Viability Of Semen Research Articles

Effect of different concentration of fish oil in skim milk-egg yolk extenders on post- thawed semen qualities of Kalang swamp buffalo bull

Objective: To explore the effect of fish oil at different concentrations on post-thawed semen of Kalang swamp buffalo. Methods: A total of 4 Kalang swamp buffalo bulls with 3-5 years of age and weighed about 340-360 kg were slected. Semen was regularly collected from these buffalo bulls once a week by an artificial vagina. Fish oil was supplementary at the dosages of 0 mg (control), 50 mg, 100 mg, 150 mg, and 200 mg to the extender (skim milk-egg yolk). Fresh, pre-freezing and frozen semen were thawed at 37 °C and evaluated for motility, viability, morphology, and plasma integrity of membrane. Results: The study results indicated that before freezing, supplementation of fish oil at the dose of 150 mg in the extender had significantly motility. And a significant (P<0.05) increase was observed in viability and motility of post-thawed semen at the dose of 150 mg fish oil, which was in difference with other treatment groups. Conclusions: Addition of 150 mg fish oil in the extender could be positive for the enhancement of the quality of post-thawed semen of Kalang swamp buffaloes.

Viability of Pony Stallion Semen in Different Temperature and Dilution

Background: Artificial insemination and transport of cooled semen has been routinely used in equine industry in the past 20 years. However, more investigations are needed regarding the methods for long time storage in pony stallion semen. The effect of dilution and cooling temperature on pH, sperm motility, membrane integrity and mitochondrial activity were investigated before and after cooling of stallion semen.Materials, Methods &amp; Results: Two ejaculates each from nine Brazilian ponies were diluted in a nonbuffered powder milk extender cooled at 5°C or 15°C for 48 h using three different dilutions (1:1, 1:2 or 1:3). Data were assessed by analysis of variance and the rate comparison was performed using the Duncan test. Samples diluted 1:1 at 5oC or 15°C showed higher pH values (7.63 ± 0.34 e 7.57 ± 0.27) and lower progressive motility (10.3 ± 11.05, 17.08 ± 9.95). All samples cooled at 15°C also showed lower incidence of morphologically altered spermatozoa (1:1 = 55.84%; 1:2 = 51.84%; 1:3 = 49.95%) [P &lt; 0.01]. Mitochondrial activity was higher on the 1:3 dilution (0.86 ± 0.19 nm) at 5°C and on the 1:1 (0.89 ± 0.23 nm), 1:2 (0.93 ± 0.2 nm) and 1:3 (0.92 ± 0.2 nm) dilutions at 15°C. Progressive motility was higher when semen was diluted 1:3 and cooled at 15°C (42.22 ± 12.38; P &lt; 0.05). Considering mitochondrial activity, similar results were observed when different dilutions of semen were used (P &gt; 0.05) despite time and temperature. The pH, progressive motility, mitochondrial activity and membrane integrity remained similar (P &gt; 0.05) on fresh semen samples independent of the dilution grade used. The best results were obtained when semen was diluted 1:3 and cooled at 15°C. All dilution grades were safe for fresh semen and pH wasincreased when semen was diluted and cooled for 48 h.Discussion: The methodology used to collect and process equine semen and semen from ponies is practically the same. Equine semen when sent for artificial insemination is usually cooled to 5°C. Our results showed that cooling reduces sperm viability, which has also been demonstrated by other studies. In contrast, the best cooling temperature was at 15°C. However, it is easier to keep the temperature at 5°C during transport, due to the large temperature oscillation that may occur during transportation. The semen of ponies can tolerate cooling at both 5 and 15°C. The 1:3 dilution cooled to 15°C provided better viability of pony sperm, and more stable pH during 48 h of cooling. Dilution 1:1 should not be used for cooling in powdered skim milk extender.

17 Relationship Between Semen Parameters and Morphometric Characteristics of Zulu Rams

Zulu sheep is an adapted breed of South Africa with desirable traits such as their ability to thrive in a harsh environment and tolerant to various diseases. However, they are endangered, with their population size not exceeding 1000, and hence are associated with high inbreeding rates. Therefore, to limit biodiversity loss, there is a need to characterise and conserve its genetic materials. The aim of the study was to evaluate the relationship between morphometric characteristics and semen parameters of Zulu rams and conserve these genetics. The study was conducted at the Agricultural Research Council. Rams (n = 6, age = 3 years and average weight = 42 kg) were fed a standard diet and water was provided ad libitum. Semen was collected twice a week, using an artificial vagina, over 4 consecutive weeks. Semen volume, spermatozoa concentration, motility parameters, and viability were evaluated immediately after arrival in the laboratory. The experiment was carried out during breeding season (March to May 2017). Body measurements (rump height, body length, scrotum circumference, body weight, scrotal width, and scrotum length) were measured in the morning following the fasting period (night) using flexible measuring tape. Pearson correlation coefficient in Minitab 17 (Minitab Inc., State College, PA, USA) was used to determine the relationship among variables. A moderate significant correlation (P &lt; 0.001) between body weight and %live spermatozoa (r = 0.46) was observed. Of note, a very weak (r = 0.28) significant correlation (P &lt; 0.05) between scrotum circumference and live spermatozoa was observed. A very weak (r = 0.06) nonsignificant correlation (P &gt; 0.05) was found between scrotum circumference and semen volume. These results indicate that body weight and scrotum circumference provide reliable spermatozoa viability estimates. Positive correlation between scrotum circumference and semen volume shows that scrotum circumference can be used to predict semen volume.

22 Comparing Three Extenders: Hashi, Green Buffer and INRA 96, for Chilled Storage of Bactrian Camel Semen

Semen preservation remains challenging in the camel industry. The objective of the present study was to compare 3 different extenders for chilled storage of Bactrian camel semen. Semen (n = 9 ejaculates) was collected from camel bulls (n = 2) manually using artificial vagina. Good neat semen, as far as mass vibration concerns, was equally distributed into 3 double-wall vessels filled with 35°C water. The 3 extenders used in the present study were Hashi, Green buffer (IMV, L’Aigle, France), and INRA 96 (IMV). The Hashi extender consisted of Tris, 2.6%; citric acid, 1.35%; glucose, 0.9%; fructose, 0.9%; penicillin G sodium, 1000 IU mL−1; streptomycin sulfate, 1000 mg mL−1 supplemented with 20% plasma egg yolk and 20% camel skim milk; osmolality of 330 and pH of 6.9). Green buffer was supplemented with 20% plasma egg yolk (osmolality of 335 and pH of 6.9). The osmolality and pH of INRA 96 were 310 and 7, respectively. Extenders at a ratio of 1:3 were added to semen followed by pipetting 10 times with an automatic pipettor. The water-jacketed extended specimen was covered with foam and transferred to individual vaccine carrier equipped with 4 ice packs. This system of cooling not only allows the specimen to cool down slowly and reach 4°C after 7 h, but also reduces the viscosity of camel semen. The assessment was carried out 7 and 24 h after semen dilution, when the specimen reached 4°C. Semen viability parameters were assessed after short-term semen preservation in different extenders. Total motility and progressive forward motility were examined subjectively by single operator using Sperm Track (ISAS, Proiser, Spain) after diluting the specimen to achieve 25 × 106 sperm mL−1. Live percentage of sperm was estimated using Eosin B Fast Green staining method. Plasma membrane integrity was assessed using the hypo-osmotic swelling (HOS) test. Following arcsin transformation, data were analysed by GLM procedure followed by Tukey test in SAS (SAS Institute Inc., Cary, NC, USA). At 7 and 24 h, there were no differences among the 3 extenders in total motility of sperm (Hashi: 73 and 67.4%; Green buffer: 71.6 and 62.1%; INRA 96: 70 and 66.2%; P &gt; 0.05), live percentage of sperm (Hashi: 76 and 73%; Green buffer: 70.5 and 65.6%; INRA 96: 77.8 and 70.7%; P &gt; 0.05), or HOS test (Hashi: 52.4 and 45.2%; Green buffer: 49.6 and 40.6%; INRA 96: 57.3 and 51.1%; P &gt; 0.05). However, at the same times, progressive forward motility was similar between Hashi (47.7 and 28.6%) and Green buffer (40 and 23.5%; P &gt; 0.05) but was different between Hashi and INRA 96 (23.6 and 16.7%; P &lt; 0.05). In conclusion, Hashi and Green buffer could be considered suitable extenders to preserve Bactrian camel semen under chilled condition. Further studies with a larger number of bulls and ejaculates are warranted.

Factors affecting storage of Slovak native rabbit semen in the gene bank.

In this study, fresh and frozen-thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen-thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen-thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen-thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen-thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

Association of novel SNPs in gonadotropin genes with sperm quality traits of Boer goats and Boer crosses

ABSTRACTThe present study aimed to investigate single-nucleotide polymorphisms (SNPs) of caprine FSHβ and caprine LHβ genes and to determine effects of the SNPs on fresh and post-thaw semen quality traits of Boer and Boer-crossbred bucks. The single-strand conformation polymorphism analysis and comparative sequencing revealed three SNPs in exon 3 of caprine follicle-stimulating hormone beta (FSHβ), including, three SNPs; 200A > G (FSHβ3-a), 226T > C (FSHβ3-b), and 237A > G (FSHβ3-c), while two of them (FSHβ3-a and FSHβ3-b) were novel. Furthermore, one SNP in exon 2 of luteinizing hormone beta (LHβ) (207T > C) (LHβ2) was detected. The associations of the four detected SNPs with quality traits of fresh and post-thaw semen were evaluated. Analyses of variance revealed significant association of the candidate genes with libido and semen quality traits. The three SNPs of FSHβ3 had significant effect on libido, progressive motility, and abnormality of fresh semen (P < .05), and on the motility, velocity, and viability traits of post-thaw semen (P < .05). LHβ2 polymorphism only showed association with sperm viability of post-thaw semen. The results of the present study suggest that the SNPs in caprine FSHβ and LHβ are associated with semen quality of male goats.

BSA 또는 Fatty Acid Free BSA 첨가가 닭 희석 정자와 동결 정자의 생존성에 미치는 영향

본 연구에서는 닭 정액의 운동성 분석에 미치는 요인을 분석하기 위하여 희석배율에 따른 생존성을 조사하고, 신선정액의 희석 시에 필요한 조건을 확보하기 위하여 희석제에 단백질원으로서 BSA와 FAF-BSA의 첨가가 닭정자의 생존성에 미치는 영향을 관찰하였다. 최종 희석율이 1/4이 되도록 닭 정액을 희석하여 생존성을 조사하면 신선정액에서 희석율이 1/8, 1/16 및 1/32로 낮아질수록 89.9%, 69.9% 및 53.2%로 생존율이 낮아지는 것을 관찰하였으며, 단백질원으로 BSA나 FAF-BSA를 이용하면 생존율을 유의적으로 증진시킬 수 있음을 증명하였다. 또한 이러한 현상은 동결정액의 희석에도 중요한 요인으로 관찰되었는데, FAF-BSA를 이용하여 희석할 때 단백질이 없는 경우보다 양호한 생존율이 나타난다는 것을 관찰하였다(17.6% 대 34.0%). 그러나 단백질원을 활용하더라도 닭정액의 희석율이 낮아질수록 생존율이 저하하는 현상은 지속적으로 나타나고 있으며, 신선정액에 비하여 동결정액은 장수성이 낮은 것으로 추정되었다. 그러므로 본 연구에서 사용된 정자의 분석방법은 닭 정액동결을 실시하여 운동성을 분석할 때 필요한 기본 정보를 제공할 수 있을 것으로 판단되며, 닭 희석배율을 낮추어 동결정액의 효율성을 높이는 연구에 기여할 것으로 추정된다. This study examined factors affecting the analysis of motility of chicken semen. The viability of spermatozoa was estimated using varying dilution ratios and supplementation with BSA or fatty acid free (FAF)-BSA as protein sources in semen diluent. Fresh semen was examined after preparing dilutions in beltsvile poultry semen extender (BPSE) of 1/8, 1/16 and 1/32 at <TEX>$25^{\circ}C$</TEX>. The motility of incubated semen at each dilution was observed at 3 min (89.9%, 69.9% and 53.2%), 30 min (86.7%, 71.4% and 51.7%), 1 h (89.5%, 74.0% and 53.5%) and 3 h (78.5%, 66.5% and 45.7%), respectively. The addition of BSA or FAF-BSA to BPSE diluent significantly increased the viability of semen in 1/32 dilution with results of 53.2% (control), 84.8% (BSA) and 92.9% (FAF-BSA) (p<0.05). This phenomenon was also observed in the dilution of frozen semen, where FAF-BSA treatment increased the viability of thawed semen from 17.6% to 34.0% in a 1/8 dilution (p<0.05). When the protein sources were used in the dilution, the survival rates of diluted chicken semen were also increased with time lapse. These results show that FAF-BSA may act to protect chicken semen and is suitable as a basic component of chicken semen diluent for the method of analyzing rooster semen after freezing.

Viability of bull semen extended with commercial semen extender and two culture media stored at 24 °C

The aim of this study was to evaluate the viability of bull spermatozoa diluted with commercial semen extender and two culture media stored at controlled room temperature (24 °C) for 72 hours. Two Nguni bulls were used for semen collection with the aid of an electro-ejaculator. After macroscopic evaluation, semen was pooled and aliquoted randomly into Triladyl, modified Ham’s F10, and TCM-199 culture media, and then stored at 24 °C. Sperm motility parameters, morphology, and viability were analysed with computer aided sperm analysis (CASA) after 0, 24, 48 and 72 hours. The study was replicated four times, and data were analysed using analysis of variance (ANOVA). Triladyl had significantly higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 hours than modified Ham’s F10 (86.8%; 26.5%) and TCM-199 (76.7%; 25.0%) culture media. Ham’s F10 had higher progressive motility rate (37.8%) than the other extenders TCM-199 (31.7%) and Triladyl (23.4%). There was no significant difference in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0 %) after 72 hours’ storage at 24 °C. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails, between the two Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media, stored at 24 °C, and stay viable for 72 hours. Keywords : Ham’s F10, TCM-199, Triladyl, sperm motility, viability

Effects of Green Tea Extract on Sperm Quality, Reactive Oxygen Species and Lipid Peroxidation in Long-term Liquid Preservation of Boar Spermatozoa

During storage, boar spermatozoa undergo several changes including diminished motility and viability and accumulated reactive oxygen species (ROS). In this study, we investigated the effects of green tea extract (GTE) supplementation in the Sui Dil extender on the sperm motility, viability, ROS and lipid peroxidation (LPO) of longterm preserved boar semen at 17℃. A total number of eight boars were used for this experiment. Pooled ejaculates were diluted to 20 × 10 6 sperm/ml in the Sui Dil extender containing 0 (control), 1, 10, 100 or 500 mg/l GTE and were preserved at 17℃ for 24, 72, 120 and 168 h, respectively. At each storage time, sperm motility and viability were estimated by microscopic examination and the fluorescent double stain Fertilight ® , respectively. Sperm ROS level and LPO were assessed using the 2’, 7’-dichlorodihydrofluorescein diacetate (H₂DCFDA)/propidium iodide (PI) and C11-BODIPY581/591/PI with flow cytometry, respectively. Compared to that of the 500 mg group, there were higher sperm motility and viability in the 1, 10 and 100 mg GTE groups during the preservation from 24 to 168 h (p < 0.05). The ROS levels of the 10 and 100 mg groups during the 168 h preservation were lower than those of the 0, 1 and 500 mg groups (p < 0.05). There were no significant differences in LPO regardless of the preservation period or the GTE concentration. In conclusion, the optimal concentrations (10 and 100 mg/l) of GTE that led to lower ROS levels may be useful for liquid boar sperm preservation at 17℃ for a period of 168 h.

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.

163 EVALUATION OF VIABILITY OF BULL SEMEN COLLECTED BY ELECTRO-EJACULATION USING COMMERCIAL SEMEN EXTENDER AND 2 CULTURE MEDIA AT CONTROLLED ROOM TEMPERATURE

Preservation of semen is an important process to ensure that semen quality is sufficient for assisted reproductive technologies. The aim of this study was to evaluate the viability of bull semen collected by electro-ejaculation using commercial semen extender and 2 modified culture media stored at controlled RT (24°C) for 72 h. Two Nguni bulls were used for semen collection; after collection, the semen was evaluated macroscopically for volume, pH, and colour, and microscopically for sperm motility, viability, and morphology. Uncontaminated semen samples with progressive motility &gt;70% and morphological defects &lt;20% were pooled after collection before being aliquoted into 3 extenders, namely Triladyl, modified Ham’s F10, and TCM-199 culture media, at a dilution ratio of 1:4 and then stored at controlled RT (24°C). Sperm motility rate was analysed using the computer-aided sperm analyser after 0, 24, 48, and 72 h of storage. Sperm morphology and viability was performed after staining the sperm cells with spermac and nigrosine-eosin stain, respectively. The study was replicated 4 times and data were analysed using ANOVA. Triladyl had a higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 h (P &lt; 0.01) compared with the 2 modified culture media, Ham’s F10 (26.5 and 86.8%) and TCM-199 (25.0 and 86.7%), respectively. However, Ham’s F10 had higher progressive motility rate (37.8%) as compared with the other extenders, TCM-199 (31.7%) and Triladyl (23.4). There was no significant difference (P &gt; 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P &gt; 0.05) in straight line velocity was observed for the three extenders. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails (P &gt; 0.05), between the 2 Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media stored at 24°C and stay viable for 72 h. Triladyl proved to be the best suitable extender of the 3 extenders, showing higher sperm viability and total motility rate as compared with Ham’s F10 and TCM-199 modified culture media.

56 THE VIABILITY AND LONGEVITY OF OGYE CHICKEN FROZEN SEMEN WITH MITOTEMPOL, MITOCHONDRIA-SPECIFIC ANTIOXIDANT

Semen from Korean Native Black roosters (Ogye) was cryopreserved with 8% N-Methylacetamide (MA) in HS-1 diluent, of the following composition (per 100 mL): glucose 0.2 g, trehalose dihydrate 3.8 g, l-glutamic acid monosodium salt 1.2 g, potassium acetate 0.3 g, magnesium acetate tetrahydrate 0.08 g, potassium citrate monohydrate 0.05 g, BES 0.4 g, Bis-Tris 0.4 g, and gentamicin sulfate 0.001 g. Ogye semen was collected 2 times a week by dorsal-abdominal massage and cooled into ice slurry. During dilution, semen was diluted by 2 steps. First, HS-1 diluent that was supplemented with respective concentration of MitoTEMPOL, mitochondria-specific antioxidant was added into fresh semen with same volume. Second, after waiting for 10 min, 16% MA containing diluent was added into first diluted semen so the final concentrations of MA were adjusted to 8%. The concentration of the MitoTEMPOL of first diluent was adjusted to 0.1, 1, 5, and 10 μM. After freezing for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, the semen was thawed 5°C for 2 min 1 to 3 weeks later. The viability and longevity of thawed Ogye semen were analysed by sperm movement methods. Briefly, 5 μL of thawed semen was placed onto a Makler chamber and the movement of spermatozoa was recorded for 10 s by digital camera and saved as movie files on the computer. With digital rewinding for 1 s of saved movies, the non-motile and motile sperm was counted using a manual counter. The resulting numbers of respective spermatozoa were analysed by Student t-test. The 0.1 and 1 μM treated semen showed significant increases in viability compared with control, 5 μM and 10 μM MitoTempol (77.3 and 84.2% v. 65.4, 53.2, and 21.5%; P &lt; 0.05). The longevity of frozen/thawed Ogye semen for 3 h was also higher in 0.1 and 1 μM treated groups than control, 5 μM, and 10 μM (67.5 and 54.2% v. 36, 5.2, and 1.2%; P &lt; 0.05). With these results, utilisation of mitochondria-specific antioxidant for freezing of Ogye spermatozoa could increase the viability and longevity of frozen-thawed semen. However, treatment with concentrations &gt;1 μM showed negative effects on freezing of Ogye chicken semen. These findings could be helpful for cryobanking of rooster semen for preservation of selected breeders from malignant viral avian disease.

Kinetin improves motility, viability and antioxidative parameters of ram semen during storage at refrigerator temperature.

The present experiment was conducted to evaluate the effect of kinetin on ram semen quality during cold storage. Ejaculates were collected using an artificial vagina from five Qezel rams. Ejaculates which met the criteria (volume of 0.75-2ml; minimum spermatozoa concentration of 2.5×109 spermatozoa/ml and forward progressive motility of 80%), were pooled, diluted with extender without kinetin (control) or enriched with 25 (K 25), 50 (K 50), 100 (K 100) and 200 (K 200) μM kinetin at a final concentration of 500×106 spermatozoa per mL. Spermatozoa motion characteristics were evaluated by computer-assisted sperm analysis. In addition, percent of viability (spermatozoa showing no color was considered to be alive) and spermatozoa with intact plasma membrane (spermatozoa with curled/swollen tail was considered healthy) were determined. Moreover, amounts of malondialdehyde (MDA), total antioxidant activity (AOA), nitric oxide (NO) and superoxide dismutase (SOD) activity were determined in the seminal plasma and spermatozoa at 0, 24, 48 and 72h of storage. Higher percent of total and forward progressive motility was observed in K 25, K 50 and K 100 groups compared to control group at 72h of storage (P<0.001). Moreover, K 25 (78.61±1.11%), K 50 (82.46±1.08%) and K 100 (82.96±1.49%) groups showed higher percentage of viability compared to control (72.38±1.49%) at 72h of storage (P<0.05). Semen enrichment with kinetin resulted in the higher percent of intact plasma membrane of spermatozoa at 48 and 72h (P<0.001). Amounts of MDA were lower and amounts of AOA were higher in K 50 and K 100 groups compared to control at 48 and 72h (P<0.05). There were no significant differences in NO levels and SOD activities of seminal plasma and spermatozoa among groups during the experiment. The present experiment revealed that kinetin at proper concentration could enhances spermatozoa kinematics, viability, spermatozoa plasma membrane functionality and amounts of AOA and reduces the level of lipid peroxidation during chilled storage of ram semen.

Preservation of honey bee spermatozoa using egg yolk and soybean lecithin-based semen extenders and a modified cryopreservation protocol

Better protocols and suitable extenders for semen handling and cryopreservation are required for honey bees to enhance sperm quality and fertility similar to that of other species. The purpose of the present research is the improvement of honey bee sperm quality using different semen extenders and a modified cryopreservation protocol. Three different extenders were used: (1) buffer and egg yolk (EY); (2) buffer and 0.5% soybean lecithin (SL0.5); and (3) buffer and 2% soybean lecithin (SL2). Semen was collected and then diluted with the experimental extenders. The diluted semen was gradually cooled in a refrigerator to 5 °C and immediately loaded into straws and frozen with liquid nitrogen. Motility and viability data analyzed using the GENMOD and GLM procedure of SAS software. The results demonstrate that the mean fresh sperm motility in EY (4.75 ± 0.14) and SL2 (4.5 ± 0.2) were significantly (p < 0.05) higher than the SL0.5 (4.12 ± 0.12). Also, the mean of cooled motile spermatozoa in EY and SL2 were significantly higher than the SL0.5 (3.87 ± 0.24). Post-thawed sperm motility in EY (3.6 ± 0.24) was significantly (p < 0.05) higher than the other extenders. Furthermore, percentage of viable spermatozoa in EY (69.75 ± 2.32%) was significantly higher than SL0.5 and SL2 (38.5 ± 2.32 and 45 ± 2.32% respectively. Therefore, according to the results of this study, use of an egg yolk-based extender could better maintain honey bee semen motility and viability after freeze-thawing process, compared to SL-based extenders. However, more advanced tests and fertility evaluation are needed to reveal the exact effects of egg yolk and lecithin based semen extenders on post-thawed drone sperm quality.

Substitution of egg yolk with different concentrations of soybean lecithin in tris-based extender during bulls' semen preservability

ObjectiveTo investigate the effect of various concentrations of soybean lecithin as an alternative for egg yolk in bull semen extender on post-chilling and post-thaw sperm quality characteristics. MethodsSemen ejaculates were collected from three mature bulls' once/week for 5 weeks. After initial evaluation the approved ejaculates were pooled and extended gradually 1:7 with tris-citrate-fructose egg yolk extender (control) and tris-citrate-fructose (TCF)+different concentration of soybean lecithin (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.0%) to ensure 60 million motile spermatozoa/mL, and then proceeded an adopted international cryopreservation protocol. Frozen straws were thawed at 37 °C for 30 s. The parameters studied were sperm motility, viability, abnormality, membrane integrity and normal intact acrosome percentages in chilled and frozen-thawed semen. ResultsThe substitution of egg yolk into TCF with 1% soya lecithin significantly (P < 0.0001) ameliorated the maintenance of semen characters motility (86.67% ± 0.80%), life (87.60% ± 0.88%), and membrane integrity (82.47% ± 0.94%), meanwhile it had significantly (P < 0.0001) reduced the abnormality (13.20% ± 0.60%) of spermatozoa to its least value compared to some other concentrations in use. Moreover, the addition of 1.5% of soya lecithin to TCF had maintained the semen characteristics compared to the control tris-citrate-fructose egg yolk. The significantly higher mean values of post-thawing sperm motility % were observed in 1% soybean lecithin (56.00% ± 1.00%) as compared to the control (41.00% ± 1.00%). Conclusion1–1.5% soya lecithin can effectively alternate egg yolk as cryoprotective additives for cryopreservation extender, without any detrimental effects on post-chilling and post-thaw semen quality in cattle bull.

Sperm pretreatment with glutathione improves IVF embryos development through increasing the viability and antioxidative capacity of sex-sorted and unsorted bull semen

The antioxidant of reduced glutathione (GSH) is the most abundant thiol in cells for the maintenance of the intracellular redox balance. The study aimed to assay the effect of sperm treatment with GSH before incubation with oocytes on the development potential of embryos obtained by in vitro fertilization (IVF). Also the mitochondrial membrane potential (ΔΦm), plasma membrane integrity (viability), DNA fragmentation, reactive oxygen species (ROS) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, methane dicarboxylic aldehyde (MDA) level as indices of lipid peroxidation in sex-sorted and unsorted sperm from three bulls were investigated using flow cytometry and enzyme-labeled instrument individually. The results showed that 2 mmol L−1 GSH increased significantly the cleavage rate (86.68% vs. 82.78%), 4- to 8-cell rate (82.30% vs. 73.43%) and blastocyst rate (43.15% vs. 35.24%) of IVF embryos compared with untreated group. Furthermore, addition of GSH increased significantly the ΔΦm and viability, decreased the ratio of DNA fragmentation in sex-sorted or unsorted semen (P<0.05), except the sex-sorted semen from bull 019. Similarly, activities of SOD, CAT and GPx were increased significantly. However, the contents of MDA were decreased significantly both in sex-sorted and unsorted semen treated with GSH (P<0.05). These results suggest that sperm pretreatment with GSH during IVF can maintain better the viability and fertility of sperm through reducing apoptosis and increasing the antioxidant capacity, which improves the IVF embryos development.

Effect of methyl-beta-cyclodextrin on the viability and acrosome damage of sex-sorted sperm in frozen-thawed bovine semen.

BackgroundThe regulation of methyl-beta-cyclodextrin (MBCD) on cryodamage on X- and Y-sperm during cryopreservation of semen was investigated. The semen was collected from ten healthy bulls of proven fertility by an artificial vagina. The bovine sperm treated with MBCD fresh solution (0, 1, 5, 10, and 20 mM). The sperms were evaluated for viability and acrosome damage using flow cytometry. Moreover, X- and Y-sperm in frozen-thawed bovine semen were sorted by flow cytometry after Hoechst 33342-dyed, and the viability and acrosome damage of sperms were analyzed.ResultsSperm viability in frozen-thawed semen was decreased by MBCD (p < 0.05), also the acrosome damage of sperm was significantly increased (p < 0.05). Moreover, we sorted X- and Y-sperm from frozen-thawed bovine semen for observing the viability and acrosome damage on the separated X- and Y-sperm after MBCD treatment. Viability of X-sperm was significantly lower than that of Y-sperm (p < 0.05). Also, acrosome damage of X-sperm was significantly higher than Y-sperm (p < 0.05).ConclusionsMethyl-beta-cyclodextrin enhances damage of sperm in frozen-thawed bovine semen, and X-sperm is more sensitive than Y-sperm in cell damage. These results demonstrate that MBCD can inhibit viability of spermatozoa in frozen-thawed bovine semen (for X-sperm, especially).

Effect of tobacco chewing on semen parameters

Background: Tobacco is a plant with green foliage and tubular flowers. Biologically titled as Nicotiana tabacum. Tobacco is evil for human health. It has been noted that 35% of death worldwide happen due to the tobacco intake only. Individual with no education is 2.69 times more likely to smoke and chew tobacco than those with a postgraduate education. Tobacco causes reduction in sperm count, motility, and concentration. Objective: To affirm the deleterious effects of tobacco abuse on various semen parameters. Materials and Methods: This cross-sectional descriptive study was conducted on 100 healthy adults in GGH Hospital, Jamnagar, Gujarat, India. Out of which, 50 were tobacco chewers and 50 were nontobacco chewers. Semen was collected in the vicinity of laboratory bathroom and immediately handover to laboratory technician for evaluation of various parameters. Result: Result shows that there is a significant decrease in semen volume, sperm concentration, motility, and viability in tobacco chewers than control group. There was progressive decrease in sperm count and liquefaction time as the duration of tobacco chewing is increase. Conclusion: There is an adverse effect on all the seminal parameters who practicing of tobacco chewing as compared to non-chewers mainly sperm concentration, total sperm count, motility, and viability of sperm are also decreases in tobacco chewer than the control group. As the frequency of tobacco chewing is increasing, it is associated with more adverse effects on semen volume, count, concentration, motility, and viability of sperm.

Effect of ficin enzyme on semen viscosity in dromedary camel

The viscous nature of camel semen limits its processing, evaluation and preservation. Present study investigated the effect of Ficin enzyme on camel semen viscosity. Semen was collected using phantom and diluted with Shotor diluent. Following initial mechanical stirring (150 rpm for 5 min), semen sample was assigned into 2 groups (Ficin and Control), with three equal fractions for each group. Ficin was added at the concentration of 0.05 mg/ml after 5 minutes initial stirring (Time 0). All samples were stirred for another 10, 20 and 30 min. At each time point, semen viability was assessed and the rest of the fraction was centrifuged at 600g for 7 min at 25°C. Semen viscosity was eliminated in all enzyme treated fractions. In all control tubes, pellet could not be separated from supernatant after 10 and 20 min; whereas, after 30 min, pellets were separated from supernatant in 50% of the samples. Treated samples displaced better total motility 20 and 30 min after adding Ficin (P<0.05). Progressive motility was also higher in treated group compared to control 20 min after adding Ficin (P<0.05). No detrimental effects on semen viability parameters were noticed following the addition of enzyme. In conclusion, Ficin could reduce semen viscosity without compromising sperm viability parameters in dromedary camel.

Introducing of a New Experimental Method in Semen Preparation: Supernatant Product of Adipose Tissue: Derived Mesenchymal Stem Cells (SPAS)

Human male infertility mostly is due to deficient semen parameters. One of the seminal plasma components providing chemical function of the ejaculate is growth factors functioning as paracrine, autocrine, and/or endocrine regulators of cell growth and differentiation. Adipose tissue-derived mesenchymal stem cells secret several proteins, such as cytokine, growth factors, and chemokine. On the other hand, due to contemporary semen preparation methods limitations, development of a new approach having least adverse effects and most efficiency is unavoidable. So, we decided to assess the effect of semen sample incubation with SPAS on semen parameters including motility, viability and DNA fragmentation. Semen sample of infertile men attending infertility treatment center whose were 30-40 years old was incubated with supernatant product of adipose tissue- derived mesenchymal Stem cells (SPAS). Motility parameters, viability and DNA fragmentation were evaluated using CASA software, eosin- nigrosine staining and halosperm kit respectively. Then conventional method of semen preparation for Intra Uterine Insemination (IUI)- Density gradient centrifugation (DGC)- was compared with SPAS method. Incubation of semen samples with SPAS (specially SP of 8×105 AD-MSCs for 40 minutes at 1:1 ratio) significantly increased motility parameters while decreased DNA fragmentation (p≤0.05) and has no effect in terms of semen viability. Also, SPAS was significantly more influential in improvement of motility parameters than DGC (p≤0.05). Since effect of growth factors in improvement of motility and DNA fragmentation is documented in previous studies, it seems that SPAS can be an effective method which provides all these factors together.