Hypo-osmotic Swelling Research Articles

Effect of vitamin E and selenium supplementation on oxidative markers and semen quality parameters in breeding bulls

The objective of the study was to evaluate the effect of vitamin E and Se supplementation on oxidative markers and semen quality parameters in breeding bulls. The study was conducted at ICAR-Central Institute for Research on Cattle, Meerut (subtropical climate), Uttar Pradesh during 2019. Eighteen bulls (six good bulls and twelve poor bulls) were divided into three groups consisting 6 bulls in each. Six good bulls of group 1 served as healthy control. Group 2 poor bulls were administered Vitamin E and Selenium @ 10 ml SC (Vitamin E 50 mg as DL-α-tocopheryl acetate and Selenium 15 mg as sodium selenite) once weekly for three weeks while group 3 poor bulls were kept as untreated control. Semen samples were collected and semen quality and biochemical parameters were done using standard procedures at fortnightly interval upto two months. Significantly low MDA and higher SOD and catalase were observed in bulls of supplemented group. Semen volume, sperm concentration, initial motility, percentage of live sperm and hypo-osmotic swelling test improved significantly in supplemented group as compared to nonsupplemented bulls. The study concludes that supplementation of vitamin E and Se causes significant improvement in oxidative stress markers and semen quality parameters in breeding bulls.

Evaluation of Soy Lecithin Efficacy in Comparison with Egg Yolk on Freezing of Epididymal Sperm in Dogs

BACKGROUND: Epididymal sperm collection is allowed the use of genetic material post-mortem or after orchiectomy from high-value animals or endangered species. OBJECTIVES: The aim of this study was to improve the accessibility of the dog epididymal sperm cryopreservation system based on the appropriate dose of lecithin. METHODS: Epididymal sperm from castrated testes of mature healthy dogs in veterinary centers were collected and divided into six groups: G1: egg yolk 20% (control), G2: lecithin 0.4% (L0.4), G3: lecithin 0.8% (L0.8), G4: lecithin 1.2% (L1.2), G5: lecithin 1.6% (L1.6), and G6: lecithin 2% (L2). Evaluation of Spermatozoa was done before freezing by Motility test, Eosin- Nigrosin vital staining and Hypo-Osmotic Swelling Test (HOST) and after thawing by Computer Assisted Semen Analysis (CASA), HOST, Eosin-Nigrosin vital staining, Mitochondrial Membrane Potential (MMP) and Intracellular Reactive Oxygen Species (ROS). RESULTS: In frozen samples, total motility and proportion of sperm with intact plasma membrane integrity based on the HOS test were lesser in all groups treated with different concentrations of lecithin as compared with the control group (P ≤ 0.05). However, beat cross frequency (BCF) was higher in all groups treated with different concentrations of lecithin as compared with the control group (P ≤ 0.05). Yet progressive motility, the proportion of live sperm based on the Eosin- Nigrosin test, VAP, VSL, VCL, STR, LIN, and ALH did not differ among various experimental groups (P > 0.05). The proportion of sperm with morphological defects did not differ between fresh and frozen samples and among various experimental groups (P > 0.05). Mitochondrial membrane potential was greater in the control than 0.4% lecithin group (P = 0.026). The proportion of sperm positive for ROS was lesser in the control than 0.4% lecithin group (P = 0.049). CONCLUSIONS: egg yolk was superior to the lecithin-based extenders to cryopreserve epididymal sperm of dogs.

Effects of insulin on sperm cell quality in ram semen cooled at 5°C

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (μm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.

Gallic acid improves the viability and mitochondrial membrane potential of post-thawed goat buck semen.

The aim of this study was to determine the effects of gallic acid (GA) on frozen-thawed goat spermatozoa. Four Honamli goat bucks were used at their breeding season, and ejaculates were collected by an electroejaculator. Mixed semen was divided into the following four groups: control (0 mM), low (L; 1 mM), medium (M; 2 mM), and high (H; 4 mM) concentration of GA. All the groups were frozen and thawed in a water bath for spermatological evaluation. The lowest motility was observed in the control group (47.60 ± 5.70%) (P < 0.05), while the highest viability (62.45 ± 1.68%), plasma membrane and acrosome integrity (44.81 ± 4.57%), and high mitochondrial membrane potential (35.96 ± 2.50%) were observed in the low GA group (P < 0.05). Also, the lowest hypo-osmotic swelling test (HOS +) value was found in the high GA group (47.60 ± 4.82%) (P < 0.05). In conclusion, supplementing a low concentration (1 mM) of GA to the Tris-based semen extender had a positive effect on spermatological parameters after freeze-thawing of Honamli goat semen. Further studies should be continued in other species with different doses and combinations using commercial and/or homemade semen extenders.

An Evaluation of Semen Quality of Donor Bulls in the Central Artificial Insemination Station (CAIS), Kundasale, Sri Lanka

Fertility assessment of semen donor bulls used in Artificial Insemination programs is an important step in improving dairy industry in Sri Lanka. A study was conducted to evaluate the fertility of semen donor bulls using semen parameters and respective pregnancy rates. The semen donor bulls (n =21) in the Central Artificial Inseminations Station, Kundasale were selected for the study, and the hypo-osmotic swelling test was performed for both fresh and frozen semen of each bull. Data were analysed using statistical function of Excel software version 2010. Presence of high percentage of hypo-osmotic swelling positive spermatozoa in both fresh and post-thawed frozen semen indicated that all bulls studied were highly fertile. A total of 49255 AIs has been performed in four provinces with an overall pregnancy rate of 28.2%. The highest pregnancy rate of 57.6% was recorded in the Northern Province and the lowest pregnancy rate of 22.5% was recorded in the Central Province. The study revealed that there was no significant difference (p&gt;0.05) in semen quality among individual bulls or among breeds. Al though the semen quality of the bulls was in the acceptable range, the pregnancy rate differed significantly (p&lt;0.05) according to the area. In addition, the overall pregnancy rate was (28.2 %) below the acceptable level. Further studies are required to investigate factors associated with semen quality and the low pregnancy rates observed in some of the areas.

Effect of Hydrated Carbon 60 Fullerene on Frozen Ram Semen Quality.

The aim of this study was to evaluate the effect of hydrated carbon 60 fullerene (C60HyFn) on ram semen quality during cryopreservation. Three ejaculates from each of seven Akkaraman rams were collected using an artificial vagina during the nonbreeding season and pooled. Pooled semen samples were divided into 10 equal parts and diluted with tris + egg yolk extender not containing (control) and containing 100, 200, 400, and 800 nM and 1, 5, 10, 20, and 40 μM C60HyFn at 37°C. After addition of 5% glycerol and an equilibration process for 3 hours, the samples were frozen in 0.25-mL straws in an automatic freezing device at -140°C and stored in a liquid nitrogen container. Straws were thawed 24 hours after freezing and analyzed immediately with no incubation period. Motility, kinematic parameters, abnormality, vitality, hypo-osmotic swelling test (HOST), and oxidative stress levels were analyzed in thawed semen. Compared with the control, 200, 400, and 800 nM and 1 and 5 μM C60HyFn doses increased motility and HOST values and decreased the dead sperm rate. When compared with the control, addition of C60HyFn significantly decreased malondialdehyde levels (between 200 nM and 40 μM doses) and significantly increased glutathione peroxidase (between 800 nM and 40 μM doses) and catalase (between 1 and 40 μM doses) activities. In conclusion, results of this study show that the C60HyFn nanoparticles are nontoxic to ram semen and their supplementation in the extender is beneficial to sperm motility and membrane integrity after freeze-thawing.

Genetic diagnosis, sperm phenotype and ICSI outcome in case of severe asthenozoospermia with multiple morphological abnormalities of the flagellum.

Are ICSI outcomes impaired in cases of severe asthenozoospermia with multiple morphological abnormalities of the flagellum (MMAF phenotype)? Despite occasional technical difficulties, ICSI outcomes for couples with MMAF do not differ from those of other couples requiring ICSI, irrespective of the genetic defect. Severe asthenozoospermia, especially when associated with the MMAF phenotype, results in male infertility. Recent findings have confirmed that a genetic aetiology is frequently responsible for this phenotype. In such situations, pregnancies can be achieved using ICSI. However, few studies to date have provided detailed analyses regarding the flagellar ultrastructural defects underlying this phenotype, its genetic aetiologies, and the results of ICSI in such cases of male infertility. We performed a retrospective study of 25 infertile men exhibiting severe asthenozoospermia associated with the MMAF phenotype identified through standard semen analysis. They were recruited at an academic centre for assisted reproduction in Paris (France) between 2009 and 2017. Transmission electron microscopy (TEM) and whole exome sequencing (WES) were performed in order to determine the sperm ultrastructural phenotype and the causal mutations, respectively. Finally 20 couples with MMAF were treated by assisted reproductive technologies based on ICSI. Patients with MMAF were recruited based on reduced sperm progressive motility and increased frequencies of absent, short, coiled or irregular flagella compared with those in sperm from fertile control men. A quantitative analysis of the several ultrastructural defects was performed for the MMAF patients and for fertile men. The ICSI results obtained for 20 couples with MMAF were compared to those of 378 men with oligoasthenoteratozoospermia but no MMAF as an ICSI control group. TEM analysis and categorisation of the flagellar anomalies found in these patients provided important information regarding the structural defects underlying asthenozoospermia and sperm tail abnormalities. In particular, the absence of the central pair of axonemal microtubules was the predominant anomaly observed more frequently than in control sperm (P < 0.01). Exome sequencing, performed for 24 of the 25 patients, identified homozygous or compound heterozygous pathogenic mutations in CFAP43, CFAP44, CFAP69, DNAH1, DNAH8, AK7, TTC29 and MAATS1 in 13 patients (54.2%) (11 affecting MMAF genes and 2 affecting primary ciliary dyskinesia (PCD)-associated genes). A total of 40 ICSI cycles were undertaken for 20 MMAF couples, including 13 cycles (for 5 couples) where a hypo-osmotic swelling (HOS) test was required due to absolute asthenozoospermia. The fertilisation rate was not statistically different between the MMAF (65.7%) and the non-MMAF (66.0%) couples and it did not differ according to the genotype or the flagellar phenotype of the subjects or use of the HOS test. The clinical pregnancy rate per embryo transfer did not differ significantly between the MMAF (23.3%) and the non-MMAF (37.1%) groups. To date, 7 of the 20 MMAF couples have achieved a live birth from the ICSI attempts, with 11 babies born without any birth defects. The ICSI procedure outcomes were assessed retrospectively on a small number of affected subjects and should be confirmed on a larger cohort. Moreover, TEM analysis could not be performed for all patients due to low sperm concentrations, and WES results are not yet available for all of the included men. An early and extensive phenotypic and genetic investigation should be considered for all men requiring ICSI for severe asthenozoospermia. Although our study did not reveal any adverse ICSI outcomes associated with MMAF, we cannot rule out that some rare genetic causes could result in low fertilisation or pregnancy rates. No external funding was used for this study and there are no competing interests. N/A.

Abstract P413: Stretch-induced Caveolae-mediated Disruption Of Cyclic AMP Microdomains Leads To Arrhythmogenic Ca 2+ Mishandling In Mouse Atrial Myocytes

Background: Atrial fibrillation (AF) often occurs during hypertension and is associated with an increase in cardiomyocyte stretch. Mechanism of ectopic beats, that trigger AF, has been linked to Ca 2+ mishandling and leaky hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain elusive. Caveolae membrane structures are involved in cell mechanosensing processes and control the cAMP signaling pathway. We hypothesized that mechanical stretch disrupts caveolae, promoting cAMP production and sarcoplasmic reticulum Ca 2+ leak via augmentation of RyRs phosphorylation. Methods and Results: Cell size analysis and Ca 2+ dynamics measurements were performed by confocal imaging of isolated mouse atrial myocytes. Cell stretch was modeled by hypoosmotic swelling (from 310 mOsM to 220 mOsM to flatten caveolae structures) resulting in a ~30% increase in cell width (p&lt;0.05) with no changes in cell length. Swelling resulted in a biphasic effect on Ca 2+ spark activity: a fast (&lt;10 min of exposure) ~50% increase (p&lt;0.001) followed by a slow decrease to the level observed in isotonic conditions (&gt;30 min of exposure). Similarly, caveolae disruption via cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) led to 2-fold increase in Ca 2+ sparks frequency (p&lt;0.001). Swelling- and MβCD-induced increases in atrial Ca 2+ spark activity were prevented via inhibition of cAMP production by adenylyl cyclases by 0.1mM SQ22536 or cAMP-dependent protein kinase A (PKA) by 1μM H-89. Then, we tested if this mechanism is present in atrial myocytes from pressure-overloaded (4-weeks transaortic constriction, TAC) mice. Atrial myocytes from TAC mice showed a 1.6 times higher Ca 2+ sparks frequency than wild-type myocytes (p&lt;0.01), which was significantly reduced (p&lt;0.01) to wild-type level after incubation with SQ22536. Conclusions: Our findings suggest that cell stretch increases spontaneous Ca 2+ spark activity through the disruption of caveolae and cAMP-mediated augmentation of PKA activity. This mechanism could be involved in the Ca 2+ mishandling and AF in pressure overloaded hearts.

Abstract P368: Caveolin-3 Prevents Swelling-induced Cell Lysis Via Regulation Of I Cl,swell Expression And Activity

Background: Caveolae membrane structures harbor mechanosensitive chloride channels (MCCs) which form a swelling-activated chloride current ( I Cl,swell ) and play an important role in cell volume regulation and mechano-electrical signal transduction. However, the role of muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCCs expression, activity, and contribution to cell viability in response to mechanical stress remains unclear. We hypothesized that Cav3-based mechano-protection is enabled by complimentary expression of MCCs. Methods and Results: Experiments were performed on native (Cav3-) and Cav3-transfected (Cav3+) HEK-293 cells. Cell stretch was mimicked by light (220 mOsM) or extreme hypoosmotic swelling (&lt;20mOsM). Cav3+ HEK-293 cells were significantly resistant to extreme hypotonic solutions (15 minute incubation) compared to Cav3- HEK-293 cells, and this mechano-protection was significantly reduced when exposed to I Cl,swell selective inhibitor DCPIB (1 μM). We found that three MCCs (ClC-2, ClC-3, and SWELL1, also known as LRRC8A) contain caveolin-binding motifs in their structure, indicating their possible localization in caveolae structures. Co-IP analysis confirmed association of SWELL1 with Cav3. Interestingly, Cav3+ HEK-293 cells showed a significant (by 2-fold) increase of SWELL1 protein level, while ClC-2/3 protein levels remained unchanged. This was accompanied by a 2-fold increase of I Cl,swell , but no change in mRNA expression levels. FRET analysis showed a &lt;10 nm membrane and intracellular association between Cav3 and tested MCCs. Furthermore, Cav3/SWELL1 membrane FRET efficiency was halved in light hypoosmotic solution, as well as after disruption of caveolae structures via cholesterol depletion by 1-hour treatment with 10 μM methyl-β-cyclodextrin. Cav3/ClC-2/3 average membrane FRET efficiency remained unchanged in hypotonic solution. Conclusions: We concluded that of MCCs tested, SWELL1 abundance and activity is regulated by Cav3 and that their association relies on membrane tension and caveolae integrity. The present study highlights the mechano-protective properties of Cav3 which are partially facilitated by complimentary SWELL1 expression and activity.

O-092 Sperm phenotype, ICSI outcome and genetic diagnosis in case of severe asthenozoospermia with multiple morphological abnormalities of the flagellum

Abstract Study question What are the feasibility and outcome of ICSI in case of presumably genetic severe asthenozoospermia with Multiple Morphological Abnormalities of the Flagellum (MMAF phenotype)? Summary answer ICSI outcome for couples with MMAF phenotype does not differ from that of other couples requiring ICSI, regardless to the genetic etiology What is known already Severe asthenozoospermia, especially when associated with multiple morphological abnormalities of the sperm flagellum (MMAF phenotype), results in male infertility. Recent findings confirm that a genetic etiology is frequently responsible for this phenotype. In such situations, pregnancies can be obtained using ICSI. However, few studies have provided detailed analyses of the flagellar ultrastructural defects underlying this phenotype, of its genetic etiologies and of the results of ICSI in such cases of male infertility. Study design, size, duration We performed a retrospective study including 25 infertile men showing severe asthenozoospermia associated with a MMAF phenotype identified through standard semen analysis. These men were recruited from an academic center for Assisted Reproduction in Paris between 2009 and 2017. Transmission electron microscopy (TEM) and Whole Exome Sequencing (WES) were performed in order to precise the sperm ultra-structural phenotype and identify causal mutations, respectively. Twenty of the 25 patients benefited from assisted reproductive therapy by ICSI. Participants/materials, setting, methods MMAF patients were recruited based on reduced sperm progressive motility and increased frequencies of absent, short, coiled or irregular flagella, in comparison with fertile control men. A quantified analysis of the ultrastructural defects was performed for the MMAF patients and for fertile control men. ICSI results for the MMAF patients were compared to those of 528 ICSI attempts performed for non-MMAF individuals considering the sperm parameters and the distribution of ultrastructural axonemal anomalies. Main results and the role of chance Thorough categorization by TEM analysis of the flagellar anomalies found in these patients brought important precisions about the structural defects underlying asthenozoospermia and sperm tail abnormalities detectable through standard microscopy. In particular, absence of the central pair of axonemal microtubules was the predominant anomaly, observed significantly more frequently than in control men (p &amp;lt; 0.01). Exome sequencing performed for 24 of the 25 patients (96%), identified in ten of them homozygous or compound heterozygous mutations that were described to be pathogenic (CFAP43, CFAP44, CFAP69, DNAH1, DNAH8, AK7, TTC29, MAATS1). A majority of those patients (55.5%,5/9) displayed the most severe ultra-structural defects of the axoneme. Forty ICSI attempts were performed for 20 MMAF patients. A hypo-osmotic swelling (HOS) test was required in 13 cycles (5 couples). Fertilization rate in MMAF group (65.7%) was not statistically different from the rate obtained for non-MMAF patients (66.0%) and did not differ according to the flagellar phenotype, nor to the use of HOS test, nor to the genotype. Clinical pregnancy rate per embryo transfer did not significantly differ between the MMAF group (23.3%) and the ICSI control group (37.1%). To date, 11 healthy babies were born among 20 MMAF patients. Limitations, reasons for caution The outcome of ICSI procedure was retrospectively assessed on a small sample and may be susceptible to recall bias. Moreover, TEM analysis was not available for some of the patients due to too low sperm concentration, and WES results are not yet available for all men included. Wider implications of the findings Couples requiring ICSI for presumably genetic severe asthenozoospermia should benefit precociously from appropriate phenotypic and genetic investigations. So far ICSI results appear similar to those observed in other ICSI indications. Identifying a genetic etiology and its mode of inheritance allows providing to these couples a most often reassuring genetic counseling. Trial registration number Not applicable

مطالعه تاثیر آنتی اکسیدانی عصاره هیدورالکلی درمنه (Artemisia incana) بر کیفیت اسپرم منجمد-یخ گشایی شده قوچ مغانی

Maintaining fertility in frozen sperm is very important. To investigate the effect of Artemisia (Artemisia incana) extract on sperm cryopservability of Moghani ram spermatozoa, different levels of the plant extract (0, 2, 4, 6, 8, 10 and 12 ml per deciliter) were added after extraction to diluted semen with an extender containing citrate buffer. After chilling, the semen was packaged in 0.5 ml straws and stored inside the liquid nitrogen until the evaluation of qualitative properties. This experiment was designed with 4 replications ­(with 10 straws in each replicate) per experimental group. Evaluation of sperm viability using eosin-nigrosine staining showed that adding 4 ml of Artemisia extract per deciliter to sheep sperm diluent had a positive effect on sperm viability. Similarly, the HOST test (hypoosmotic swelling test) to examine the integrity of the plasma membrane showed that the treatment had the highest membrane integrity (P<0.05). The highest and lowest Total Motility (TM) was observed in experimental groups 4 and 10 ml of extract in diluted semen, respectively (P<0.05). Examination of peroxidative damage by measuring malondialdehyde showed the lowest amount of malondialdehyde production was seen in the treatment with 12 mg of extract per deciliter diluent that was significantly different from other experimental groups (P<0.05). According to the results of the present experiment, it seems that the use of 4 ml Artemisia extract per deciliter of the diluent has positive effects on the quality of sperm after freezing.

Non-enzymatic extraction of spermatozoa from alpaca ejaculates by pipetting followed by colloid centrifugation

Viscous camelid ejaculates present problems for sperm handling and sperm preservation. In the present study, a technique that had been used for dromedary camel semen was tested with alpaca semen. Ejaculates (n=9) were collected by artificial vagina at San Marcos University, Lima, and were liquefied by gentle pipetting in tris-citrate-fructose. Half of the sample was prepared by Single Layer Centrifugation (SLC) through a colloid; the other half was centrifuged without colloid as a control. Each control and SLC sample was then split into two parts; one part was stored cooled for 24 h at 5 °C and the other part was frozen, resulting in 4 treatments for each ejaculate. All samples were evaluated for sperm motility, hypoosmotic swelling test (HOST), plasma membrane integrity, and morphology, immediately after centrifugation and again after storage Total motility and plasma membrane integrity were greater in samples prepared by SLC than controls (motility 72±13% vs. 57±7%; plasma membrane integrity 63±13% vs. 54±8%, for SLC and controls respectively). Normal morphology and HOST were not different between treatments (65±13 vs. 61±13% and 42±6 vs. 39±10%, for SLC and controls respectively). After 24 h cooled storage, motility and plasma membrane integrity were greater for SLC samples (motility: 51±16 vs. 34±15%; p<0.001; membrane integrity: 51±15 vs. 40±18%; P < 0.05 for SLC and controls, respectively); HOST (40±14 vs. 34±11%) and normal morphology (67±13 vs. 63±14%) were not different between treatments. Sperm quality decreased considerably after cryopreservation (P<0.001 for all parameters); however, motility (P<0.01), plasma membrane integrity (P<0.05) and morphology (P<0.05) were higher for SLC than for controls. These results indicate that alpaca spermatozoa can be extracted from semen using a combination of pipetting and SLC, potentially with a beneficial effect on sperm quality. Samples could be stored cooled for 24 h, retaining better motility than controls; motility and plasma membrane integrity were greater in SLC samples than controls after freezing and thawing but the freezing protocol requires improvement.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&amp;gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&amp;gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

Detailed cell-level analysis of sperm nuclear quality among the different hypo-osmotic swelling test (HOST) classes

We studied the quality differences between the different hypo-osmotic swelling test (HOST) classes, as measured by criteria of DNA fragmentation, DNA decondensation, and nuclear architecture. The aim was to find particular HOST classes associated with good-quality metrics, which may be potentially used in ICSI (intra-cytoplasmic sperm injection). Ten patients from the Department of Reproductive Medicine at Tenon Hospital (Paris, France) were included. Their semen samples were collected and divided into two fractions: one was incubated in a hypo-osmotic solution as per HOST protocol and sorted by sperm morphology, and a second was incubated without undergoing the HOST protocol to serve as an unsorted baseline. Three parameters were assessed: DNA fragmentation (TUNEL assay), DNA decondensation (chromomycin A3 assay), and nuclear architecture (FISH, with telomeric and whole chromosome painting probes). The different HOST classes were evaluated for these three parameters, and statistical analysis was performed for each class versus the unsorted non-HOST-treated sperm. Results with p<0.05 were considered statistically significant. For each of the parameters evaluated, we found significant differences between HOST-selected spermatozoa and non-selected spermatozoa. Overall, spermatozoa of HOST classes B and B+ exhibited the highest quality based on four metrics (low DNA fragmentation, low DNA decondensation, short inter-telomeric distance, and small chromosome 1 territory area), while spermatozoa of HOST classes A and G exhibited the poorest quality by these metrics. In addition to their pathophysiological interest, our results open possibilities of sperm selection prior to ICSI, which may allow for optimization of reproductive outcomes in heretofore unstudied patient populations.

The quality of intact plasma membrane of bull frozen sperm in different breeds

Hypoosmotic Swelling (HOS) Test is a technique to evaluate the intact plasma membrane of spermatozoa. The purpose of this study was to determine the level of intact plasma membrane in various breeds of cattle regardless of age, feed, environment and maintenance management. Semen examination was carried out microscopically. Valuable semen assessment parameter was the percentage of intact plasma membrane in the cows of Bali, Limosin, FH, Brahman, Angus and Simental. Cattles with the highest intact plasma membrane values were Simental and the lowest was FH. The results of statistical analysis on the intact plasma membrane spermatozoa showed a significant difference (P<0.05) between cattle breeds. The intact plasma membrane in Simental was significantly different (P<0.05) compared to Bali, Limousine and FH. However, it is not significantly different from the Brahman and Brangus cattle. Different breeds of cattle can affect the level of intact plasma membrane.

Type III antifreeze protein (AFP) improves the post-thaw quality and in vivo fertility of rooster spermatozoa

Antifreeze proteins (AFP) have the potential for improving sperm cryopreservation. We have applied Type III antifreeze protein (AFP3) on the cryopreservation of spermatozoa from broiler breeder roosters, aiming to enhance post-thawing quality and fertility. Semen was extended at 37°C in Lake's extender containing AFP3 at 0.01, 0.1, 1, 5, and 10 µg/mL (no AFP3 as control). Post-thawing sperm assessment included sperm motility (CASA), morphology, membrane functionality by hypoosmotic swelling test (HOST), lipoperoxidation as malondialdehyde (MDA) production, and sperm viability, early apoptosis (phosphatidylserine exposure as annexin V-positive staining in viable spermatozoa), and mitochondrial activity by flow cytometry. Fertility was assessed after artificial insemination (30 hens/treatment). Total and progressive motility, membrane functionality, and mitochondrial activity increased in 0.1 and 1 µg/mL AFP, compared to control and other concentrations, whereas apoptosis was significantly lower. VAP, VSL, and viability were significantly higher for 1 µg/mL AFP3 than with the other treatments except for 0.1 µg/mL (which was not always significantly different from the control or other concentrations), and with abnormal forms being significantly lower. The proportion of fertilized and hatched eggs was also higher for 1 µg/mL AFP3, with 0.1 µg/mL also showing significantly higher results than the control, and no differences with other concentrations). In conclusion, 1 µg/mL AFP3 could improve the post-thawing results of rooster spermatozoa frozen in Lake's extender. According to our results, concentrations between 1 and 0.1 µg/mL could be similarly efficient.

Comparative evidence support better antioxidant efficacy of mitochondrial-targeted (Mitoquinone) than cytosolic (Resveratrol) antioxidant in improving in-vitro sperm functions of cryopreserved buffalo (Bubalus bubalis) semen

The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 μM, 25 μM, 50 μM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P < 0.05) in 200 nM MitoQ and 50 μM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 μM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P < 0.05) in MitoQ (200 nM) than RESV (50 μM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P < 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P < 0.05) and Bovine cervical mucus penetration was higher (P < 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P < 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P < 0.01) sperm with a higher reduction (P < 0.05) in membrane scrambling. MitoQ improved (P < 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P < 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.

Semen quality and production potential of Indian buffalo breeds

The objective of the study was to evaluate non-genetic factors on semen traits. The overall means for the semen quality traits: ejaculate volume, sperm concentration, total sperm, mass activity, initial and post thaw motility, hypoosmotic swelling test, acrosome integrity of fresh and frozen test were 4.39±0.01 ml, 1.29±0.001 billion, 5.48±0.01 billion, 2.55±0.003, 73.41±0.0004%, 55.9±0.0002%, 59.01±0.001%, 84.98±0.002% and 71.13±0.002%, respectively. Breeds were found to differ in all the semen production and quality traits studied except acrosome integrity of fresh semen. Summer for semen production, and winter and monsoon for semen quality traits were comparatively better season. The quality traits improved with age and declined slightly in the later life.

The Effects of Cytopathic and Non-cytopathic Biotypes of Bovine Viral Diarrhea Virus on Sperm Vitality and Viability of Holstein Dairy Bulls in Vitro

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the most important pathogens. OBJECTIVES: The aim of this study was to investigate the effects of cytopathic (CP) and non-cytopathic (NCP) biotypes of BVDV on vital status, membrane integrity, and motility of sperm cells in Holstein dairy bulls in vitro. METHODS: BVDV-free frozen semen samples were counted after thawing and centrifuged to separate live sperms. A sample containing 105 spermatozoa/mL was prepared. CP and NCP BVDV with 3 different doses of 105 (high dose), 104 (medium dose), and 103 (low dose) tissue culture infectious dose (TCID) 50/mL were challenged to sperm cells. After 2 hours of incubation at 38.5°C, eosin-nigrosine staining and hypoosmotic swelling (HOS) test were performed to assess the sperm viability and plasma membrane integrity. Computer assisted semen analysis (CASA) was used to evaluate the sperm motility parameters. The obtained data were analyzed using GLM method in SAS software. RESULTS: The percentage of live spermatozoa in the control group was 72±3.60%. However, it decreased significantly with the increase of virus concentration in both groups (p ≤0.05). Sperm integrity in the control group showed that the quality of semen was 65± 3.21. But the effect of virus biotypes resulted in a significant decrease in both high (105) and low (103) concentrations (p ≤0.05). BVDV biotypes are able to reduce different sperm movements as their concentration-increases. CONCLUSIONS: We concluded that CP and NCP biotypes of BVDV had a significant effect (p ≤0.05) on survival, plasma membrane integrity, and motility of sperm cells in vitro.

In-vitro embryo production in caprine using slaughterhouse ovaries

The slaughterhouse Caprine ovaries were the cheap source of Caprine germplasm, and it was collected in a hygienic method within a short period. The slicing was the method of oocyte isolation followed in this study. The grade A and B quality oocytes were isolated, washed, and matured in a prepared media. The does in standing heat was identified and fresh buck semen was collected from trained buck available in the animal shed. The collected semen was evaluated for physical attributes, mass motility, live percentage of spermatozoa, individual motility, acrosomal integrity, and the Hypoosmotic swelling test (HOST). The semen from proven buck was used for the successful in-vitro fertilization (IVF) of oocyte and production of Caprine embryos. Various factors were affected the successful production of cleaved embryos in Caprine and were well explained by various authors. The stepwise precautions needed to be taken to successful in vitro embryo production were the main objective of this study.