility, livability with acrosomal status and normality, enzymatic actions of AST, ALT, LDH, and ALP, and sperm penetration ability for both bull and ram diluted semen. Concerning the sperm characteristics of the commercial Triladyl® extend-er, it was better than those of TGY without any supplementation for both bull and ram diluted semen.In conclusion, the obtained results suggest that obtaining a reasonable and advantageous impact for the preservation of bull and ram spermatozoa could be acquired by using diluents containing a sugar blend. However, as these results depend on in vitro assessments, there is a need for further fertil-ity trials to confirm it.

Quality assessment of cryopreserved New Zealand white rabbit spermatozoa in INRA-82 extender containing different cryoprotectants

&lt;p&gt;The present study aimed to evaluate the effect of supplementation of INRA-82 semen extender with different cryoprotectants (dimethyl sulphoxide; DMSO vs. dimethyl formamide; DMF) on the quality of white New Zealand rabbit buck spermatozoa. We also investigated the possible association between the synergistic action of DMSO and DMF and their relation with INRA-82 extender composition. Semen was collected and pooled from 8 adult rabbit bucks. Pooled semen samples were diluted 1:1 with INRA-82 extender supplemented with DMSO 8%, DMF 8% or a combination of DMSO 4% and DMF 4%. The diluted semen samples were cryopreserved in 0.25 plastic straws. After thawing, progressive motility, sperm viability, sperm abnormalities, membrane integrity, acrosome status, viability index and DNA integrity were evaluated. The results showed that dilution of rabbit buck semen in INRA-82 supplemented with DMSO and DMF (4% each) before freezing significantly (&lt;em&gt;P&lt;/em&gt;&amp;lt;0.05) improved sperm motility (42.00%), percentage of live spermatozoa (45.30%), proportions of spermatozoa with intact acrosome (59.75%) and percentage of spermatozoa with non-fragmented DNA (86.04%), compared to those diluted in INRA-82 supplemented either with DMSO 8% (+9, +10, +5 and +7 percentage points, respectively) or with DMF 8% alone (+18 +18, +12 and +9 percentage points, respectively). In conclusion, dilution of rabbit buck semen before freezing with INRA-82 extender supplemented with a combination of DMSO 4% and DMF 4% improved quality of frozen-thawed New Zealand White rabbit spermatozoa. Furthermore, our results also suggest that supplementation of INRA-82 with DMSO or with DMF alone at higher concentrations deteriorates the sperm quality.&lt;/p&gt;

Does conventional freezing affect sperm DNA fragmentation?

ObjectiveSperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test.MethodsIn total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups.ResultsThere was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability.ConclusionConventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

Morphological indices for canine spermatozoa based on the World Health Organization laboratory manual for human semen.

Attempting to contribute to the development of a more objective morphological evaluation of dog spermatozoa, in this study the indices of multiple sperm defects (multiple abnormalities index [MAI]; teratozoospermic index [TZI]; sperm deformity index [SDI]) were calculated following the World Health Organization (WHO) guidelines. In Experiment I, the concordance of MAI, TZI and SDI with the proportions of morphologically normal spermatozoa (MNS) was evaluated in fresh ejaculated spermatozoa (dogs=47). In Experiment II, the potential role of indices as prognostic values was assessed in spermatozoa of different origin and treatment (fresh ejaculated: n=6; fresh epididymal: n=6; frozen-thawed ejaculated: n=6) by their correlation with different semen parameters (motility, membrane integrity and acrosome status) and with an in vitro sperm function test. Samples with different proportions of MNS showed different values of SDI, the index that better represented the decline of sperm morphology in both fresh and frozen-thawed samples (Exp. I and II; p<0.05). No correlations between indices and semen parameters were observed (Exp. II), but when samples were evaluated collectively, negative correlations (SDI-motility, p=0.01; SDI-acrosome integrity, p=0.002) were found. Including all the defects of each spermatozoon, SDI might be a useful index during morphological analysis and better discriminates the increase in multiple defects. A more objective morphological evaluation for dog spermatozoa was achieved by the WHO method, and in vitro tests allowed to elucidate the validity of SDI as prognostic indicator of in vitro fertilizing potential.

Molecular markers of sperm quality

Light microscopic semen evaluation provides useful information about a given sperm sample, but due to its subjective nature has limited prognostic value for the reproductive performance of males or the outcome of assisted fertilization. Cryptic sperm abnormalities (occurring at the molecular level) are not easily detectable by light microscopy, but can be revealed by an array of biomarkers. The latter include fluorescent markers of acrosomal status, fluorochromes detecting altered sperm chromatin or DNA integrity, vital dyes revealing sperm mitochondrial activity, probes detecting apoptotic events, and antibodies detecting proteins that are either up- or down-regulated in defective spermatozoa. Many of the above biomarkers are best tested by flow cytometry, permitting rapid, automated, high throughput, objective measurement of the relative abundance of these biomarkers in semen. This review summarizes a strategy for the identification of novel male fertility/sperm quality biomarkers based on proteomic, biochemical and immunocytochemical analyses of defective spermatozoa. This approach identifies proteins or ligands uniquely associated with defective spermatozoa, regardless of whether they carry gross morphological defects or subtle, but critical hidden defects (e.g. DNA strand breaks) not detected with conventional, light microscopic analysis. Such markers, including ubiquitin, sperm thioredoxin SPTRX3/TXNDC8, 15LOX, and Lewis(y)-terminated N-glycans, are associated with poor semen quality and reduced fertility, warranting a designation of negative markers of fertility. The significance of sperm cytoplasmic droplet, a structure that accumulates several of the discussed biomarker proteins, is also discussed with regard to sperm quality and fertility.

Disruption of bovine sperm functions in the presence of aplastic midpiece defect.

Bulls are of great importance in the productive chain and for this reason they should have a good semen quality. There is no doubt that sperm morphology is very important to bull fertility, although little is known about how exactly the abnormal morphologies may affect sperm functions. To detail the morphological description of the aplastic midpiece defect (AMD), as well as to understand its consequences for male fertility based on membrane and acrosome status, mitochondrial membrane potential and DNA integrity parameters. The bulls were divided into two groups: control, consisting of satisfactory potential breeders (n=3); and AMD, consisting of unsatisfactory potential breeders with a high percentage of AMD (n=3). Bulls were evaluated by the breeding soundness evaluation; five ejaculates were collected from each animal and analyzed by flow cytometry. Spermatozoa from AMD group exhibited lower sperm motility and vigor (p<0.05). In addition, it also exhibited lower mitochondrial membrane potential (p<0.05), a higher percentage of spermatozoa with DNA fragmentation (p<0.05), lower acrosome and plasma membrane integrity (p<0.05), and higher lipid bilayer sperm membrane disorganization (p<0.05) in comparison with control bulls. These findings may be due to oxidative stress and a reduction of the energy production capacity in addition to an alteration in the structural composition of the sperm cell. Moreover, semen with a high percentage of AMD may also be undergoing apoptosis. Bulls with a high percentage of AMD in their semen are not suitable for reproduction. Furthermore, it suggests there is a putative genetic basis for this sperm defect.

In Vitro Effects of Tea Tree Oil (Melaleuca Alternifolia Essential Oil) and its Principal Component Terpinen-4-ol on Swine Spermatozoa.

The growing interest towards essential oils stems from their biological capabilities that include antibacterial and antioxidant effects. Such properties may be extremely useful in the reproductive field; nonetheless essential oils show toxic effects that can lead to cell disruption. The present study aimed to evaluate and compare the effects of tea tree oil (TTO) and its principal component terpinen-4-ol (TER) on the morpho-functional parameters of swine spermatozoa. Experimental samples were prepared by suspending 15 × 107 spermatozoa in 5 mL of medium with different concentrations of the above-mentioned compounds: from 0.2 to 2 mg/mL at an interval of 0.2 for TTO, while TER concentrations were adjusted according to its presence in TTO (41.5%). After 3 h incubation at 16 °C, samples were analyzed for pH, viability, acrosome status, and objective motility. The results highlighted a concentration-dependent effect of TTO with total motility as the most sensitive parameter. TER was better tolerated, and the most sensitive parameters were related to membrane integrity, suggesting a different pattern of interaction. The study confirms the importance of evaluating the effects of natural compounds on spermatozoa before exploiting their beneficial effects. Spermatozoa seem to be good candidates for preliminary toxicological screenings in the light of their peculiar properties.

The importance of trace minerals copper, manganese, selenium and zinc in bovine sperm-zona pellucida binding.

SummarySperm-zona pellucida (ZP) binding is a necessary event for successful fertilization. The aim of this study was to determine the effect of trace minerals such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) on bovine spermatozoa binding to ZP. Sperm viability, functional membrane integrity, acrosomal status (AS), total antioxidant capacity (TAC) and sperm lipid peroxidation (LPO) were also evaluated. For the present study, in vitro fertilization (IVF) medium was supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), all minerals (Cu+Mn+Se+Zn), or tested without supplement (Control). Considerably more sperm bound to ZP when Cu, Se or Zn were added to the IVF medium, but there were no difference compared with the Control, Mn and Cu+Mn+Se+Zn groups. After 1 h of incubation, viability was increased by the addition of Cu, Mn and Se with respect to the Control but, after 2 h, viability was higher only with the addition of Mn to IVF medium. Functional membrane integrity improved in sperm treated with Cu. Acrosome integrity was higher in sperm treated with Zn after 1 h of incubation. LPO was significantly higher in sperm treated with Cu or Cu+Mn+Se+Zn. The mean TACs of sperm treated with Cu, Mn, Zn or Cu+Mn+Se+Zn were lower than in the Control. In conclusion, the results obtained in the present study determined that the presence of Cu, Se and Zn in the IVF medium increased the number of spermatozoa bound to the ZP, highlighting the importance of these minerals in the fertilization process.