Total Motility Of Spermatozoa Research Articles

Biogenic and risk elements in wild boar testes and relation to spermatozoa motility

Environmental pollution results in serious health hazards to animals, reflected in biogenic and risk element concentrations in animal tissues. The objective of this study was to examinate concentration of selected elements in testes, and epididymal spermatozoa motility of wild boars. Wild boars were hunted in region Žuhračka - Levice, Slovak Republic. Testes were removed postmortem, spermatozoa were collected from cauda epididymis and assessed by CASA system. Elements concentrations were measured by ICP and by CV-AAS. Spermatozoa motility was 44.29% and progressive motility 18.47%. Concentration of elements in testes was in following order: K > Na > Mg > Ca > Fe > Zn > Al > Cu > Se > Mn > As > Cr > Pb > Mo > Sr > Ni > Ba > Cd > Li > Hg. The most notable correlations indicate association between Se and total spermatozoa motility, as well as with progressive motility, furthermore between As and velocity curved line and beat cross frequency. A high positive significant correlation was found between mercury and beat cross frequency. The data may serve as a fine control indicator to detect potentially toxic elements accumulated from polluted environment that can affect reproduction of wild animals.

Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen.

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0,50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.

Antioxidant effects of zinc-oxide nanoparticles on post-thaw quality and in vivo fertility of Beetal buck spermatozoa

Metallic nanoparticles are generally considered potential cryoprotectants during semen cryopreservation owing to their ability to improve the spermatozoa quality following cryopreservation when added in cryo-diluents. The present study aimed at investigating the effects of incorporating zinc oxide nanoparticles (ZnO-NPs) in the cryo-diluent on in vitro post-thaw sperm quality attributes, oxidant-antioxidant profile, and in vivo fertility rate in Beetal buck. After characterization, ZnO-NPs were added to the Tris-citric acid-fructose yolk extender. Semen samples, collected from five breeding bucks (5 ejaculates per buck) using an artificial vagina, were pooled. The pooled semen samples, distributed into five equal aliquots, were diluted in the Tris-citric acid cryo-diluent containing five different concentrations of ZnO-NPs (0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, and 0.0 mg/ml called control group), and were cryopreserved for 24 h. At post-thaw, various in vitro sperm quality parameters, and antioxidant enzyme profiles like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and reduced glutathione (GSH) levels were determined. For the in vivo fertility experiment, breeding does (n = 152), aging from 1.5 to 4 years, were artificially inseminated with spermatozoa with intact acrosome containing the same five concentrations of ZnO-NPs. Results demonstrated that the 1.0 mg/ml ZnO-NPs (crystallite size of 19.73 nm) improved (p < 0.01) the activities of antioxidant enzymes (SOD, CAT, and POD) with concomitantly decreased levels (p < 0.001) of lipid peroxidation (LPO) and reactive oxygen species (ROS) when compared with the other groups. Total spermatozoa motility, rapid velocity, and average path velocity were higher (p < 0.05) with the 1.0 mg/ml ZnO-NPs when compared with the control group. Progressive motility and straight-line velocity were higher (p < 0.05) with the 0.5, 1.0, and 1.5 mg/ml of ZnO-NPs addition compared with the 2.0 mg/ml and control groups. The sperm kinematics parameters; the amplitude of the lateral head displacement, the beat cross frequency, the straightness, and the linearity were not influenced by the ZnO-NPs inclusion. Supra-vital plasma membrane integrity was higher (p < 0.001) with the 0.5 mg/ml and 1.0 mg/ml of ZnO-NPs added groups when compared with either 2.0 mg/ml ZnO-NPs or the control group. The viable sperm with intact acrosome was higher (p < 0.05) in the 1.0 mg/ml ZnO-NPs group compared with the other groups. Low supplementation rates improved (p < 0.05) the DNA integrity when compared with the higher dose (2.0 mg/ml ZnO-NPs). The pregnancy per AI was higher (59.3 %; P < 0.05) in the does inseminated with semen added with the 0.5 mg/ml ZnO-NPs followed by 1.0 mg/ml (58.8 %), 1.5 mg/ml (58.6 %), and 2.0 mg/ml (46.6 %) when compared with the control group (33.3 %). We concluded that the 1.0 mg/ml ZnO-NPs added in the citric acid-fructose yolk extender improved the post-thaw quality of Beetal buck spermatozoa in terms of elevated activities of endogenous antioxidant enzymes, improved structural and functional integrity along with higher fertility.

Evaluation of semen parameters from Fleckvieh-Simmental bulls and the influence of age and season of collection.

The aim of this paper was to evaluate semen parameters from Czech Fleckvieh (dual-purpose breed) bulls used in artificial insemination in the Fleckvieh population. The ejaculate was collected from bulls only once a week, which is not usual. Respectively, the effects of age and season of collection on semen parameters were tested. The average volume of ejaculate by Fleckvieh bulls was 8.72 mL, which is higher than results in studies of bulls which were collected usually more than once a week. The average total motility was 72.82 % and progressive motility was 67.99 %. Sperm concentration reached on average . The total motility of spermatozoa after thawing was 40.88 %. A significant influence of age was observed on ejaculate volume, total and progressive motility, and total motility after thawing ( ). The season of collection had a significant influence on the volume of ejaculate, total motility, progressive motility, concentration of spermatozoa, and total count of spermatozoa ( ).

Influence of Sperm Number and Antioxidant Melatonin in Extender on the Quality of Post-Thawing Sheep Spermatozoa

This study aimed to examine the effect of spermatozoa concentration and the effectiveness of melatonin supplementation in diluent on the quality of post-thawing semen. Ejaculated semen was collected using the artificial vaginal method (MVB). The study was carried out in two stages, firstly semen was frozen in andromed diluent with different concentrations in one straw (50, 100, 200 million per straw), and the second was frozen semen in diluent supplemented with melatonin with different doses (0, 0,5, 1, 0 and 1.5 mM melatonin). Parameters observed were the movement of spermatozoa using Computer Assisted Sperm Analysis, membrane integrity, and acrosome integrity. Data were analyzed using ANOVA and further tested using Duncan's test. The results showed no significant difference in the quality and movement pattern of sheep semen when frozen at concentrations of 50, 100, or 200 million per straw (P 0.05). Furthermore, adding melatonin to the diluent in this study affected spermatozoa's total motility and progressive motility at a concentration of 1.0 mM (P0.05) but did not significantly affect the percentage of spermatozoa motility pattern characteristics. From the results, it can be concluded that the concentration of per straw spermatozoa does not affect the quality of sheep semen, and melatonin supplementation in diluent can reduce the effects of the frozen-thawed process on motility, acrosome cap, and plasma membrane integrity in sheep semen. Melatonin supplementation with a concentration of 1 mM in the extender was the highest quality concentration in this study.

COMBINED TREATMENT OF PATHOSOSPERMIA WITH ADJUVANT THERAPY WITH SPEROTON COM- PLEX IN PATIENTS WITH VARICOCELE

OBJECTIVE&#x0D; To evaluate the effectiveness of combined treatment of pathozoospermia in patients with varicocele by performing micro- surgical phlebectomy of varicose veins of the spermatic cord in combination with adjuvant therapy with the Speroton complex&#x0D; MATERIAL AND METHODS&#x0D; Twenty microsurgical phlebectomies of varicose veins of the spermatic cord were performed in patients with pathozoo- spermia (oligoasthenospermia) against the background of varicocele with the appointment of the Speroton complex in the postoperative period for 3 months with an assessment of spermogram parameters before and after the operation in comparison with a group of 20 men with pathozocelemia without the use of adjuvant therapy. The study of morphological and functional parameters of spermogram of men of reproductive age (18-49 years) without signs of immune infertility (MAR-test IgG &lt;10%) before and after the start of treatment was carried out. The average age of men was 33 ± 2.3 years (M ± m).&#x0D; RESULTS&#x0D; All patients with infertility in combination with varicocele had oligoasthenozoospermia before treatment. Three months after the start of treatment in the study groups, there was a statistically significant improvement in sperm counts due to an increase in the concentration of spermatozoa in 1 ml of ejaculate, an increase in the number of progressively mobile forms and total motility of spermatozoa by 50-68%, an increase in the number of live sperm by 19-20% and a decrease in the num- ber of dead forms of spermatozoa by 30-35%. A statistically significant decrease in the DNA fragmentation index by 34-52% (p &lt;0.05) was found in all study groups. It was found that combination therapy, including microsurgical phlebectomy of the spermatic cord veins using the Speroton complex for 3 months, statistically significantly led to an increase in the concentration of spermatozoa in 1 ml of ejaculate (38.6 [38.2; 69.4]), an increase in progressively mobile forms (49.2 [26.7; 47.2]%) and general motility (72.2 [35.3; 64.7]%) of spermatozoa and viable forms (67.8 [39.3; 81.7] %), a decrease in the DNA fragmenta- tion index compared with the spermogram indicators of patients who underwent only microsurgical phlebectomy (p &lt;0.05 for all study indicators). In the group of patients after combined treatment with the inclusion of adjuvant therapy of the Speroton complex, there was a statistically significant decrease in the sperm DNA fragmentation index to almost normal values (12.1 ± 1.1%) compared with the group of patients who underwent only surgery (17.2 ± 0.8%) (p = 0.01). Combined treatment (micro- surgical phlebectomy of the veins of the spermatic cord in combination with adjuvant therapy Speroton) of pathozoospermia in men of reproductive age with infertility on the background of varicocele can significantly improve the morphofunctional parameters of spermogram after 3 months from the start of treatment compared to men who underwent only surgical correction of varicocele.

Effect of the oral intake of astaxanthin on semen parameters in patients with oligo-astheno-teratozoospermia: a randomized double-blind placebo-controlled trial.

BackgroundHigher concentrations of seminal reactive oxygen species may be related to male infertility. Astaxanthin with high antioxidant activity can have an impact on the prevention and treatment of various health conditions, including cancer. However, efficacy studies on astaxanthin in patients with oligospermia with/without astheno- or teratozoospermia (O±A±T) have not yet been reported. Our aim was to evaluate the effect of the oral intake of astaxanthin on semen parameters.Patients and methodsIn a randomized double-blind trial, 80 men with O±A±T were allocated to intervention with 16 mg astaxanthin orally daily or placebo. At baseline and after three months basic semen parameters, sperm deoxyribonucleic acid (DNA) fragmentation and mitochondrial membrane potential (MMP) of spermatozoa and serum follicle-stimulating hormone (FSH) value were measured.ResultsAnalysis of the results of 72 patients completing the study (37 in the study group, 35 in the placebo group) did not show any statistically significant change, in the astaxanthin group no improvements in the total number of spermatozoa, concentration of spermatozoa, total motility of spermatozoa, morphology of spermatozoa, DNA fragmentation and mitochondrial membrane potential of spermatozoa or serum FSH were determined. In the placebo group, statistically significant changes in the total number and concentration of spermatozoa were determined.ConclusionsThe oral intake of astaxanthin did not affect any semen parameters in patients with O±A±T.

Ethanolic extract of Dutch eggplants (Solanum betaceum) protects spermatozoa motility exposed to lead acetate

Lead is a toxic material that can have negative effects on reproductive organs. Lead exposure reduces the activity of endogenous antioxidant enzymes and increases the number of oxidants in the body. High free radicals will affect spermatogenesis and result in decreased motile spermatozoa. Antioxidants are known to protect the motility of spermatozoa, and adequate antioxidants can be found in Dutch eggplants (Solanumbetaceum). This study aimed to evaluate the effect of S. betaceum on spermatozoa motility after exposure to lead acetate. This study is a true experimental design with a randomized post-test-only control group design. Forty male Balb/C mice 12 weeks old were randomly divided into 5 groups: two control groups (C0, C1) and three treatment groups (T1, T2, T3).The C0 received distilled water, and the C1 received 75 mg/kg BW lead acetate. The T1, T2 and T3 received 100, 200 and 400 mg/kg BW of S. betaceum, respectively, an hour before exposed lead acetate. The data were analyzed using one-way ANOVA with a significant level of p &lt;0.05. A significantly increasein the mean total motility of spermatozoa in T1, T2, and T3 was reported.This study indicates that S. betaceum have a protective effect on spermatozoa motility when exposed to lead acetate.

Effect of alpha-lipoic acid on oxidative stress, viability and motility in the common carp (Cyprinus carpio) spermatozoa after short-term storage and cryopreservation

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.

165 Comparison of spermatozoa traits and testosterone levels, seminal plasma constituents, and lipid peroxidation of Large White×Landrace and Kolbroek boars

Reproductive traits in domestic pigs are one of the most important economic characteristics for improved pig production. Seminal plasma (SP) constituents provide a better indicator of male fertility as measured by semen traits. The objective of this study was to compare spermatozoa traits, testosterone concentration, SP constituents, and lipid peroxidation in Large White×Landrace (LW×LR) and Kolbroek boars aged between 8 and 9 months. Kolbroek (n=12) and LW×LR (n=12) boars were used in this study. Semen was collected using the gloved-hand technique. Blood samples were collected from each boar monthly to determine testosterone concentrations. Spermatozoa motility traits was evaluated using computer-aided sperm analysis. Spermatozoa viability was evaluated using the hyperosmotic swelling test, lipid peroxidation was evaluated using the malondialdehyde (MDA) test, and SP protein was evaluated using atomic absorption spectrophotometric analysis. Analysis of variance was used to test the difference between the breeds. The LW×LR boars had higher ejaculation volume and testosterone concentrations (P&amp;lt;0.05) than Kolbroek boars. The average spermatozoa concentration for Kolbroek and LW×LR boars was 1.8±1.0 and 1.9±1.1×109mL−1, respectively (P&amp;gt;0.05). The average genotype spermatozoa total motility was 91.7±4.0 and 90.6±4.1%, respectively (P&amp;gt;0.05). No significant differences were found between genotype in abnormal spermatozoa morphology, rapid spermatozoa, and total spermatozoa motility, spermatozoa velocity on the curve line, and hyperosmotic swelling test. Spermatozoa with distal, head, and midpiece abnormalities were significantly different in Kolbroek boars (17.1±1.4, 5.0±2.0, and 7.0±2.6%, respectively) and LW×LR boars (9.2±0.1, 3.5±1.1, and 2.6±1.2%, respectively). The LW×LR boars had lower levels of MDA than Kolbroek boars (P&amp;lt;0.05). The SP constituents with selenium, sodium, magnesium, and calcium were significantly different in Kolbroek boars (166.6±1.0, 167.2±0.2, 5.0±0.8, and 10.2±0.3%, respectively) and LW×LR boars (155.0±0.0, 140.0±0.5, 12.0±0.2, and 22.3±0.0%, respectively). In conclusion, spermatozoa parameters of Kolbroek boars were similar to those of LW×LR boars except for semen volume, head and distal spermatozoa abnormalities, testosterone concentration, MDA, and seminal plasma. Further studies need to be conducted on the major seminal plasma constituents as well as their physiological roles.

140 The relationship between morphometric characteristics and semen parameters in Bapedi rams

Bapedi is a fat-tailed, hardy, and disease-tolerant breed of sheep that is indigenous to South Africa. The Bapedi sheep breed is facing genetic degradation due to nonselective cross-breeding and irregular mating; therefore, breeding strategies for urgent conservation of Bapedi sheep are crucial. The aim of the present study was to investigate the relationship between body measurements and semen traits of Bapedi rams conserved in situ (conserved in their natural habitat) and ex situ invivo (live animals conserved outside their natural habitat). Semen was collected twice per week from 33 rams aged 2-4 years (four ejaculates per ram) using an electroejaculator. Body measurements (rump height, body length, scrotum circumference (SC), scrotal width, and scrotum length (cm)), body condition score (BCS; scale of 1-5), bodyweight (BW; kg), rectal temperature (°C), semen volume (mL), spermatozoa concentration (billionsmL−1), and motility parameters were measured using a computer-aided semen analysis system. Data were analysed using analysis of variance and the general linear models of SAS (SAS Institute Inc.). The Pearson correlation coefficient in SAS was used to determine the relationship among variables. There was uniformity in all body measurements of Bapedi sheep regardless of the conservation method (P&amp;gt;0.05). There were no significant differences in body temperature during semen collection, SC, semen volume, semen pH, semen concentration, spermatozoa total motility, and kinematics in Bapedi rams between methods of conservation (P&amp;gt;0.05). Pearson correlation revealed moderate significant relationships between semen volume and BW, BCS, and SC (r=0.315, 0.638, and 0.381, respectively). Rump length had a moderate correlation (0.566) with normal spermatozoa (P&amp;lt;0.05). It was concluded that Bapedi sheep are still a uniform breed regardless of their decreasing numbers. The BW, BCS, and SC must be considered in the selection criteria for improving the reproductive performance of Bapedi breeding rams because they were positively correlated with semen volume. It is recommended that more studies be done to correlate body measurements, testicular morphometry, and semen parameters in this breed.

Time and Dose-Dependent Effects of Viscum Album Quercus on Rabbit Spermatozoa Motility and Viability in Vitro.

The target of this study was to evaluate the effect of extract of the European mistletoe - Viscum album quercus L. on spermatozoa motility and viability in vitro. The CASA system was used to determine the spermatozoa motility parameters at different time intervals (0, 1, 2 and 3 h) and spermatozoa viability was determined in five different doses of Viscum album quercus L [10 (QA), 6.6 (QB), 3.3 (QC), 2.5 (QD) and 2 (QE) mg/ml]. Results in experimental groups detected a significant deterioration on rabbit spermatozoa after 1, 2 and 3 hours, compared to the control. The initial total spermatozoa motility showed increased value for all doses of Viscum album quercus in comparison to control. After in vitro culture a dose-dependent decrease (QA: reduction of 69.7 %, QB: reduction of 40.9 %) was found. For the progressive spermatozoa most significant decrease (86.8 % for QA vs. 48.5 % for QB) was detected compared to the control after 3 hours of culture. Spermatozoa viability (MTT test) was decreased in all experiment groups at the end of experiment, but the differences were not significant. Significant alterations of membrane integrity were found in groups with the highest Viscum album quercus concentration (QA, QB), but acrosome integrity showed no significant changes. Results suggest negative dose- and time-dependent effect of Viscum album quercus at higher doses on spermatozoa motility and viability parameters in vitro.

Trace elements content in semen and their interactions with sperm quality and RedOx status in freshwater fish Cyprinus carpio: A correlation study

Objective of the present study was to investigate interactions between trace elements content and RedOx status, as well as sperm quality parameters (motility features, DNA fragmentation) in fish spermatozoa in natural conditions. Reproductively mature male freshwater fish (n = 16) of Cyprinus carpio breed were used in the study. Trace elements content was determined in fish milt samples by inductively-coupled plasma optical emission spectrometry (ICP-OES) and by cold-vapor atomic absorption spectroscopy (CV-AAS). Sperm quality evaluation was realized by computer-assisted sperm analysis (CASA) quantifying several parameters: concentration, total motility, progressive motility, distance average path, distance curved line, distance straight line, velocity average path, velocity curved line, velocity straight line, straightness, linearity, amplitude of lateral head displacement and beat cross frequency. The general scheme of descending concentrations of trace metals in semen samples was following: Zn > Fe > Cu > As > Sr > Ni > Mn > Se > Pb > Cr > Cd > Hg. Total motility of spermatozoa was relatively high (91.45%), however progressive motility was not even half of this value (39.47%). Sperm DNA fragmentation values were relatively low (4.00–6.29%). The percentage of immotile spermatozoa showed a significant correlation with all RedOx status parameters and also with DNA fragmentation. Positive statistically significant correlations were observed between trace elements (Mn, Se, Sr, and Zn) and some qualitative spermatozoa parameters (velocity and distance parameters). Cu and Hg content shows similar negative associations with progressive motility. Hg also interacted with production of malondialdehyde. Overall, the present study suggests application of multi-component mixtures of environmentally related trace elements concentrations when assessing the potential reproductive risk.

21 Comparative Study on Epididymal Spermatozoa Traits of Large White×Landrace and Kolbroek Boars

The Kolbroek pig is an early maturing breed (maturing at 4 to 5 months) that grows more slowly than modern pig breeds. There is general concern that the genetic variation within Kolbroek pig breed is becoming extinct. The aim of the study was to compare semen quality of epididymal spermatozoa derived from slaughtered Large White × Landrace and Kolbroek boars aged between 7 and 9 months. Kolbroek (n = 10) and Large White × Landrace (n = 10) boars were used in this study. Semen was collected from the head of epididymis of Kolbroek and Large White × Landrace testicles. Spermatozoa samples were extracted from head of epididymis by making incision with a razor. Semen samples were then evaluated for macroscopic (semen volume, pH, and concentration) and microscopic characteristics (spermatozoa motility and morphology). Spermatozoa motility was evaluated using computer-aided sperm analysis. Analysis of variance was used to test the difference between the breeds. The average percentage of Kolbroek and Large White × Landrace spermatozoa total motility was 92.4 ± 4.0 and 94.0 ± 4.1%, respectively (P &gt; 0.05). The spermatozoa velocity on the curve line for Kolbroek was lower (147.2 ± 39.2) compared with the Large White × Landrace (178.3 ± 30.1 μm/s; P &gt; 0.05). However, no significant difference was observed between the 2 breeds for rapid spermatozoa and total spermatozoa motility. No significant differences were observed in Kolbroek and Large White × Landrace boar semen volume (8 and 9 mL, respectively) or semen pH (7.0). The average spermatozoa concentration for Kolbroek and Large White × Landrace was 2.5 ± 1.2 and 1.1 ± 1.0 (×109 mL−1), respectively (P &gt; 0.05). There were no significant differences between Kolbroek and Large White × Landrace in abnormal spermatozoa morphology. However, spermatozoa with distal, head, and midpiece abnormalities were significantly different in Kolbroek (14.0 ± 3.6; 3.6 ± 6.0 and 3.4 ± 3.7%) and Large White × Landrace breed (5.4 ± 4.7; 4.7 ± 1.0 and 0.4 ± 1.0%), respectively. In conclusion, macroscopic and microscopic epididymal spermatozoa characteristics of Kolbroek were similar to those of Large White × Landrace boars, except for head and distal spermatozoa abnormalities.

KUALITAS SPERMATOZOA SWIM UP KAMBING PERANAKAN ETAWAH HASIL PEMBEKUAN MENGGUNAKAN METODE VITRIFIKASI DENGAN PERSENTASE GLISEROL YANG BERBEDA

The objective of this research was to examine the effect of glycerol addition in different percentage to quality of swim up spermatozoa from Etawah Grade buck that frozen with vitrification method. This research was carried out at Frozen Semen Laboratory, Sumbersekar, Faculty of Animal Husbandry, Brawijaya University from February 2016 to March 2016. This research design was experimental laboratory using completely randomized design and the data were analyzed using Analysis of Variance (ANOVA) one way pattern, followed by the multiple test Duncan. There were four treatments in this research (P0= Tris-Yolk + 0% Glycerol, P1= Tris-Yolk + 7% Glycerol, P2= Tris-Yolk + 14% Glycerol, P3= Tris – Yolk + 21% Glycerol). Each treatment consisted of ten replications. The results showed that addition of glycerol was significantly different (P&lt;0,01) on the percentage of post thawing motility, viability, and total spermatozoa motility. It was concluded that addition of glycerol 7% gave the best result on spermatozoa quality. Keywords : swim up, glycerol, frozen semen, vitrification, post thawing motility

The effect of diluent, temperature and age on turkey spermatozoa motility in vitro

The aim of this study was to evaluate the turkey spermatozoa motility in in vitro conditions and to prove the effect of different conditions of incubation – diluents, temperature and age of birds. Spermatozoa were obtained from adult turkey's line of Big 6, and spermatozoa motility parameters were evaluated using a computer-assisted semen analyzer (CASA) system. Significant decrease of spermatozoa motility at laboratory temperature (22°C) was detected from time 0 (94.15%) till 180 minutes of incubation (53.91%). At the cool media incubation (5°C), this difference was lower (95.41 and 78.86%, respectively), and the differences were significant from 30 minutes of incubation till 180 minutes. Progressive spermatozoa motility replicated the tendency of total spermatozoa motility. When the physiological solution to commercial diluent at 5°C was compared, the spermatozoa motility and progressive motility in both groups were very consistent for 90 minutes of incubation. Subsequently, significantly higher spermatozoa motility was detected at time periods 120, 150 and 180 minutes of incubation in commercial diluent. Motility was also higher in this group after 24 hours. Influence of age on spermatozoa motility parameters was analysed at 22°C at the time 0 and after 30 minutes of incubation. Analysis of spermatozoa motility as well as progressive spermatozoa motility proved higher values in Group A (aged 35–42 weeks) compared to Group B (aged 63–73 weeks). These results clearly suggest that low temperature and commercial diluents maintain motility parameters during longer time periods and the increasing age of birds has negative impact on motility parameters.

Pentoxifylline added to freezing or post-thaw extenders does not improve the survival or in vitro fertilising capacity of boar spermatozoa

This study evaluated whether pentoxifylline added to freezing and thawing extenders influenced the function of boar spermatozoa. In Experiment 1, pooled ejaculated sperm-rich fractions were frozen in 0.5 ml straws after dilution in extender supplemented with pentoxifylline to a final concentration of 0, 2, 4, 8, 16 or 32 mM. The addition of 4, 8, 16 and 32 mM pentoxifylline to the freezing extender significantly decreased the progressive and total motility of spermatozoa. The percentage of viable spermatozoa with intact acrosomes as well as the penetration rate and the efficiency of fertilisation were significantly lower in pentoxifylline-treated groups compared with the untreated control. In Experiment 2, a pool of three straws with 'good' post-thaw sperm quality parameters and another three straws with 'poor' sperm quality were diluted in extender with 0, 1, 2, 4, 8, 16 or 32 mM pentoxifylline. Post-thaw samples with both 'good' and 'poor' sperm quality with 0, 2, 4, 8 and 16 mM were used to assess IVF parameters. The addition of pentoxifylline to post-thaw extender did not improve the post-thaw motility or viability of spermatozoa compared with the control. The in vitro penetration was higher (P<0.05) than the control for oocytes fertilised with spermatozoa that were thawed and incubated in extender with 4, 8 and 16 mM pentoxifylline. However, no differences were observed in the efficiency of fertilisation. We conclude that pentoxifylline, as a supplement added to the freezing extender, has a deleterious effect and that it does not improve the survival or in vitro fertilising efficiency of frozen-thawed boar spermatozoa when added after thawing.

Treatment of asthenozoospermia with HCG.

30 patients with isolated asthenozoospermia received a treatment with 5000 I.U. Human-Chorionic-Gonadotropin (HCG) a week intramuscularly for twelve weeks. Pretreatment basal levels of FSH, LH and testosterone were in normal or low normal range. 16 males responded to therapy, the total motility of spermatozoa increased significantly from 34% to 40%. In spite of a decreased sperm density and morphologic quality 6 pregnancies occurred. This result strengthens the interpretation, that sperm motility is the most important factor for fertilization. HCG therapy must be considered as an additional approach for treating motility disturbances, especially in cases with high sperm densities.

Influence of IGF-I on buffalo ( Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro

The objective of the present experiment was to examine the influence of mean physiological concentration of insulin-like growth factor-I (IGF-I) on frozen–thawed Surti buffalo ( Bubalus bubalis) spermatozoa functional parameters, i.e., motility, plasmalemma integrity, acrosomal integrity, functional membrane integrity, lipid peroxidation and fructose uptake in vitro. Frozen–thawed semen samples ( n = 6) were washed in tris buffer and divided into two equal parts (control and IGF-I groups). Only in the IGF-I group, IGF-I (rhIGF-I analogue) was added to a final concentration of 100 ng/ml. The samples were incubated at 37 °C for 2 h and the assessments were made at 0, 30, 60, 90 and 120 min of incubation. The mean concentration of the buffalo seminal plasma ( n = 17) IGF-I was 116.83 ± 28.34 ng/ml (range 41.4–198.95). IGF-I had significant effect on the total motility ( P < 0.01), progressive forward motility ( P < 0.01), functional membrane integrity ( P < 0.05) and lipid peroxidation levels ( P < 0.05) during the 120-min study period as assessed by area under curve. Treatment with IGF-I increased ( P < 0.01) the total spermatozoa motility at 30, 60 and 90 min as compared to the control. The progressive forward motility was significantly ( P < 0.01) higher at 60 and 90 min of incubation. The addition of IGF-I resulted in significant ( P < 0.01) increase in straight-line velocity (VSL, μm/s) as compared to the control at 60 and 90 min of incubation. The linearity (%) was significantly ( P < 0.01) higher in IGF-I treated semen as compared to control at 60 min of incubation. Plasmalemma integrity in IGF-I group was significantly ( P < 0.05) higher than control at 30 and 60 min of incubation. The functional membrane integrity differed significantly ( P < 0.01) between groups (control and IGF-I) at 60 and 90 min of incubation. The percentage of acrosomal intact spermatozoa decreased continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant ( P < 0.05) loss of acrosome was observed at 60 and 90 min of incubation in control (63.87 ± 3.17 vs. 58.52 ± 2.54) and IGF-I (61.60 ± 2.26 vs. 56.11 ± 2.12) groups, respectively. Lipid peroxidation levels were significantly lower in IGF-I group at 90 min ( P < 0.05) and 120 min ( P < 0.01) of incubation than the control group. Fructose utilization was significantly higher in IGF-I group as compared to control at 30 min ( P < 0.05) and 60 min ( P < 0.01) of incubation. The present study suggests that addition of IGF-I improve spermatozoa functional parameters by reducing lipid peroxidation levels.

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.