Quality Of Frozen-thawed Semen Research Articles

Comparison of computer-assisted sperm analysis and smartphone-applied sperm analysis for evaluation of frozen-thawed bull semen.

This study aims to compare the efficacy of computer-assisted sperm analysis (CASA) and smartphone-applied sperm analysis (SASA) in assessing the quality of frozen-thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA-37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA-25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA-37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen-thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p > .05). However, significant differences among the groups were observed for total motile spermatozoa values (p < .001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p < .001). The immotile spermatozoa values were significant between the groups (p < .001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen-thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis.

Influence of linoleic acid supplementation on the quality of frozen-thawed ram semen

Influence of linoleic acid supplementation on the quality of frozen-thawed ram semen

Supplementation with MitoTEMPO before cryopreservation improves sperm quality and fertility potential of Piedmontese beef bull semen.

The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 μM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 μM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 μM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 μM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 μM and the mitochondrial membrane potentials was increased at 80 μM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 μM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 μM as an additive in the cryopreservation media of bull semen to improve sperm quality.

The Effect of κ-Carrageenan on Porcine Sperm Cryo-Survival.

κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen-thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress.

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.

Effects of semen quality on pregnancy rate in artificially inseminated dairy cows

The study aimed to evaluate the quality of fresh and frozen-thawed semen of five adult Holstein-Friesian crossbred bulls and the pregnancy rate of cows inseminated with frozen semen of those bulls. The fresh semen of breeding bulls collected for artificial insemination (AI) programme in the field was of good quality with volume (6.7 ± 0.2 ml - 8.9 ± 0.5 ml), concentration (904.2 ± 56.4 million/ml), mass activity (3.3 ± 0.2-3.6 ± 0.2), total motility (77.0 ± 1.1% – 92.1 ± 0.6%), progressive motility (67.0 ± 1.2% – 87.4 ± 0.6%) and semen viability (73.0 ± 0.6% to 85.4 ± 0.7%). The computer assisted sperm analysis (CASA) results showed that diluted pre-freezing semen had good sperm total motility (50.1 ± 3.8 % to 59.0 ± 4.7%), progressive motility (30.0 ± 1.2%-39.0 ± 1.6%), the velocity traits of straight linear velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP) of sperm ranged from 48.0 ± 1.3 - 71.3 ± 0.7 μm/s,118.1 ± 2.8-181.3 ± 10.9 μm/s and 68.4 ± 2.5 to 91.0 ± 2.9 μm/s, respectively. Bull 1 showed significantly higher VSL (71.3 ± 0.7 μm/s), VCL (181.3 ± 10.9 μm/s) and VAP (91.0 ± 2.9 μm/s) compared to others. Viability of frozen-thawed semen was lower in Bull 5 (73.0 ± 1.71%) compared to others. Although in frozen-thawed semen these parameters declined, the semen was sufficiently good to be used in AI in the field. The overall pregnancy rate using frozen semen was 55.6% and the highest pregnancy rate (62%) was in cows that were inseminated with frozen semen of Bull 1, but the differences between bulls was not significant. The pregnancy rate had positive correlation with sperm count, total motility, progressive motility, VCL, VSL, VAP, amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), straightness (STR), sperm viability. In artificially inseminated cows, the intensity of oestrus of cows, timing of AI, site of semen deposition and season had a significant effect on pregnancy rate. In conclusion, the fresh and frozen-thawed semen of breeding bulls supplied in North-East Bangladesh for AI programme were good quality. Heat detection and insemination timing need to be improved to increase the pregnancy rate.&#x0D; Bangl. vet. 2022. Vol. 39, No. 1-2, 1–15

Quality of frozen equine semen stored in different shipping containers

The use of cryopreserved semen in horses requires sophisticated equipment, including containers maintaining the temperature of liquid nitrogen (LN). Simpler and less expensive devices for transport of frozen semen may help breeding programs in developing countries and reduce the costly return of empty containers. Veterinarians sometimes question the quality of frozen-thawed shipped semen because of potential fluctuations in shipping temperatures. This study aimed to investigate the effects of shipping duration and temperature on post-thaw quality of frozen semen stored either in a dry shipper (≤ -150°C) or on dry ice (≤ -78°C) for 14 days. Straws (n=165) containing cryopreserved semen from five stallions stored in liquid nitrogen were transferred (D0) to a dry shipper (DS) and to a styrofoam box with dry ice (DI). Straws from DS and DI were returned to LN on D1, D3, D7, D10, and D14. All semen doses were evaluated at each time point by CASA for total (TMot) and progressive motility (PMot), and kinematic parameters such as average path velocity (VAP), curve-linear velocity (VCL) and straight-line velocity (VSL), by fluorescence microscopy for the percentage of membrane-intact spermatozoa (SYBR14/PI stain), and high mitochondrial membrane potential (HMMP; JC-1 stain). Data were analyzed by repeated measures ANOVA with both storage time and storage device as within subject factors. Thetemperature inside the DS and DI containers was monitored continuously with a Tempod 200XB data logger. There were no temperature fluctuations during the 14-day storage in both containers. Until D7 of storage, no changes were observed in TMot, PMot, and membrane-intact spermatozoa (%). Thereafter all three parameters decreased in semen stored in DI but not in DS (storage device at least p<0.05, time p<0.01, time x storage device p<0.001; e.g., TMot D7 on DI and in the DS both 59±14 versus 34±10 and 60±13%, respectively, on D14; membrane-intact spermatozoa D7 on DI 50±13 and in the DS 52±12 versus 28±7 and 53±19% on D14). The HMMP percent decreased continuously during storage in both containers (p<0.001) and this decrease was more pronounced for DI compared to DS (time x storage device p<0.05; DI from 43±4 on D1 to 26±3% on D14, DS from 43±5 on D1 to 30±4% on D14). All velocity parameters remained constant with storage in both devices. In conclusion, cryopreserved frozen stallion semen can be stored for up to 7 days on dry ice without negative effects on post-thaw characteristics. Dry ice may thus be an alternative to liquid nitrogen containers for the short-term transport of frozen stallion semen. Controlled insemination trials to confirm the fertility of frozen stallion semen shipped on dry ice are justified.

Assessment of the Quality of Frozen-Thawed Semen or Epididymal Sperm in Three Native Vietnamese Pig Breeds

Assessment of the Quality of Frozen-Thawed Semen or Epididymal Sperm in Three Native Vietnamese Pig Breeds

Качественные характеристики замороженно-оттаянного семени (обычное и разделенное по полу ) у быков-производителей голштинской черно-пестрой породы и возраст полового созревания полученных от них телочек

A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS‑500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 μm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 μm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.

Идентификация популяций клеток крови рыб на основе сверточной нейронной сети для составления лейкограммы

A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS‑500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 μm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 μm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.

Cryoprotectant effects of natural honey on spermatozoa quality of pre-freezing and frozen-thawed boar semen.

Natural honey has been successfully used in the preservation of mammalian gametes because of its beneficial properties. The objectives of this study were to determine the inclusion level of honey in extender for improving boar semen quality before freezing and to investigate the effects of honey inclusion in extender and freezing media on post-thaw quality of frozen-thawed boar semen samples. Ejaculates from six terminally crossbred boars were collected using the gloved-hand technique for two experiments. Experiment 1 was a randomized block design, evaluating four inclusion levels of honey in boar semen extender [Control (0H)-Androhep Plus or Androhep Plus with 0.25%, 0.50%, and 0.75% honey (0.25H, 0.50H, and 0.75H respectively)]. Ejaculates were pooled, aliquoted according to treatments, and cooled for 24 h at 17 ºC. The results of this experiment were used to determine inclusion levels in exp. 2. Experiment 2 was a 2 x ×3 factorial design, evaluating the inclusion of honey in boar semen extender and freezing media. Semen samples from individual boars were cooled in extender with or without honey (C0: Androhep Plus; C1: Androhep Plus + 0.25% honey). After 24 h, semen samples were evaluated, diluted in lactose-egg yolk (LEY) media, and one of three freezing media types; F0: 93% LEY + 6% glycerol + 1% Equex-STM Paste (ESP); F1: 93% LEY + (3% glycerol and 3% honey) + 1% ESP; and F2: 93% LEY + 6% glycerol + (0.5% ESP and 0.5% honey). Samples were frozen in 0.5 mL straws using a controlled-rate freezer and stored in liquid nitrogen. In exp. 1, 0.25H and 0.50H improved motility (P = 0.033) and progressive motility (P = 0.001) of cooled boar semen. Nevertheless, 0.25H was selected for exp. 2. In exp. 2, post-thaw motility and progressive motility were highest (P < 0.05) in C0F2 but not different from C1F2. Morphologically normal cells and acrosomes were higher with all inclusion levels of honey (P < 0.05). In conclusion, 0.25% and 0.50% inclusion of honey in Androhep Plus improves motility and progressive motility of cooled boar semen samples after 24 h. Supplementing Androhep Plus with 0.25% honey maintains higher normal sperm cells and acrosomes of cryopreserved boar semen. Replacing 50% Equex-STM paste with honey in freezing media improves post-thaw sperm motility, progressive motility, percentage of normal sperm, and acrosome of cryopreserved boar semen.

The proAKAP4 concentrations in Nelore bull sperm and their relation to FTAI conception rate results

The AKAP4 protein has an essential role in sperm motility, and its precursor, the proAKAP4, is considered a biomarker for sperm quality and fertility. Despite this, proAKAP4 concentration was never evaluated in Bos indicus sperm. Therefore, this study aims to determine the proAKAP4 concentration in the sperm of Nellore bulls and its association with sperm kinetics, morpho-functionality, morphology, and conception rates after fixed-time artificial insemination (FTAI). The bulls (n = 9) used in our study presented sperm characteristics above expected standards for quality. Sperm from each bull was evaluated regarding kinetics by the CASA system, and assessed for mitochondrial functionality, sperm membrane integrity, and morphology. For the FTAI protocol, multiparous Nelore cows (n = 1507) were inseminated with frozen-thawed sperm from the same batches used in the laboratory analysis. Additionally, the sperm proAKAP4 concentration was determined using an ELISA kit. The data were analyzed by linear regression and the significance level was set to 5%. The mean conception rate was 52.52%. The mean proAKAP4 concentration was 25.75 ng/106 spermatozoa. The proAKAP4 concentration positively affected the sperm total and progressive motilities, and conception rates after FTAI (respectively, r2 = 0.49, P = 0.03; r2 = 0.63, P = 0.01; and r2 = 0.51, P = 0.03). Based on the relationship detected in this study, we conclude that the sperm proAKAP4 concentration is a suitable biomarker to predict the quality and fertility of frozen-thawed Bos indicus semen.

Evaluation of Chicken Egg Yolk Plasma and Low-Density Lipoprotein Alone or Enriched with Ewe or Cow Skim Milk in Tris-Citric Acid-Based Diluent for Cryostorage of Ram Semen.

The main purpose of the current study was to find suitable and optimum levels of protectants among chicken egg yolk plasma (CEYP) and low-density lipoproteins (LDLs), alone or supplemented with ewe or cow skim milk, for cryopreservation of ram semen. In Experiments 1 and 2, the CEYP (28%) freezing extender was enriched with ewe or cow milk (2.5%, 5%, 10%, or 20%; v/v), respectively. In Experiments 3 and 4, the semen extender was prepared by varying the amounts of fresh or lyophilized LDL (lyo-LDL), respectively. Finally, ewe or cow skim milk was added to the freshly extracted LDL extender and the quality of frozen-thawed semen was examined (Experiments 5 and 6). Kinematics of spermatozoa (assessed using a computer-assisted sperm analysis system), viability, functionality of the plasma membrane, and levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were evaluated. Results revealed that addition of ewe or cow skim milk (5%, 10%, or 20%; v/v) to the CEYP diluent enhanced kinematics, viability, and membrane integrity of spermatozoa compared with the control (p < 0.05). Moreover, fresh LDL diluent was more effective than lyo-LDL in the cryosurvival of ram spermatozoa. In addition, enrichment of fresh LDL diluent with ewe or cow skim milk improved different variables of spermatozoa compared with the control (p < 0.05). Levels of MDA and TAC were not affected by adding ewe or cow milk to the diluents (p > 0.05). In conclusion, enrichment of fresh LDL extenders with ewe or cow milk also is proposed as an approach to preserve ram semen quality against cold shock and cryodamage injuries.

Effect of supplementation of freezing media with isoespintanol on functional integrity of equine semen

Abstract Adding antioxidants to semen extender has become one of the major strategies to reduce the damage caused by freezing to stallion sperm. Isoespintanol (Iso) is a natural antioxidant that is known as a good free radical scavenger. The aim of this research was to evaluate the effect of supplementation of freezing media with Iso on functional integrity of equine semen. Semen from five Colombian Creole horses was frozen with 0, 10, 20, 30, 40 and 50 µM Iso. Post-thawing motility and kinetics, structural membrane integrity (SMI), normal morphology (NM) and functional membrane integrity (FMI) of sperm were evaluated. Seminal quality (SQi) and seminal velocity (SVi) indexes were calculated. Through a flow cytometric analysis, mitochondrial membrane potential (ΔΨM), viability and DNA integrity of frozen-thawed semen were assessed. General linear models were adjusted, and means comparisons were performed using Tukey’s test. The significance level was P&lt;0.05. No differences for post-thaw sperm quality parameters were found, except for total motility, which showed a reduction by adding 40 µM Iso (P&lt;0.05). However, the estimation of indexes, showed that supplementation with 20 or 30 µM Iso before semen freezing, can increase post-thaw SQi and SVi values of some horses. A higher proportion of high-∆ΨM sperm for 20 µM Iso was found. It is concluded that Iso can improve the quality and mitochondrial activity of frozen-thawed equine semen, however its effect can vary according to the horse and in high concentrations it could have detrimental effects.

Frequency of Semen Collection Affects Ram Sperm Cryoresistance.

Simple SummarySperm cryopreservation is an important step to optimize artificial insemination protocols for rams. Traditionally, the improvement of ram sperm cryopreservation protocols has been addressed through different methods: formulation of different cryoprotectants, supplementation with antioxidants or carrying out different freezing-thawing rates. However, the influence of the male sexual regime as a factor for the improvement of freezability has not been studied until now. The frequency of sperm collection in the work protocols of reproduction centers can be variable depending on the dose demand. To corroborate the variability of the quality of thawed semen according to the frequency of collection, in addition to traditional analyses, new tests based on the redox balance of spermatozoa were performed. All these aspects have been applied and discussed extensively throughout this manuscript. In addition, new approaches, such as antioxidant activity of some enzymes have been incorporated to provide more precision in integrative studies of the redox state in spermatozoa. The deleterious effect triggered by a high dose demand should be demonstrated as a consequence of a redox imbalance.The improvement of frozen-thawed sperm quality has been mostly approached from the view of cryopreservation protocol optimization in terms of cryoprotectant solutions, freezing-thawing rates and antioxidant supplementation, while the impact of sperm collection frequency remains unknown in rams. In this work, a multiparametric study was carried out in cooled and frozen-thawed semen to evaluate sperm quality after different semen collection frequencies during a month: zero sperm collection (0 CW), four sperm collections per week (4 CW), and ten sperm collections per week (10 CW). Traditional analyses have been applied, in combination with novel technologies related to redox balance. Frozen-thawed semen quality showed a significant decrease (p < 0.05) in 0 CW and 10 CW in comparison to 4 CW, concerning motility and kinetics parameters. However, apoptosis showed a significant increase (p < 0.05) in 10 CW in comparison to 0 CW and 4 CW. The employment methods related to redox balance provided us with the definitive probe to ensure the influence of collection frequency on balance redox after thawing. Specifically, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activity showed a significant decrease (p < 0.05) in 10 CW compared to 0 CW and 4 CW. The characterization of alternative strategies to sperm cryopreservation based on consideration of male sexual regimes, could improve the quality of frozen-thawed sperm.

Temperature-processing effect on equine semen cryopreservation

Advances in semen cryopreservation, alongside the implementation of new laboratory techniques to better protect sperm cells viability, have allowed increased quality and fertility of frozen semen in mammals. However, this process can impair the acrosomal integrity, viability, and motility of spermatozoa to some degree. Deleterious effects of cryopreservation are largely due to shock temperature during the freezing process that causes osmotic and oxidative stress. The aim of the study was to assess whether an early semen cooling could influence main parameters of equine frozen-thawed semen. Eight healthy and fertile Sicilian Oriental Purebred stallions underwent semen collection by using a Missouri artificial vagina. Semen samples were processed according to two different freezing protocols. Semen was extended 1:1 ratio (semen:extender) in INRA-96 extender prewarmed at 37°C; thereafter in protocol A diluted samples were placed into conical tubes with 2ml Maxifreeze cushion and centrifuged at room temperature for 10 min at 600g. After removal of supernatant and cushion, the sperm pellet was resuspended with INRA-freeze to a final concentration of <200 × 10 6 spermatozoa/ml. The obtained semen was then drawn at room temperature into 0.5-ml straws (Minitube, Germany), sealed thermally, and cooled at 5°C over one hour. Protocol B differed from protocol A as diluted samples were cooled at 5°C for one hour before being centrifuged at 5°C and all other steps of semen processing were performed at 5°C. Straws obtained with both protocols were placed 4 cm above liquid nitrogen in the vapor phase in a foam box for 10 min before being plunged into the liquid phase. The straws were then stored in goblets and kept immersed in liquid nitrogen until computer-assisted semen analysis (CASA). Student's T-test was performed to highlight differences in total, progressive, and immotile sperm, p ≤ 0.05 was considered statistically significant. After thawing, equine semen frozen according to protocol A showed higher total (p=0.03) and progressive (p=0.01) motility, in comparison with protocol B. In addition, immotile sperm were significantly lower in protocol A than B (p=0.03). Stallions show high individual variation in semen freezability, and equine spermatozoa are known to be very sensitive to shock temperature, hence several studies focused on exploration of temperature drop rates and freezing protocols. In this study, a higher quality of frozen-thawed semen was observed with room temperature centrifugation and packaging, rather than after one-hour semen cooling at 5°C before refrigerated centrifugation.

Evaluation of the effects of hesperidin on fresh and frozen-thawed semen quality using two different cryopreservation methods in Simmental bull.

In the industry of bull semen freezing centers, one-step and two-step semen dilution protocols are two standard and well-known methods in semen freezing process. As the freezing/thawing processes cause detrimental effects on sperm function, the addition of antioxidants can improve sperm characteristics. Hesperidin (Hesp) is an antioxidant used as the male reproductive protective agent. Therefore, the aim of this study was to investigate two different dilution methods, as well as to evaluate Hesp supplementation influence on sperm characteristics in fresh and frozen thawed semen. Semen samples were collected from 12 Simmental bulls. Two separate examinations were conducted in, with and without Hesp supplementation groups. Statistical analysis was performed by an independent t-test, Mann Whitny test, MANOVA and ANOVA tests. In comparison to the one and two-step dilution protocols without Hesp supplementation, the two-step dilution showed greater cryoprotective potential. In the Hesp supplemented group, each semen sample was divided into six equal parts for experimental groups (dilution step method/µM of Hesp). In the both one and two step dilution protocols, significant improvements were detected in semen motility parameters by Hesp administration. Also, oxidative stress status was reduced in seminal plasma of Hesp treatment groups. Interestingly, in comparison with Hesp dosage, 1µM was shown to have greater semen cryoprotective potential. In conclusion,

Combination of nanoparticle green tea extract in tris-egg yolk extender and 39 °c thawing temperatures improve the sperm quality of post-thawed Kacang goat semen.

Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.

The Use of κ-Carrageenan in Egg Yolk Free Extender Improves the Efficiency of Canine Semen Cryopreservation.

Simple SummarySemen cryopreservation generates sperm damage and reduces fertilization capacity as a consequence of reactive oxygen species formation, a known trigger of the apoptosis cascade. The present study aimed to examine the effects of κ-carrageenan supplementation to the freezing medium on canine sperm cryo-survival. To this end, the sperm was frozen in egg-yolk-free diluent containing different κ-carrageenan concentrations (0.0%, 0.1%, 0.2%, 0.3%, and 0.5%). The addition of κ-carrageenan to the extender at a 0.2% concentration resulted in a significant increase in the total motility and rapid progressive motility. Increasing levels of κ-carrageenan increased the spermatozoa acrosome integrity (p < 0.05). Apoptosis levels were significantly lower in the 0.1% and 0.2% κ-carrageenan treatment. Moreover, the κ-carrageenan supplemented group exhibited an upregulation of the relative expression of anti-apoptotic and down-regulation of oxidative state-related genes (p < 0.05). Overall, the introduction of κ-carrageenan as a component of egg-yolk-free freezing diluent could be useful for defining the cryopreservation media and improving the overall efficiency of cryopreserved canine semen.κ-Carrageenan is a plant polysaccharide derived from red seaweeds reported to possess potential medicinal and antioxidants activities. The present study aimed to identify the cryoprotective effects of κ-carrageenan on the quality of frozen-thawed canine semen. Twenty-eight ejaculates were collected and diluted in a Tris egg-yolk-free extender supplemented with various concentrations of κ-carrageenan (0.0%, 0.1%, 0.2%, 0.3%, and 0.5%). The addition of κ-carrageenan to the extender at a 0.2% concentration induced a significant increase in the total motility (TM) and the rapid progressive motility (RPM) of canine sperm. Among the experimental groups, the highest percentage of sperms with intact acrosomes was found in the 0.5% κ-carrageenan group (p < 0.05). Apoptosis levels were significantly lower in the 0.1% and 0.2% κ-carrageenan treatment. Moreover, sperm in the κ-carrageenan supplemented group showed a significantly higher expression of antiapoptotic (Bcl-2) and lower expression of NADPH oxidase (NOX5), spermine synthase (SMS), and spermine oxidase (SMOX) genes than those in the control group. In conclusion, the addition of κ-carrageenan to the freezing extender improved the overall efficiency of frozen-thawed dog spermatozoa.

Comparative ejaculatory response, fresh and frozen semen quality and fertility to artificial vagina vs electroejaculation method of semen collection in mithun (Bos frontalis) bulls.

The study aimed to describe a comparative ejaculatory response, fresh and frozen-thawed semen quality and fertility of semen collected by artificial vagina and electroejaculation in mithun. Experimental bulls were divided into two groups, G-I: young bulls (n=4; 4-5years of age) and G-II: older bulls (n=4; 8-10years of age). Sixteen ejaculates were collected from each group G-I (AV1 ) and G-II (AV2 ) by artificial vagina method (control). Thirty-six ejaculates were collected from the same bulls from each group G-I (EE1 ) and G-II (EE2 ) by electroejaculation method (treatment). The study did not reveal any significant (p>0.05) difference in the ejaculatory responses between EE1 and EE2 . Mann-Whitney U test indicated that salivation discomfort sign score was significantly (p<0.05) higher in EE1 . Fresh and frozen-thawed semen quality parameters, and motility and velocity profiles recorded by computer-assisted sperm analyser were significantly (p<0.05) lower in electroejaculation than the artificial vagina. The conception rates (AV1 vs EE1 & AV2 vs EE2 ) at day 35-45 post insemination were nonsignificantly higher (p>0.05) in the artificial vagina group. It concluded that, although artificial vagina method has better semen quality, nevertheless, electroejaculation has the potential for semen collection from free-range mithun bulls to incorporate in assisted reproductive technology procedures.