Hypo-osmotic Swelling Test Research Articles

Relação da qualidade do sêmen com endogamia e marcha de cavalos crioulos colombianos

ABSTRACT: High consanguinity among equines has negative effects on semen quality, thus resulting in low motility and high levels of abnormality in the spermatozoa. However, such a relationship has not been studied in Colombian Creole horses, which have been subjected to particular selection practices focusing mainly on their gait. This research assessed the relationship of semen quality to inbreeding and gait of Colombian Creole horses. Semen was collected from 50 horses using the artificial vagina method. Sperm motility and kinematics were assessed with a computerized analysis system (SCA®). Sperm vitality (SV) and abnormal morphology (AM) were assessed via the eosin-nigrosin staining test. Functional membrane integrity (FMI) was assessed via the hypo-osmotic swelling test (HOST). Genealogies and consanguinity analysis was conducted using the Breeders Assistant for Horses program. An average of 3.6 ± 0.4 % was reported for the inbreeding coefficient (Ft). A decrease in sperm motility and kinematics was reported, which was associated with an increase in consanguinity (P < 0.05). Furthermore, differences in consanguinity were found based on gait. Similarly, a relationship between horse gait and semen quality (P < 0.05) was found. Authors concluded that semen quality of Colombian Creole horses has been affected by inbreeding and its relationship with genetic selection based on gait.

Semen cryopreservation in Bornean Sun Bear (Helarctos malayanus euryspilus)

Semen cryopreservation is one common tool for extended genome storage and serves as insurance for declining biodiversity. In the endangered Bornean sun bear, semen was collected with electroejaculation and this is the first attempt at semen cryopreservation for this subspecies. Chilled semen with motility of more than 50 % was extended with Caniplus freezing medium, and cryopreserved with slow freezing protocol. A total of 22 cryopreserved semen straws were evaluated with computer-assisted sperm analysis and conventional semen evaluation tests as well as additional functional tests including evaluation of the acrosome (Rose Bengal Fast Green stain), plasma membrane (hypoosmotic swelling test), and chromatin (toluidine blue stain). Post-thaw semen quality was compromised with poor viability (27.6 ± 11.2 %), motility (8.3 ± 7.1 %), and progressive movement (1.3 ± 2.0 %) but maintained high integrity for the acrosome (71.1 ± 10.8 %), plasma membrane (54.7 ± 9.6 %), and chromatin (89.7 ± 7.6 %). The total post-thaw sperm abnormality was 51.7 ± 10.7 %, predominantly bent tail (28.0 ± 7.6 %) and proximal droplets (20.0 ± 11.8 %). Although this current cryopreservation was not a success, the compromised frozen-thawed semen may be a valuable resource in assisted reproductive technologies such as intracytoplasmic sperm injection. The three additional functional tests demonstrated were simple and affordable and hence are recommended to be part of the routine semen evaluation. Further research is required to develop species-specific cryopreservation protocols and to explore other assisted reproductive technologies in the Bornean sun bear.

Development of assays for the characterization of sperm motility parameters, viability, and membrane integrity in the epididymis and vas deferens of the greater rhea (Rhea americana)

Development of assays for the characterization of sperm motility parameters, viability, and membrane integrity in the epididymis and vas deferens of the greater rhea (Rhea americana)

Effects of extender filtration and egg yolk concentration on canine semen cryopreservation.

The semen cooling and freezing extenders commonly contain the chicken egg yolk (EY) as the main sperm cryoprotectant. Besides its advantages, the EY has large lipoprotein granules that cause several physical and biological interferences. The previous studies have proposed several methods to resolve the problems with the EY-based semen extenders, including mechanical agitation, EY fractionation, replacing the EY with purified EY LDL, and ultrasonication. In the current research, we aimed to evaluate the syringe filtration (220 nm) of an EY-based canine semen freezing extender as a simple and cheap method to remove the EY granules. We also studied the possibility of re-aggregation of EY granules after cooling, freeze/thawing, and lyophilization/rehydration of the filtered extenders. Additionally, we compared the effects of the filtration on lipid profile, turbidity, EY particle size distribution, and osmolality of the EY-based extenders. Next, we examined the effects of filtered extenders containing different levels of EY (5%, 10%, 15%, 20%, and 25%) versus the control extender (20% EY, unfiltered) on post-thaw sperm quality traits. We collected the semen samples from seven clinically healthy mixed-breed adult dogs and pooled them for sperm freezing procedures. Samplings were repeated at least five times, independently. Our results indicated that the syringe filtration could remove the large EY particles and reduce the extender turbidity without affecting the lipid profile of the whole extender solution. The filtered extender supplemented with 25% (v/v) EY led to the best post-thaw canine spermatozoa quality markers. The frozen-thawed spermatozoa evaluations included motility parameters (computer-assisted semen analysis system), membrane and acrosome integrity (hypo-osmotic swelling test, chlortetracycline binding assay), DNA fragmentation (sperm chromatin dispersion assay), membrane lipid peroxidation (MDA levels), apoptosis (Annexin V/propidium iodide assay), and fertility-associated sperm mRNA transcript abundance (protamine 2 and 3). In conclusion, the syringe filtration of the EY-based semen extenders was a simple and cheap method that could effectively remove large EY lipoprotein granules and possibly prevent EY-origin bacterial contamination of the final extender solution. The EY at 25% (v/v) concentration in the filtered extenders resulted in the highest canine spermatozoa cryo-tolerance.

Linseed oil in boar's diet improved in vivo fertility and antioxidant status.

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography-mass spectrometry (GC-MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.

Comparative Effects of Deoxynivalenol, Zearalenone and Its Modified Forms De-Epoxy-Deoxynivalenol and Hydrolyzed Zearalenone on Boar Semen In Vitro

Deoxynivalenol (DON) and zearalenone (ZEN) are described as detrimental factors to sow and boar fertility. In comparison, literature reports on the impact of modified forms of DON and ZEN, such as de-epoxy-DON (DOM-1) and hydrolyzed ZEN (HZEN), on swine reproduction are scarce. The aim of our study was to compare the effects of DON, DOM-1, ZEN and HZEN on boar semen in vitro. To this end, pooled boar semen ejaculates from two adult boars were treated with either 50.6 μM DON, 62.8 μM ZEN or equimolar concentrations of DOM-1 and HZEN, respectively (dilution volume of v/v 0.7% DMSO in all cases). Effects on semen motility, morphology, viability, hypo-osmotic swelling test reaction and DNA integrity were investigated hourly up to four hours of incubation. DON negatively affected particular parameters evaluated with a computer-assisted sperm analysis system (CASA), such as immotile spermatozoa and progressive motile spermatozoa, whereas those effects were absent in the case of DOM-1 treatment. In contrast to HZEN, ZEN affected almost all CASA parameters. Furthermore, only ZEN decreased the proportion of viable spermatozoa and increased the proportion of spermatozoa with abnormalities. In conclusion, DON and ZEN negatively affected boar semen in vitro, whereas equimolar concentrations of DOM-1 and HZEN did not induce harmful effects.

Sodium dodecyl sulphate, N-octyl β-D glucopyranoside and 4-methoxy phenyl β-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls

Sodium Dodecyl Sulphate (SDS), N-Octyl β-D Glucopyranoside (NOG), 4-Methoxy Phenyl β-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −40 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); ii) Moderate P25 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −50 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); and iii) Rapid P26 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −60 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

Effect of Myo-inositol on sperm quality and biochemical factors in cryopreserved semen of patients with Asthenospermia.

In this study, the influence of myoinositol (MYO) as an antioxidant on the inhibition of the negative impacts of cryopreservation on sperm quality in men with Asthenospermia was investigated. In this prospective study, each semen sample from 25 cases was separated into three groups: Fresh, Control (with freezing medium), Myoinositol (2 mg/ml). According to the World Health Organization criteria (WHO) (2010), total motility, progressive sperm motility, viability, normal morphology, and DNA integrity were assessed. In addition, the hypo-osmotic swelling (HOS) test and mitochondrial membrane potential (MMP) were used. Total antioxidant capacity (TAC), malondialdehyde (MDA), and antioxidant enzyme activity were determined by the ELISA method. In contrast to the fresh samples, lipid peroxidation, DNA integrity damage, DNA fragmentation, HOST, and MMP had significant enhancement in the control samples. Sperm quality was significantly decreased (p < 0.05). Mean percentage viability, normal morphology, total motility, progressive motility, and DNA integrity were significantly enhanced in the MYO group in comparison to the control group (p < 0.05). The MDA and TAC levels and DNA damage in the MYO group were significantly lower compared to the control group (p < 0.05). The findings confirm that sperm quality in patients with Asthenospermia is improved by the administration of 2mg/ml of myoinositol together with the freezing medium after sperm cryopreservation.

Photoperiod and Melatonin Supplementation: Variable Effects on the Quality of Chilled Dog Semen.

The addition of melatonin in seminal extenders due to its antioxidant properties and its beneficial role in sperm preservation has been previously described, especially in seasonal species. The aim of this study was to study a potential seasonal effect based on photoperiod duration when adding a physiological concentration of melatonin in the canine ejaculate. A total of 24 ejaculates were obtained from 10 healthy dogs during the increasing photoperiod (from December 21 to June 21), whereas 12 ejaculates were collected from five healthy individuals during the decreasing photoperiod (from June 22 to December 20). Each ejaculate was separated into two aliquots, and one of them remained as a control, whereas melatonin (100 pM) was added to the other one (C and M treatment groups, respectively). Diluted semen was refrigerated at 5°C. On days 0, 1, 2, 3, and 6, sperm motility analyses were performed using a CASA system and hypoosmotic swelling test (HOST), osmotic resistance test (ORT), and flow cytometry analysis. No effect of melatonin on motility was detected in either photoperiod. Negative effects of melatonin were found for acrosomal defects, apoptosis, and viability in the decreasing photoperiod. The addition of melatonin to sperm in the decreasing photoperiod could create such a high level that it would cause the described negative effects. We found a beneficial effect of melatonin in the increasing photoperiod on acrosomal defects and apoptosis during 0–6 days. Melatonin treatment also increased viability in the short term (days 1 and 2) for both photoperiods. Also, melatonin can provide certain beneficial effects on mitochondrial activity in the medium term (days 2 and 3) in the decreasing photoperiod.

The Correlation Between Plasma Membrane Integrity and Sperm Parameters for the Arrabi Rams Semen

The study was executed to investigate the different cooling periods (0, 24, and 48 hours) effects on plasma membrane integrity depending on the hypo osmotic swelling test (HOST), spermatozoa motility, acrosome integrity, live sperm, and spermatozoa abnormalities, and correlation among the Host and other rams sperm parameters This study was done at an animal farm in Al-Kut city (southern Baghdad city\\ Iraq), from 5th January to 5th February 2021 The semen was collected from four mature Arrabi rams (2-4 years old) by using an artificial vagina to the evaluation of semen characteristics The results exhibited a significant (p≤005) effect of the cooling times of the rams semen parameters, on the other hand, there is a highly significant (P≤001) correlation (positive) among host, acrosome integrity, live sperm, and spermatozoa motility (r = 100, 099, 095) respectively However, the results explained the significant (P≤005) correlation (negative) between host and spermatozoa abnormalities (r = -100) In conclusion, there are a relatively significant effect of the cooling periods and Arrabi ram semen parameters and there is a significant correlation between host and other sperm characteristics during different times of cooling

Effect of Adding Different Vitamin D3 Concentrations on Chilled Storage of Awassi Ram Semen

Prolong semen quality following the chilled process is important for assistant reproductive techniques such as artificial insemination. The objective of the current study is to evaluate the effect of adding different concentrations of vitamin D3 to the Tris-egg yolk extender on some semen parameters stored at 5ºC. Three Awassi rams were used in this study. Semen samples were collected pooled and diluted with an egg-yolk extender (1:10). Four Aliquots were divided into three treatments with vitamin D3 (T1= 0.02, T2=0.004, T3= 0.002 gm) and one control (T0). All samples were chilled to 5ºC. Samples were estimated at time h0 and after 24, 48,72, and 96 hours following treatment. Individual motility, dead sperm, abnormal sperm, and hypo-osmotic swelling test (HOST) was used in this study. The individual motility significantly decreased in T1 compared to T0, T2, and T3 in all studded hours. Also, the individual motility in T3 was significantly greater at h48, h72, and h96, compared to T0, T1. The dead sperm was significantly greater in T1 on h0 compared to T0, T2, and T3. The HOST test was significantly greater in T0, T2, and T3 on h0 compared to T1. In conclusion, low vitamin D3 supplementation to the Awassi semen might potentiate the individual motility for up to 72 hours and trend to enhance some semen parameters.

P-093 A comparison of the effects of IMSI and HOST for sperm selection on morphokinetic parameters and ploidy status in sibling embryos

Abstract Study question Are hypo-osmotic swelling test (HOST) and intracytoplasmic morphologically selected sperm injection (IMSI) both effective alternative methods for sperm selection? Summary answer Similar results were obtained in embryo morphokinetics between HOST and IMSI. What is known already HOST is a simple, low-cost and rapid test based on the swelling of the tail of the sperm in a hypoosmotic environment. Less DNA fragmentation, membrane integrity and aneuploidy rates have been found in sperm selected by the HOST method. It also allows identification of viable sperm in a totally immotile sperm sample. IMSI, on the other hand, is a method for identifying the morphologically best sperm using high-magnification microscope. In studies, IMSI has been associated with high fertilization rates. However, it is a time-consuming method that requires expensive equipment. Study design, size, duration Time-Lapse Monitoring (TLM)-incubated sibling embryos of 25 male factor patients who applied for ART treatment to Istanbul, Sisli Memorial ART and Genetics Center in 2021 were included into the study. Sibling embryos were grouped according to their sperm selection method. In the IMSI group 170 embryos, in the HOST group 173 embryos were evaluated retrospectively in terms of the morphokinetic parameters, the rates of blastulation, Top- Quality (TQ) and Good Quality (GQ) embryos and euploidy. Participants/materials, setting, methods Blastocyst grading was performed according to Gardner’s classification. Blastocysts 3AA,4AA,4AB,4BA,4BB,5AA,5AB,5BA,5BB were accepted as TQ+GQ. Blastulation rates was determined including the embryos at least 2 expansion. PGT-A results were obtained by Next Generation Sequencing(NGS) method. The comparison of the TLM parameters and clinical features of the patients were performed using Student’s t-test. Chi-square test was used for comparing the rates of fertilization, blastulation, TQ+GQ embryo and euploidy in each group. Main results and the role of chance No statistical difference was observed between IMSI and HOST groups in terms of morphokinetic parameters (tPN, tPNf, t2, t3, t4, t5, t6, t7, t8, t9, tSC, tM, tSB, tB, tEB) (p &amp;gt; 0.05). There were higher rates of normal fertilization in the HOST group (90.8%) than in the IMSI group (82.4%) and of TQ+GQ embryo (HOST: 50%, IMSI: 47.3%). However, these diffrences were not statistically significant (p &amp;gt; 0.05). There was statistically lower abnormal fertilization rate was found in HOST group (2.3% vs 7.1%, p = 0.038). When PGT-A results were compared (HOST:19 embryos, IMSI: 19), slightly higher results were observed in IMSI group than HOST group ( 63%, 58% respectively) (p &amp;gt; 0.05). Limitations, reasons for caution This was a retrospective of a small number of cases. Wider implications of the findings The similarity of the outcome in these initial findings studying a small number of cases, suggest that this study should be extended. Trial registration number None

Study on cryopreservation of Guanzhong dairy goat semen with bovine semen seminal plasma

There are phospholipase and yolk agglutinase in the seminal plasma of dairy goat. The existence of these enzymes will hydrolyze lecithin in yolk into lysophosphatides and cause the change of pH value of diluent, resulting in toxic and side effects on sperm. In order to explore the effect of bovine seminal plasma replacing different doses of dairy goat seminal plasma (0%, 25%, 50%, 100%) on semen freezing quality of Guanzhong dairy goat, we used computer-aided sperm analysis (CASA) system, specific fluorescent probe detection technology, hypo osmotic swelling test (HOST), oxidation index determination and in vivo fertilization verification test to study the semen cryopreservation of Guanzhong dairy goat. The results showed that if we used bovine seminal plasma instead of 100% dairy goat seminal plasma, the sperm motility, plasma membrane functionality and acrosome integrity rate were the highest after thawing, reaching 67.04%, 38.12% and 68.83% respectively, which was significantly higher than that of other groups (P < 0.05); In terms of oxidation indexes, the values of SOD and GSH-PX in sperm of dairy goats were 199.78 U/mL and 133.55 U/L, respectively, which were significantly higher than those in other groups (P < 0.05), and the values of ROS and MDA were 397.58 U/mL and 5.16 nmol/L, respectively, which were significantly lower than those in other groups (P < 0.05); The results of artificial insemination also showed that using bovine seminal plasma instead of 100% of dairy goat seminal plasma could significantly improve the fertilization ability of sperm. The quality of frozen Guanzhong dairy goat sperm can be improved by using bovine seminal plasma instead of dairy goat seminal plasma and using bovine diluent.

Linseed oil in boar’s diet during high temperature humidity index (THI) period improves sperm quality characteristics, antioxidant status and fatty acid composition of sperm under hot humid sub-tropical climate

Environmental heat stress in sub-tropical climates negatively impacts boar semen production and its quality. The present study aimed to examine the heat stress alleviating effects of dietary linseed oil on semen quality and antioxidant status of boar, in the summer and winter seasons in sub-tropical climate. Six Hampshire crossbreed boars were fed with 90 mL linseed oil (treatment) whereas six boars of the same breed were fed 90 mL vegetable oil (control) for sixteen weeks during both season. Sperm quality was assessed for motility, viability, abnormality, acrosomal integrity, and Hypo-osmotic swelling test (HOST). Sperm velocity attributes were assessed by computer-assisted semen analysis (CASA). Antioxidants (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC and nitric oxide; NO) and lipid peroxidation (malondialdehyde; MDA) were measured in seminal plasma and serum. Gas chromatography-mass spectrometry (GC-MS) was used for the estimation of fatty acid composition of seminal plasma and spermatozoa. Feeding linseed oil to the boars significantly (p < 0.05) improved sperm quality at the fresh stage and after 72 h of liquid storage in both season. There was a significant (p < 0.01) effect of treatment and season on semen quality parameters. Significant boar (p < 0.05) effect was recorded on reaction time, semen volume, sperm abnormality, acrosomal integrity and HOST reactive sperm. There was a significant (p < 0.01) effect of treatment and season on the velocity attributes viz. VAP, VSL, VCL, ALH, BCF and STR%. Linseed oil supplementation significantly (p < 0.01) enhanced antioxidant and lowered MDA levels in serum as well as seminal plasma. The concentration of alpha-linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids were significantly (p < 0.01) increased in seminal plasma and sperm after linseed oil supplementation. In conclusion, linseed oil supplementation to boar during high THI months improved the semen quality parameters viz. semen volume, sperm concentration, and progressive motile sperm, along with enhanced antioxidant capacity.

Effects of post-freezing addition of relaxin on the fertility parameters of equine semen

The cryopreservation of equine semen, although advantageous, has critical points: the spermatozoa undergo structural and physico-chemical changes which cause alterations of flagellum, plasma membrane and cellular organelles, compromising the fertilizing potential (Freitas et al. J Equine Vet Sci. 2016; 46: 1–6). The aim of the work was to evaluate whether the post-thawing addition of relaxin, a pleiotropic hormone, had positive effects on motility, velocity, viability, membrane and DNA integrity, mitochondrial activity and apoptosis rate of the equine semen. The data were compared with a previous work, in which relaxin had been added before cryopreservation to the same semen batches (Elkhawagah et al. Anim Reprod Sci. 2020; 106351). The semen of 3 stallions of proven fertility was collected, pooled, and frozen using standard procedures, with relaxin supplementation (0, 12.5, 25, 50, 100 ng/ml). In this work, relaxin was added after thawing to the row semen at the same concentrations. Immediately after the addition of relaxin and after 15’, 30′, 60′, 90′ and 120′ of incubation (37°C, CO2 5%), sperm motility and velocity were evaluated with CASA system, viability was assessed through Hancok test and membrane integrity with Hypoosmotic Swelling test. DNA integrity, mitochondrial activity and apoptosis rate were assessed by acridine orange staining, JC-1 and annexin V-PI (flow cytometricanalyses). Viability, mitochondrial activity, DNA integrity and percentage of apoptotic spermatozoa were not significantly improved after post-thawing addition of relaxin, but it seemed to exert a positive effect on the mitochondrial membrane potential and on the prevention of apoptosis, in accordance with Ferlin et al. (J Androl. 2012; 33: 474–482). The percentage of spermatozoa with intact membrane significantly increased in the samples with the post-thawing addition of 12.5, 25 and 50 ng/ml relaxin, but not in those supplemented with relaxin before freezing. The post-thawing addition of 50 ng/ml relaxin implemented and maintained sperm motility and velocity over time, particularly after 90 min of incubation, and the same results were obtained after 30 minutes of pre-freeze relaxin supplementation at 12.5 ng/ml. It was concluded that one protocol was no better than the other, but the pre-freeze addition of relaxin is more suitable for classical AI, while the post-freeze supplementation is better for deep AI, endoscopic AI or ICSI, as it also seems to activate capacitation. In the future, establishing the metabolic pathways through which relaxin acts on the male gamete will be essential to fully exploit the potential of a hormone that, only recently, has been found to be extremely important for male fertility.

Evaluation of sperm membrane functionality during epididymal transit in red-rumped agouti (Dasyprocta leporina).

We studied the sperm membrane functionality through the epididymal transit by comparing different hypoosmotic solutions and verifying possible associations among osmotic response and functional parameters of sperm in red-rumped agouti (Dasyprocta leporina). For this purpose, epididymal sperm from six sexually mature male agoutis were collected via flotation. Then, analyses of sperm parameters and hypoosmotic swelling test using different hypoosmotic solutions (0, 50 and 200mOsm/L) in different regions of the epididymis (caput, corpus and cauda) were performed. There was an increase (p<.05) in the values for sperm concentration, the total number of sperm recovered, total and progressive motility, average path velocity, straight-line velocity, curvilinear velocity, and rapid and medium subpopulations following the caput-corpus-cauda direction. Regardless of the hypoosmotic solution, the agouti sperm membrane presented similar functional integrity in all the epididymal regions. Moreover, the highest (p<.05) osmotic responses were reached with the use of 50mOsm/L solution in comparison to 0 and 200mOsm/L for all the regions. Significant correlations among osmotic response and some sperm kinetic parameters were observed, especially in epididymal caput, while no correlations were found in the region of the cauda. In summary, red-rumped agouti sperm present similar membrane functionality during epididymal transit, but there are evident correlations among such functionality and sperm kinetic parameters, especially in the caput region. Moreover, we indicate the use of a 50mOsm/L hypoosmotic solution for the analysis of this parameter through the hypoosmotic swelling test.

Hypo-Osmotic Swelling Test (HOST) for Feline Spermatozoa: The Simplified Procedure and the Aspect of Sperm Morphology

Simple SummaryOne of the methods used to assess semen quality is the hypo-osmotic swelling test (HOST). Different protocols for this test are available, including a very simple one: swelling of spermatozoa is induced by incubation in distilled water for 5 min. Such a protocol could have easily been introduced in clinical practice, but it has not yet been evaluated for feline sperm cells. The objectives of this study were: (1) to check if a simplified HOST procedure can be applied for feline semen and (2) to check if sperm abnormalities may influence the results of this test. The results proved that the simplified HOST gives the same results as the standard one and that the presence of live, abnormal spermatozoa does not interfere with the test results.Hypo-osmotic swelling test (HOST) is used to assess the functional integrity of sperm plasma membranes in many species. The primary aim of this study was to test a simplified HOST procedure for the evaluation of feline semen. The second objective was to check if sperm abnormalities can influence the results of this test. Urethral semen was collected from 19 male, domestic cats. In Exp. 1, HOST was performed in different media (50 mOsm/kg fructose or distilled water), temperature (37 °C or room temperature) and time (5 and 30 min). In Exp. 2, the potential effect of sperm abnormalities on HOST results was assessed by observing individual normal and abnormal spermatozoa microinjected into droplets of distilled water. The results showed no differences between the HOST results performed in different media, temperature and time. Viable abnormal spermatozoa were able to swell under hypo-osmotic conditions in the same manner as normal ones, except spermatozoa with distal droplets, which showed a higher frequency of ‘despiralization’. In conclusion, HOST can be reliably performed at 0 mOsm/kg for 5 min at room temperature, which may contribute to a wider use of this test under clinical environments. Viable abnormal spermatozoa are able to swell under hypo-osmotic conditions; therefore, their presence in the ejaculate would not bias the results of HOST when total coiling is calculated.

Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo (Bubalus bubalis) bulls

Graphene oxide (GO) greatly suppresses the growth and recrystallization by curving the hexagonal shape of ice crystals. Study was conducted to evaluate effect of GO as cryoprotectant in semen extender for augmenting sperm viability in dairy (cattle and buffalo) animals. In experiment one, semen was extended with TRIS Egg Yolk Glycerol (TEYG) extender supplemented with different concentrations of GO: 0.0125, 0.25, 0.5, 0.1 and 0.2 mg ml−1. Freezing of semen samples was conducted at 30 °C min−1 from temperature drop from 4 °C to −15 °C and −15 °C to – 60 °C followed by 50 °C min−1 from – 60 °C to −140 °C, and the semen straws were plunged in liquid nitrogen. Second experiment evaluated the performance of TEYG extender supplemented with combinations of GO (G05 as 0.05 and G10 as 0.1 mg ml−1) and glycerol (T48 as 4.8 and T64 as 6.4%) in four groups as G05T48, G05T64, G10T48 and G10T64. Freezing rates of 30 °C min-1[Protocol (PRT) I], 40 °C min−1 (PRT II) and 50 °C min−1 (PRT III) in the critical temperature fall zone of −15 °C to −60 °C were evaluated for semen extender supplemented with glycerol 6.4% and GO 0.05 mg ml−1 in the third experiment. Cattle (n = 3) and buffalo (n = 3) bulls were chosen for the study taking six ejaculates per bull per treatment. Post-thaw sperm motility, membrane integrity, viability and abnormalities were observed by means of CASA, Hypo-osmotic swelling test (HOST), Eosin-Nigrosin stain and Rose Bengal stain procedures, respectively. Post-thaw total motility (TM), progressive motility (PM), VCL, VSL, VAP, HOST response and viability increased significantly in extender with GO concentrations of 0.1 and 0.05 mg ml−1 as compared to control. Per cent abnormalities were significantly (p < .05) lower in group with GO 0.025 and 0.0125 mg ml−1 as compared to control. Results from the second experiment showed higher post-thaw TM, PM, VCL, VAP, VSL, HOST response, viability increased significantly (p < .05) in G05T64 and G05T48 as compared to G10T64. Sperm abnormalities did not vary among the groups as compared to control for cattle spermatozoa. In the third experiment post-thaw TM, PM, VCL, VSL, VAP, HOS response and sperm viability increased significantly (p < .05) in PRT III as compared to PRT I for buffalo and cattle spermatozoa. Sperm abnormalities were significantly (p < .05) lower in PRT II and PRT III as compared to PRT I for buffalo, whereas, lower in PRT II as compared to PRTI for cattle spermatozoa. GO as cryoprotectant when added to semen extender at the rate of 0.05 and 0.1 mg ml−1, resulted in better plasma membrane function and viability. Glycerol concentration below 6.4% in buffalo semen extender reduced post-thaw quality of sperm even when GO was added to the extender. Higher freezing rate of 50 °C min−1 in the critical temperature fall zone of −15 to −60 °C perform better than the freezing rate of 30 °C min−1. It is concluded that TEYG extender having glycerol 6.4% and GO 0.05 mg ml−1 improved post-thaw semen quality of cattle and buffalo.

Effect of Ascorbic Acid and Proline Amino Acid Supplementations on Cryosurvival and Fertility Rates of Cryopreserved Honey Bee (Apis mellifera) Semen.

This study investigated the effect of ascorbic acid (vitamin C) and proline amino acid alone or together on the quality and fertility of frozen/thawed honey bee spermatozoa. The experiments were designed to compare a single ascorbic acid, a single proline amino acid, and different combinations of ascorbic acid with proline amino acid on the cryopreservation of honey bee semen based on sperm motility, viability, intact membrane (hypo-osmotic swelling test), and fertility rates. Eight cryopreserved study groups comprised Control II with no supplement, along with groups with ascorbic acid (2 mg), proline 25 mM, proline 50 mM, proline 100 mM, and combination groups of both ascorbic acid (2 mg) and proline 25 mM, proline 50 mM, and lastly proline 100 mM groups, respectively. Using 50 mM proline in the tested groups had the greatest impact on sperm motility, viability, the percentage of spermatozoa with intact membrane, and fertility. The cryopreservation process caused a gradual decrease in motility, viability, intact membrane (p < 0.05), and fertility rates (p < 0.01) in all the tested research groups as against the fresh semen control group. Successful honey bee sperm cryopreservation and fertility are achievable when using an appropriate sperm freezing protocol and antioxidant. Proline amino acid as an antioxidant in semen extender had a more beneficial influence on sperm quality parameters and fertility. The success of cryopreservation with antioxidants is related to the chosen antioxidant in a dose-dependent manner.

Protective Effects of Trehalose and Pentoxifylline on Goat Sperm Exposed to Chilling-Freezing Process.

Freezing of sperm is known as an important part of assisted reproduction. However, many studies have illustrated that cryopreservation negatively affects the quality and fertility rate of sperm. This study aimed to evaluate the effects of trehalose and pentoxifylline (PTX) in diluents on cooled and frozen-thawed Markhoz goat sperm. Preassessed samples were pooled and diluted with a basic diluent using trehalose and PTX. The cooled sperm showed significant improvement. The motion characteristics of cryopreserved sperm were evaluated based on computer-assisted system analysis. In this study, we investigated the viability, membrane integrity, malondialdehyde concentration, total abnormality, acrosome integrity, and seminal hyaluronidase enzyme. Also, the hypo-osmotic swelling test, mitochondrial activity, apoptotic features, caspase activity, chromatin dispersion test, active mitochondria, and reactive oxygen species (ROS) activity were assessed as complementary parameters. The data illustrate that the total motility, progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and the ratio of sperm chromatin dispersion, viable sperm were improved significantly (p < 0.05) using 3 mM PTX alone or 3 mM PTX plus 50 mM trehalose, while other characteristics indicate significant enhancement by 3 and 6 mM PTX and 50 and 70 mM trehalose alone or in combination, except amplitude of lateral head displacement (ALH), beat/cross frequency (BCF), and intracellular ROS-(O-), which demonstrate no significant difference among treatments. In conclusion, this study indicates that addition of 3 and 6 mM PTX alone or with 50 and 70 mM trehalose seems to reduce the damage caused by cooling and cryopreservation processes.