Pooled Semen Samples Research Articles

Bolstering Buck Fertility: The Impact of Asparagus racemosus Aqueous Extract on Semen Cryopreservation and Antioxidant Defense System.

Importance of Study: Semen cryopreservation results in sperm damage due to lipid peroxidation or oxidative stress, leading to a decrease in conception rate. The sperm damage during cryopreservation can be minimized with the use of suitable antioxidant supplements in semen diluent. Some herbs have potent antioxidant potential and can be used in semen diluent to protect the spermatozoa. Objective: Hence, the investigation was planned to evaluate the effect of Asparagus racemosus (A. racemosus) aqueous extract on buck semen quality during cryopreservation. Methodology: In the current study, semen was collected from eight Sirohi bucks, and from each buck, 8 ejaculates were collected. Good-quality semen samples were pooled during each collection. Pooled semen samples were then divided into four equal parts and diluted in TRIS buffer containing different concentrations of A. racemosus aqueous extract (different groups, i.e., G I -5 mg, G II -2.5 mg, G III -1.25 mg, and G IV -0 mg of A. racemosus aqueous extract in 1 mL TRIS buffer). All the diluted semen samples were kept at equilibration temperature (5°C) for 2 hours and then cryopreserved by the manual method. Semen samples were evaluated for various sperm characteristics and antioxidant status before and after cryopreservation. Results: Asparagus racemosus aqueous extract showed significant (p < 0.05) enhancement of sperm viability, sperm motility, acrosomal integrity, and plasma membrane integrity, whereas it reduced sperm abnormality. Furthermore, in the experimental groups, the antioxidant gene expression was found to be increased compared to that of the treatment group. G III (p < 0.05) showed significantly better results in terms of sperm viability, sperm motility, acrosomal integrity, and plasma membrane integrity. Conclusion: Asparagus racemosus aqueous extract has the antioxidant potential to protect buck spermatozoa during semen cryopreservation.

Impact of varying doses of Moringa leaf extract supplementation in the cryopreservation media on sperm quality, antioxidant capacity and antimicrobial activity of frozen-thawed buck spermatozoa

The current investigation was planned to evaluate the effect of Moringa leaves also called Moringa oleifera (M. oleifera) aqueous extract on buck semen quality during cryopreservation, and its antimicrobial potential against the Escherichia coli (E. coli) isolated from the semen samples. Semen was collected from 8 Jakhrana bucks, and from each buck, 8 ejaculates were collected. Semen samples were subjected to bacteriological studies and pathogenic coli were isolated from semen samples. Then, various concentrations of M. oleifera extract were evaluated for its antimicrobial potential. Good quality semen samples were pooled during each collection. Pooled semen samples were then divided into 4 equal parts, and diluted in TRIS buffer containing different concentration of M. oleifera leaf aqueous extract (Different groups, i.e. T1-50 mg, T2-100 mg, T3-200 mg, C-0 mg of M. oleifera aqueous extract in 100 ml TRIS Buffer). Semen samples were evaluated for various sperm characteristics after cryopreservation. oleifera aqueous extract supplemented groups showed significant enhancement in sperm viability, sperm motility, acrosomal integrity and plasma membrane integrity. The treatment significantly reduced the lipid peroxidation in supplemented groups and M. oleifera extract shows the potent antimicrobial activity against the E. coli isolated from buck semen.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P &lt; 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.

Reduction of cryopreservation-induced structural, functional and molecular damages in ram sperm by hydrated C60 fullerene.

This study aimed to investigate the morphological, functional and molecular changes in frozen-thawed ram sperm using an extender containing different concentrations of hydrated carbon 60 fullerene (C60 HyFn), a nanotechnological product. Semen taken from each of the seven Akkaraman rams were pooled. Semen collection was done twice a week and it continued for 3 weeks. Each pooled semen sample was divided into six equal groups and diluted with tris + egg yolk extender including 0 (control), 200, 400, 800 nM, 1 and 5 μM concentrations of C60 HyFn at 37°C. They were then frozen in liquid nitrogen vapour at -140°C, stored in liquid nitrogen container (-196°C) and thawed at 37°C for 25 s before analysis. In comparison with control, C60 HyFn addition prior to freezing procedure provided significant increases in total and progressive motility rates, glutathione peroxidase, catalase activities and percentage of highly active mitochondria, and significant decreases in dead and abnormal sperm rates, lipid peroxidation, caspase-3 and DNA fragmentation levels in frozen-thawed ram semen. When compared to control, C60 HyFn supplementation significantly down-regulated the expression levels of miR-200a and KCNJ11, and significantly up-regulated the expression levels of miR-3958-3p (at the concentrations of 200, 400, 800 nM and 1 μM), CatSper1 (at the concentrations of 200, 400 nM and 5 μM), CatSper2 (at the concentrations of 1 and 5 μM), CatSper3 (at the concentrations of 200, 400 nM, 1 and 5 μM), CatSper4 (at all concentrations), ANO1 (at the concentrations of 800 nM, 1 and 5 μM) and TRPV5 (at the concentrations of 200, 400 and 800 nM). The addition of C60 HyFn had no effect on global DNA methylation rates. As a result, C60 HyFn supplementation to ram semen extenders may be beneficial in reducing some of the functional, structural and molecular damages in sperm induced by the freeze-thawing procedure.

Asparagus racemosus improves seminal antioxidant status and sperm characteristics in buck semen at refrigeration temperature

The present investigation was carried out to evaluate the effect of Asparagus racemosus (Shatavari) aqueous extract on buck semen quality during preservation. In the current study, 8 ejaculates from 8 Jakhrana bucks maintained at Jakhrana unit of ICAR-CIRG (semi-arid region) were collected (total 64 ejaculates) during the period from April to June, 2022. Good quality semen samples were pooled during each collection. Pooled semen samples were then divided into 4 equal parts, and diluted in TRIS buffer containing different concentration of Shatavari aqueous extract (Different groups, i.e. Gr1-5 mg, Gr2-2.5 mg, Gr3-1.25 mg, Gr4-0 mg of Shatavari aqueous extract/ml of semen diluent). All the diluted semen samples were kept at refrigerated temperature (5°C) for seven days, and on each day, diluted semen was evaluated for various sperm characteristics and antioxidant status. Gr3 showed significantly better results in terms of sperm viability, sperm motility, acrosomal integrity and plasma membrane integrity. Along with this, the longevity of sperm was also enhanced in Shatavari supplemented group.

Synergistic effect of extracellular adenosine triphosphate and quercetin on post-thaw quality and fertilization potential of Lohi ram sperm

This study determined the individual and combined effects of extracellular adenosine triphosphate (ATP) and quercetin (QUE) on the quality of post-thawed sperm and the fertilization potential of Lohi rams. In experiment 1, semen samples from four Lohi rams were pooled and extended with different concentrations of ATP or QUE (control; no ATP or QUE, 1 or 2 mM ATP and 10 or 20 μM QUE). In experiment 2, pooled semen samples were extended with various combinations of ATP and QUE (control; no ATP and QUE, 1 mM ATP + 10 μM QUE, 1 mM ATP + 20 μM QUE, 2 mM ATP + 10 μM QUE and 2 mM ATP + 20 μM QUE). All samples in both experiments were cryopreserved and analyzed for post-thawed sperm quality. In experiment 3, the best combination of ATP and QUE from experiment 2 was to extend semen, which was then used for laparoscopic insemination in estrus-synchronized ewes (n = 83). The results of experiment 1 showed that 1 mM ATP and 20 μM QUE treatments resulted in higher total motility, progressive motility, viability, plasma membrane intactness (PMI), and motion kinetics (VCL, VSL, VAP, LIN, and STR) compared to other treatments (p < 0.05). In experiment 2, the 1 mM ATP +10 μM QUE-treated group exhibited significantly higher total and progressive motility, PMI, and motion kinetics (VSL, VCL, VAP, STR, and BCF) compared to the control group (p < 0.05). In experiment 3, the fertilizing potential of sperms treated with 1 mM ATP +10 μM QUE was greater than that of untreated controls (58.1% vs. 27.5%, respectively, p-value = 0.012). In conclusion, the quality of post-thawed ram semen is enhanced when the extender is supplemented with extracellular 1 mM ATP and 20 μM QUE, whether used separately or in combination with 1 mM ATP and 10 μM QUE. Furthermore, the inclusion of 1 mM ATP and 10 μM QUE together in the extender significantly improves in vivo fertility in Lohi ram.

Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

The addition of egg yolk to the physiological saline extender improved the motility and viability of kampung rooster spermatozoa at cool temperatures

The purpose of this study was to determine the motility and viability of kampung rooster (Gallus gallus domesticus) spermatozoa in 0.9% Sodium chloride (NaCl) solution as an extender with the addition of egg yolk at different concentrations at cool temperature (5°C). This study was performed using two 1.5 years old healthy local roosters. Semen was collected through massage in the morning. The pooled semen sample was divided into four treatment groups. In the T0 group the semen was diluted in 0.9% NaCl, while in the T1, T2 and T3 groups the semen was diluted in 0.9% NaCl added with 5, 10 and 15% egg yolk. The results showed that the motility and viability of spermatozoa decreased when stored at cool temperatures for ten hours (p &lt;0.05). Semen of roosters stored at 5°C in saline solution without the addition of egg yolks showed the lowest motility and viability of spermatozoa (p &lt;0.05). The addition of egg yolk into the saline extender increased the motility and viability of spermatozoa. Concentration of 15% egg yolk in saline solution resulted in the highest spermatozoa motility and viability when stored for up to 8 hours (p &lt;0.05). However, motility and viability of spermatozoa at 10 hours of storage were not significantly different (p &gt;0.05) with the addition of 10% and 15% egg yolks. Therefore, it could be concluded that the addition of 15% egg yolk into a saline solution as an extender could maintain the motility and viability of kampung rooster spermatozoa when stored at 5°C for 10 hours.

The addition of egg yolk to the physiological saline extender improved the motility and viability of kampung rooster spermatozoa at cool temperatures

The purpose of this study was to determine the motility and viability of kampung rooster (Gallus gallus domesticus) spermatozoa in 0.9% Sodium chloride (NaCl) solution as an extender with the addition of egg yolk at different concentrations at cool temperature (5°C). This study was performed using two 1.5 years old healthy local roosters. Semen was collected through massage in the morning. The pooled semen sample was divided into four treatment groups. In the T0 group the semen was diluted in 0.9% NaCl, while in the T1, T2 and T3 groups the semen was diluted in 0.9% NaCl added with 5, 10 and 15% egg yolk. The results showed that the motility and viability of spermatozoa decreased when stored at cool temperatures for ten hours (p &lt;0.05). Semen of roosters stored at 5°C in saline solution without the addition of egg yolks showed the lowest motility and viability of spermatozoa (p &lt;0.05). The addition of egg yolk into the saline extender increased the motility and viability of spermatozoa. Concentration of 15% egg yolk in saline solution resulted in the highest spermatozoa motility and viability when stored for up to 8 hours (p &lt;0.05). However, motility and viability of spermatozoa at 10 hours of storage were not significantly different (p &gt;0.05) with the addition of 10% and 15% egg yolks. Therefore, it could be concluded that the addition of 15% egg yolk into a saline solution as an extender could maintain the motility and viability of kampung rooster spermatozoa when stored at 5°C for 10 hours.

Effects of Commercial and Homemade Extenders on Post-thaw Sperm Quality and Fertility of Semen from Ethiopian Indigenous Horro Chicken Breed

Cryopreservation of spermatozoa represents an important strategy for in vitro programs designed for the conservation of the genetic material of livestock populations. The objective of this study was to evaluate the effects of homemade tris-egg yolk-based and commercial poultry semen extenders on post-thaw sperm quality, fertility, and hatchability of semen from the Ethiopian Indigenous Horro chicken breed. A total of 30 roosters were used for semen collection, and 160 adult hens were inseminated artificially. The collected, qualified, and pooled semen samples were divided into three groups. Each semen sample was diluted with a homemade tris-egg yolk-based extender, Dimethyl-formamide commercial extender, and Commercial Beltsville Poultry Extender. Each extended semen was further divided into 20 sterile tubes as replicates. The extended semen samples were cryopreserved in liquid nitrogen using standard procedures. Changes in post-thaw spermatozoa mass and progressive motility, in vitro viability, morphological abnormality, fertility, and hatchability were evaluated. The post-thaw evaluation showed a decrease in the mass and progressive motility, morphologically normal spermatozoa, and an increase in dead spermatozoa and spermatozoa with bent necks, compared to fresh semen. There were significant differences in progressive sperm motility, motility, and in vitro viability between commercial and homemade cryoprotectants. However, no significant difference was observed in mass motility across the extenders. The commercial Dimethyl-formamide extender was found to be the most suitable regarding the proportion of morphologically normal sperm and in vitro viability rate of cryopreserved sperm samples. There were no significant differences across all treatments in terms of fertility and hatchability rate. However, there was a significant difference between the control treatment and commercial extenders in terms of fertility and hatchability. The findings indicated favorable outcomes for a tris-egg yolk-based extender that was prepared locally with regard to the cryopreservation of poultry semen. Additional investigations are recommended to enhance the fertility and hatchability of the semen.

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.

Antioxidants and Oxidants in Boar Spermatozoa and Their Surrounding Environment Are Associated with AMPK Activation during Liquid Storage.

Activation of the AMP-activated protein kinase (AMPK) has been demonstrated to be beneficial for boar sperm quality and functionality, while the underlying mechanism of AMPK activation of boar spermatozoa remains obscure. This study aimed to explore the effect of antioxidants and oxidants in boar spermatozoa and their surrounding fluid (SF) on the activation of AMPK during the liquid storage. Ejaculates from Duroc boars, routinely used for semen production, were collected and diluted to a final concentration of 25 × 106/mL. In experiment 1, twenty-five semen samples from eighteen boars were stored at 17 °C for 7 days. In experiment 2, three pooled semen samples created from nine ejaculates of nine boars were used, and each sample was treated with 0, 0.1, 0.2, and 0.4 μM/L H2O2 and stored at 17 °C for 3 h. Sperm quality and functionality, antioxidants and oxidants in boar spermatozoa and SF, the intracellular AMP/ATP ratio, and the expression levels of the phosphorylated AMPK (Thr172) were determined. Sperm quality significantly decreased with storage time in terms of viability (p < 0.05). Antioxidant and oxidant levels were markedly affected with storage time, with a decline in the SF total antioxidant capacity (TAC) (p < 0.05), SF malondialdehyde (MDA) (p < 0.05), and the sperm's total oxidant status (TOS), as well as a fluctuation in sperm superoxidase dismutase-like (SOD-like) activity (p < 0.05). The intracellular AMP/ATP ratio increased (p < 0.05) on day 4 and subsequently decreased to its lowest value on days 6 and 7 (p < 0.05). The phosphorylated AMPK levels increased from day 2 to day 7 (p < 0.05). Correlation analyses indicate that sperm quality during liquid storage was correlated to antioxidants and oxidants in spermatozoa and SF (p < 0.05), which were correlated to the phosphorylation of sperm AMPK (p < 0.05). Treatment with H2O2 induced damages in sperm quality (p < 0.05), a decline in antioxidant levels (SF TAC, p < 0.05; sperm SOD-like activity, p < 0.01), an increase in oxidant levels (SF MDA, p < 0.05; intracellular ROS production, p < 0.05), a higher AMP/ATP ratio (p < 0.05), and phosphorylated AMPK levels (p < 0.05) in comparison with the control. The results suggest that antioxidants and oxidants in boar spermatozoa and SF are involved in AMPK activation during liquid storage.

Facile fabrication of zinc oxide nanoparticles for enhanced buffalo sperm parameters during cryopreservation

BackgroundSemen cryopreservation could be enhanced by integration of biocompatible nanomaterials. Conditions in which nanoparticles are synthesized should be taken in consideration to avoid divergences in research results concerning beneficial or toxic effects. The objective of this study was to examine the potential of zinc oxide nanoparticles (ZnONPs) synthesized by simple co-precipitation method for providing protection to buffalo sperm against cryo-damage. MethodsZnONPs were characterized by means of X-ray diffraction, high resolution transmission electron microscope (HR-TEM), zetasizer and Raman spectroscopy. Semen was collected from six healthy buffalo bulls using artificial vagina and pooled semen samples were diluted with tris-egg yolk-fructose extender only (control) or extender supplemented with 10, 25, 50, 100 and 200 µg/ml of ZnONPs. ResultsSperm motility, membrane integrity and acrosome defects were measured. DNA damage of spermatozoa was evaluated using comet assay. To determine antioxidative role of ZnONPs, malondialdehyde (MDA) level as a biomarker of lipid peroxidation was measured in seminal plasma. Sperm motility, viability index as well as membrane integrity increased with ZnONPs concentrations up to 100 μg/ml. Likewise, no adverse effect was reported on sperm DNA integrity with all concentrations. MDA decreased up to concentration of 100 μg/ml ZnONPs. Notably, adding 25, and 50 μg/ml ZnONPs to extender produced best significant positive effects on semen parameters compared to ZnONPs free group. ConclusionThe results confirm that low concentrations of ZnONPs fabricated by co-precipitation method using water as exchange media could effectively preserve the quality of buffalo bull frozen-thawed sperm by reduction of cryoinjury.

EFFECTS OF ADDITIN OF TAURINE AND MANGANESE IN EXTENDER ON POST THAW SEMEN QUALITY OF SAHIWAL BULLS

Effect of two antioxidants (Taurine and Manganese) in extender on different spermparameters (sperm motility, live sperm percentage, membrane and acrosome integrity) of Sahiwal bullswas evaluated. Semen was collected twice a week for 8 weeks from six adult, regular donor andclinically healthy Sahiwal bulls. A total of 96 ejaculates were collected and pooled after initialevaluation. The pooled semen samples were diluted at with tris-citric acid,fructose, egg yolk andglycerol extender either containing different level of taurine (20.0, 40.0 and 60.0 mM), manganese(100.0, 150.0 and 200.0 µM), or combinations of taurine and manganese (20.0 mM + 100.0 µM, 40.0mM + 150.0 µM and 60 mM + 200 µM) at a temperature of 37oCand without antioxidants as control.Semen samples were loaded in 0.5 mL French straws and cooled to 4°C. Samples were frozen andstored in liquid nitrogen. These samples were thawed and anlayzed for different parameters. The postthaw sperm motility (%) was significantly (P&lt;0.01) higher in treatment groups containing 20.0 mM oftaurine, 100.0 and 150.0 µM of manganese and 20.0 mM + 100.0 µM of taurine and manganeserespectively. Likewise, the live sperm percentage was higher (P&lt;0.01) in 20.0 mM of taurine and100.0, 150.0 µM of manganese supplemented groups. The maximal sperm acrosome integrity of 20.0mm was observed in taurine group. A higher proportion (P&lt;0.01) of sperm with intact membrane wasobserved in 20.0 mM of taurine and 150.0 µM in manganese containing groups. It was concluded thataddition of taurine and manganese in cryodiluents with a concentration of 20 mM and 150 µMrespectively could improve the post-thaw sperm quality in Sahiwal bulls.

The effect of Caulerpa sertularioides extract on bull sperm freezablity and subsequent embryo development

Artificial insemination is a valuable and essential tool in genetic improvement programs, and its success requires proper semen collection, freezing, and thawing procedures. Nowadays, despite applying of advanced protocols for semen cryopreservation, post-thawing sperm quantitative and qualitative parameters are not satisfactorily comparable to fresh sperm. The present study was designed to evaluate the effects of the supplementation of an alcoholic extract of Caulerpa sertolarioides alga into the tris-egg yolk-based Simmental bull sperm freezing media. The pooled semen samples were divided into five groups, of which four were supplemented with 500, 1000, 1500, and 2000 ppm alga extract and one allocated as a control. Total motility, progressive motility, plasma membrane integrity, DNA integrity, apoptosis, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, and total antioxidant capacity (TAC) of sperm were measured. The frozen sperm from each group were used for IVF on the slaughterhouse-derived oocytes. Fertilization, cleavage, and blastocyst rates were assessed for all groups. Total motility, progressive motility, and velocity curvilinear (VCL) parameters were higher (p ≤ 0.05) in group 1000 ppm than the control group. Velocity in a straight path (VSL) was higher (p ≤ 0.05) in all treatment groups except in 500 ppm compared to the control group. Average path velocity (VAP) was higher (p ≤ 0.05) in 1000 and 1500 ppm groups than in the control group. Straightness (STR) showed a higher value (p ≤ 0.05) in 1000 and 2000 ppm than the control group. Groups 500 and 1000 ppm showed more viable sperm than the control group (p ≤ 0.05). DNA damage was lower (p ≤ 0.05) in group 1000 ppm than in the control group. HOST was higher (p ≤ 0.05) in all groups than in the control group. SOD, GPx, and TAC were higher (p ≤ 0.05) in 1000 ppm than the control and all other groups. Apoptosis was not significantly different among the treatment and control groups. In conclusion, supplementation of alcoholic extract of Caulerpa sertularioides into the Simmental bull freezing extender ameliorated the sperm parameters after the freeze-thawing process. Moreover, the results of this study indicated that the best dose to achieve the antioxidant properties of the alga extract in Simmental bull sperm freezing media was 1000 ppm. It was also evident that 1000 ppm alga extract supplementation into the bull sperm improved fertilization, cleavage, and blastocyst rates.

Effect of Different Extenders on Sperm Motility and Vitality in Goose Semen Cryopreservation

This study aimed to investigate the usability of different diluents containing 6% Dimethylformamide (DMF) for cryo-preservation of the semen of geese (Anser cygnoides). The diluents of Glucose (G), Tris-Glucose (T), Lactated Ringer’s-Glucose (LG), and Lactated Ringer’s (L), all of which contained 6% DMF, were used as cryoprotectants. The researchers collected semen samples from four geese, twice a week over a four-week period, by means of abdominal massage; they then calculated how much sperm each goose ejaculated. Next, the semen samples were pooled and their spermatological parameters were determined. Their volume (4x (mL)), concentration (×108/mL), pH, motility (%), and vitality (%) rates were 0.31±0.01, 3.49±0.32, 7.13±1.06, 67.75±1.28, and 70.00±2.03, respectively. Then, these pooled semen samples were equally divided into four groups. Once they were frozen and thawed, the researchers discovered that the diluent L had the highest motility rate: 40.12% ± 1.35. The motility rates of the other diluents were as follows: LG (28.25%± 1.48), G (21.50% ± 1.41), and T (5.12% ± 0.83). Likewise, the vitality rates (%) of the diluents were as follows: L (41.93% ±1.87), LG (31.50%±1.88), G (29.43% ±1.45), and T (10.56%±1.34), respectively. Freezing and thawing appeared to lower each diluent’s vitality and motility rates. However, for the Lactated Ringer’s (L), this decrease was predictable. Therefore, Lactated Ringer’s diluent containing 6% DMF can be used in cryo-preservation of goose semen.

A new approach for forensic analysis of saliva-containing body fluid mixtures based on SNPs and methylation patterns of nearby CpGs

Saliva samples obtained from crime scenes often contain body fluids from other people, which makes it difficult to not only interpret the obtained DNA profiles, but also interpret saliva identification test results. α-amylase activity, an indicator of most saliva identification methods, can be slightly detected in other body fluids. This study aimed to overcome these difficulties. Here, we identified 13 saliva-specific methylated regions and five saliva-specific unmethylated regions neighboring common single nucleotide polymorphisms (SNPs) by array-based genome-wide methylation analysis of pooled saliva, blood, semen, or vaginal swab samples. Bisulfite sequencing by massively parallel sequencing (MPS) technology was then performed using individual body fluid samples to evaluate the saliva-specificity of each CpG of the three regions selected from the identified candidates. Although no single CpG demonstrated complete saliva-specificity, we found that the reads that were simultaneously (un)methylated at the selected neighboring two to three CpGs of each region were highly specific for saliva DNA. Based on these findings, we then designed MPS-based bisulfite sequencing assays for each region to analyze the selected CpGs and SNP(s) on the same read. These assays could identify the saliva of a target person from body fluid mixtures of known contributors (individual-specific saliva identification) by calculating the ratios of simultaneous (un)methylation at the selected CpGs within the reads containing SNP alleles unique to the target person. Moreover, these assays could indicate the SNP types of saliva DNA (saliva-specific genotyping) from body fluid mixtures by analyzing the alleles of the reads simultaneously (un)methylated at the selected CpGs, while careful attention should be paid to interpret the results of heterologous genotypes. Although further regions should be identified, especially for saliva-specific individual identification, the CpG-SNP approach may be an effective method to interpret the complicated results obtained from saliva-containing body fluid mixtures.

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

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

Synergistic effect of royal jelly in combination with glycerol and dimethyl sulfoxide on cryoprotection of Romanov ram sperm

Sperm fertility decreases significantly after freezing. Providing a suitable and useful diluent compound for freezing ram sperm can increase the efficiency of artificial insemination and consequently, the reproductive performance of sheep. Various biological properties such as antibacterial, anti-cancer, immunosuppressive, antioxidant and reproductive properties of royal jelly (RJ) are well known. The aim of this study was to investigate the possible synergistic effect of royal jelly in combination with glycerol and dimethyl sulfoxide (DMSO) in sperm cryopreservation extender of Romanov ram. The pooled semen samples from 5 Romanov rams were allocated into 3 experiments. The effect of 6% DMSO, 6% glycerol and a combination of 3% DMSO +3% glycerol co-supplemented with 1, 2 and 3% RJ was evaluated in 3 experiments. Samples were frozen by conventional slow freezing method and post-thaw parameters of total motility, progressive motility, plasma membrane integrity, DNA damage, apoptosis, enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), total antioxidant capacity (TAC) and malondialdehyde (MDA) were evaluated. The results showed that the percentage of motility, progressive motility, TAC, GPx, SOD and all sperm kinematic parameters except LIN in the group containing 2% RJ + 6% DMSO was higher than the control group (p < 0.05). Some parameters such as progressive motility, sperm membrane integrity, TAC, GPx, VAP, VCL, STR and SRT in the group containing 2% RJ + 6% DMSO were more than (p < 0.05) in the sperm group containing 1% RJ + 6% DMSO. MDA values in sperm groups containing 2% RJ + 6% DMSO were significantly (p < 0.05) lower than the sperm containing 1% royal jelly and the control group. In the sperm group containing 2% RJ + 6% glycerol, sperm membrane integrity, TAC, GPx, SOD, progressive motility and all sperm kinematic parameters except VAP were higher and MDA values and sperm abnormalities were lower than the control group (p < 0.05). The sperm group containing 1% RJ and 3% DMSO +3% glycerol had higher motility, progressive motility, membrane integrity, and all sperm kinematic parameters except VSL; and lower sperm abnormalities, DNA damage, apoptosis and MDA than the control group (p < 0.05). As a general conclusion of this study, the addition of 2% RJ + 3% DMSO and 3% glycerol to the freezing extender improved microscopic and biochemical ram sperm parameters after the freeze-thaw process. Hence, moderate concentrations of royal jelly (2%) are sufficient to protect sperm from freezing damage, and high (3%) and low (1%) concentrations do not have a good cryoprotective effect.

Evaluation of dextran for rabbit sperm cryopreservation: Effect on frozen–thawed rabbit sperm quality variables and reproductive performance

Effects were analysed of dextran supplementation to Me2SO and acetamide rabbit semen freezing extenders on quality characteristics of rabbit spermatozoa and reproductive performance. The final concentration of cryoprotectants in pooled semen samples was 12.4 % Me2SO for the A extenders, 10.7 % Me2SO and 2.9 % acetamide for the D extenders and 8.9 % Me2SO and 2.9 % acetamide in F extenders, with a supplementation of 1.7 % sucrose in all cases. There was not inclusion of dextran in the A0, D0, F0; while 5 % dextran was included in A5, D5, F5 and 10 % dextran in A10, D10 and F10 extenders. Sperm motility and viability rates were similar with use of the different extenders. Acrosome integrity after the freeze-thawing processes, however, was markedly greater when there was dextran supplementation of D and F extenders. Prolificacy was affected by extender composition. When there was artificial insemination (AI) using semen cryopreserved in the A extenders, number of kits born was similar to when there was AI with fresh semen when there was inclusion of 5% dextran for cryopreservation, while there was no effect on prolificacy when there was cryopreservation of semen using the D and F extenders. In conclusion, dextran supplementation of extenders containing Me2SO and acetamide resulted in greater acrosome integrity. Furthermore, when there was AI using sperm preserved in cryo-diluents containing an intermediate concentration of Me2SO, combined with inclusion of 5 % dextran, there was a marked beneficial effect on rabbit doe reproductive performance.