Sperm Kinetic Parameters Research Articles

Fertility comparison of frozen bull semen stored in cryogenic deep freezer (−152 °C) and LN2 container

The present study aimed to use cryogenic deep freezers that could be a feasible alternative for cryopreserved semen storage. A total of 284 straws from three Simmental bulls and 272 Simmental cows were used. The experimental group consisted of 151 semen straws that were stored at −152 °C for a week. Moreover, the control group consisted of 133 semen straws that were stored at −196 °C. Firstly, two samples per bull (n = 6) were examined in terms of sperm kinetic parameters by CASA. Furthermore, plasma membrane, acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were analyzed by flow cytometry. Then, artificial inseminations were performed on Simmental cows with 272 straws belonging to two groups. Then, 56th-day Non-return Rate (NRR56) was determined. All spermatological data were subjected to a linear mixed model. Chi-Square test was performed to NRR56 between storage temperature groups. Also, logistic regression analysis was used to examine the effect of bull, storage temperature and age of cows on pregnancy status. While age of cows was included in the final logistic regression model, effect of bull x storage temperature was not included because it was found as non-significant. The post-thaw PMOT and STR of cryopreserved bull semen, which was stored at −152 °C, had lower and statistically significant values (p < 0.05). However, frozen bull semen, which were stored at −152 °C, kept its fertility ability as which stored at −196 °C. Besides, NRR56 of semen stored at −152 °C and −196 °C were detected as 57.24% (83/145) and 55.91% (71/127), respectively (p > 0.05). Nevertheless, these results should be supplemented with more pre-freezing and post-thaw sperm quality analyses and more fertility data for increasing the accuracy of the method.

Use of Equine Sperm Cryopreservation Techniques as a Conservation Method of Donkey Germplasm

The aim of this study was to test equine semen cryopreservation techniques for the conservation of donkey germplasm. Ejaculates of three male donkeys were used (n = 18; six ejaculates per donkey; six repetitions), collected by the artificial vagina method. To remove the seminal plasma, the ejaculates were split and submitted to filtration or centrifugation methods. To assess the freezing method, each fraction was submitted to the automated system or the conventional system, and groups were formed: automated centrifuge, automated filtrate, conventional centrifuge and conventional filtrate. After thawing (37°C/30 seconds), were analyzed the sperm kinetic parameters, integrity and functionality of the plasma membrane and mitochondrial membrane potential. Highest sperm concentration (P < .05) was observed in the filtrate groups; the conventional filtrate group presented lower (P < .05) progressive motility and curvilinear velocity compared to the other groups; no difference was observed (P > .05) among the groups for the membrane integrity and functionality, and mitochondrial membrane potential. Thus, centrifugation is the most indicated technique to remove donkey seminal plasma and the automated and conventional freezing methods can be used in donkey semen conservation.

Coenzyme Q10 improves the quality of sheep sperm stored at room temperature by mitigating oxidative stress.

The purpose of this experiment was to explore whether coenzyme Q10 (CoQ10 ) improves the quality of sheep semen stored at room temperature by attenuating oxidative stress. Semen was diluted without (control group), and with antioxidants (5, 50, 250, and 500 μmol/L CoQ10 ). Sperm kinetic parameters and plasma membrane integrity were determined, and the reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), total antioxidant capacity (TAOC), catalase (CAT), and superoxide dismutase (SOD) activity were evaluated on the fifth day of semen preservation. The results showed that compared with the control group, the progressive motility in the 50 μmol/L group was higher (p < 0.05) within 2-5 days, and the plasma membrane integrity of sperm was higher in the 50 μmol/L group. The ROS content in the 5 and 50 μmol/L groups was reduced. The MDA level was reduced in the CoQ10 supplementation groups (p < 0.05). Additionally, the CAT, SOD, TAOC, ATP and MMP levels in the 50 μmol/L group were higher than those in the control group (p < 0.05). In conclusion, CoQ10 improved the quality of ram semen by alleviating oxidative stress, and 50 μmol/L CoQ10 was the optimum concentration.

Characterization of sperm quality in Brycon hilarii: How does morphology affect sperm movement?

Brycon hilarii is a neotropical fish whose population in rivers is being threatened by overfishing, construction of hydroelectric dams, transposition of riverbeds, and pollution. Fundamental knowledge of the reproductive characteristics of this species is necessary to enable its breeding in captivity, for aquaculture as well as for biological conservation. The aim of this study was to investigate and describe the sperm parameters, their correlations, and relevant factors, and how these issues can affect sperm quality in B. hilarii. Sperm was collected after hormonal induction using carp pituitary homogenate. Spermatozoa concentration, total length, motility, and percentage of normal cells were measured to be 2.85 × 109 cells mL−1, 40.28 µm, 68.1%, and 51.6%, respectively. Tail abnormalities were most frequent; however, head abnormalities were closely related to practically all sperm kinetic parameters. Present results suggest that these abnormalities affect motility and movement kinetics, and modifying the pattern of sperm cell movement, sinuosity of movement and vigor. The potential application of this study in aquaculture management of B. hilarii is crucial for the artificial reproduction of fish and represents a significant contribution to the knowledge of its reproductive biology, facilitating the reproduction of the species.

The effect of age and abstinence time on semen quality: a retrospective study.

This study analyzed the effects of male age and abstinence time on semen quality and explored the best abstinence time for Chinese males among different age groups. Semen parameters, including sperm kinetics, morphology, and DNA fragmentation index (DFI), were reviewed from 2952 men. Samples were divided into six age groups (≤25 years, 26–30 years, 31–35 years, 36–40 years, 41–45 years, and >45 years) and were divided into six groups according to different abstinence time (2 days, 3 days, 4 days, 5 days, 6 days, and 7 days). The differences in semen quality between the groups were compared, and the effect of age and abstinence time on semen quality was analyzed. Significant differences were observed in semen volume, progressive motility (PR), and DFI among the age groups (all P < 0.05), and no significant differences were observed in sperm morphological parameters (all P > 0.05). There were significant differences in semen volume, PR, and DFI among different abstinence time groups (all P < 0.05) and no significant differences in sperm morphological parameters (all P > 0.05). Pearson analysis showed that male age and abstinence time were both significantly correlated with sperm kinetics and DFI (both P < 0.05), while no significant correlation was found with sperm morphological parameters (all P > 0.05). The box plots and histograms of men's age, abstinence time, and semen quality show that most semen quality parameters differ significantly between the 2 days and 7 days abstinence groups and other groups at different ages. Except for the sperm morphology parameters, sperm kinetic parameters and sperm DFI are linearly related to male age and abstinence time.

Application of artificial seminal plasma to short-term storage of a large volume of common carp (Cyprinus carpio) sperm for two weeks under controlled conditions

We previously demonstrated that artificial seminal plasma (ASP), developed for common carp (Cyprinus carpio), enabled considerable improvement of the fertilizing capacity of sperm in this species. Here, for the first time, the effectiveness of using ASP (ASP: 2 mM CaCl2, 1 mM MgSO4, 40 mM KCl, 100 mM NaCl, 20 mM Tris; pH 7.5 and 310 mOsm kg−1) for short-term storage of tenfold diluted sperm for up to two weeks at 10 °C was evaluated. Additionally, the fertilizing capacity of stored sperm was investigated. Sperm were stored in a volume of 5 ml in containers equipped with ventilation plugs enabling gas exchange of the stored sperm. During the study, the quality of the sperm was verified using the computer-assisted sperm analysis (CASA) system after 1, 2, 5, 9, 11 and 14 days of storage. After 14 days of common carp sperm storage in ASP sperm motility (MOT, %), the progressive motile sperm (PRG, %) and curvilinear velocity of sperm (VCL, μm s−1) were confirmed to be 55%, 25% and 120 μm s−1, respectively. At the same time, in undiluted sperm (Control), we did not observe sperm motility. Only until the 2nd day of storage in undiluted samples motility and sperm kinetic parameters remained high level (84%, 34% and 144 μm s−1 for MOT, PRG and VCL, respectively). The fertilization of eggs and hatching rate after 14 days of sperm storage in ASP were 80% and 40%, respectively, and did not differ significantly from the fertilization and hatching rate determined after fresh sperm was used for fertilization. In summary, for the first time, the fertilization capacity of common carp sperm after 14 days of short-term storage under ASP conditions was confirmed, indicating the possibility of using previously collected and diluted ASP sperm in reproduction at a time convenient for the breeder. Thus, it is possible to reduce the stressful and time-consuming handling of fish during breeding under controlled conditions, which has an impact on the welfare of spawners.

Seminal attributes of Vechur bulls (Bos indicus), the smallest humped dairy cattle

The study was undertaken at Mattupatti semen station to objectively evaluate the seminal attributes of frozenthawed semen of Vechur bulls, the smallest humped dairy cattle in the world, using the computer assisted semen analyzer (CASA) and Flow cytometer. The mean motility and progressive motility of the frozen-thawed semenestimated via CASA was 77.25±9.56 and 64.0±4.21% respectively. Mean values obtained for the sperm kinetic parameters like path velocity (VAP), progressive velocity (VSL), track speed (VCL), lateral head amplitude (ALH),beat cross frequency (BCF), straightness (STR) and linearity (LIN) were 82.56±12.12, 76.22±10.38, 107.77±21.42, 4.39±0.91, 20.87±3.81, 92.37±3.25 and 72.87±6.64% respectively. Flow cytometry analysis of the functional sperm parameters like sperm viability, live sperms with intact acrosome, sperms with normal mitochondrial membranepotential (MMP) and sperms with altered DNA were 81.67±4.55, 76.16±9.76, 82.40±5.61 and 11.75±0.3% respectively. The mean value of the HOS reactive sperms was 71.81±5.71%. Mean values for the length and width of the sperm head, length of mid-piece, tail and overall sperm length measured by computer image analysis was 8.99±0.36, 5.07±0.23, 14.21±0.35, 45.12±1.53 and 68.32±1.41 μm respectively. Study concluded that seminal attributes of Vechur bulls could be estimated with CASA and Flow cytometer and the parameters were comparable to other indigenous, crossbreds and exotic dairy cattle.

The effect of adding pentoxifylline to the Ross rooster semen on quality parameters of spermatozoa during 6 hours of storage at cooling temperature

The most important factors involved in sperm fertility are motility, morphology and sperm membrane integrity. The studies show that, the addition of Pentoxifylline(PTX) can increase sperm motility by increasing energy production. The aim of the present study was to investigate the effect of adding Pentoxifylline to rooster semen on qualitative parameters of rooster sperm after 6 hours of storage at 4°C. Semen samples were collected from 15 Ross 308 roosters at 28 weeks of age. After initial evaluations, semen samples were mixed together to eliminate individual differences. After diluting the semen and adding different levels of Pentoxifylline (0, 0.25, 0.5, 0.75 and 1 μM), the samples were kept at 4°C for 6 hours. Then, sperm kinetic parameters were evaluated. The results showed that the addition of 0.75 μM PTX significantly improved progressive mobility, curvilinear velocity, viability and plasma membrane integrity of spermatozoa compared to the control group (P<0.05). In overall, it can be concluded that methylxanthine-derived drugs such as Pentoxifylline can have a significant effect on the motility, viability and plasma membrane integrity of rooster sperm in the short period of time.

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P&lt;0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P&lt;0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus(R) extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.

Cryopreservation of Spix's yellow-toothed cavy epididymal sperm using Tris- and coconut water-based extenders supplemented with egg yolk or Aloe vera

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.

Docosahexaenoic acid associated vitamin E in the diluent for cryopreservation of goat semen

The objective of this study was to evaluate the effect of and determine the optimum level of inclusion of docosahexaenoic acid (DHA) in the diluent for goat semen cryopreservation. Five Boer males underwent semen collection, totaling 10 viable collections per animal. After evaluation, the ejaculates were pooled and fractionated in Tris-yolk medium with the addition of 0; 30; 45; or 60ng mL-1 of DHA and 0.4 mmol of alpha-tocopherol (Vitamin E). The semen was cryopreserved in a freezing machine (TK 3000TM) and placed in a cryogenic cylinder for subsequent analysis. Data were evaluated by regression analysis at 5% significance. There were no differences (P &gt; 0.05) in sperm kinetic parameters evaluated by computer assisted sperm analysis: total motility (79.17 ± 17.31%), progressive motility (14.04 ± 5.73%), curvilinear speed (58.82 ± 6.35µm/s), progressive linear speed (22.49 ± 3.63µm/s), mean path speed (35.17 ± 4.52µm/s), linearity (38.69 ± 5.79%), rectilinearity (63.99 ± 6.64%), and oscillation index (59.68 ± 2.99%). There were no differences (P &gt; 0.05) found from the membrane functional integrity test for reactive spermatozoa (69.66 ± 9.76%), plasma and acrosomal membrane integrity of intact spermatozoa (29.86 ± 7.57%), mitochondrial potential of Class I cryopreserved goat semen (72.75 ± 9.81%), and chromatin compaction of intact chromatin (96.87 ± 4.37%). Thus, the inclusion of up to 60ng mL-1 of DHA did not promote any improvement in the seminal quality parameters of post-thawed goat semen.

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen.

The aim of this study was to evaluate the effects of adding different concentrations of edible bird’s nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.

61 Swim-up and microfluidic techniques improve the kinetic parameters of selected bovine spermatozoa for invitro fertilization: Preliminary results

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL=curvilinear velocity, VSL=straight line velocity, VAP=average path velocity, ALH=lateral displacement of sperm head, BCF=beat frequency cross, STR=path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2×106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization=Day 0). The results were calculated with analysis of variance and Tukey’s test (P&amp;lt;0.05). The values of sperm kinetic parameters obtained with swim-up (VCL 132.5µm/s; VSL 73.5µm/s) and microfluidic technique (VCL 129.5µm/s; VSL 64.4µm/s) were significantly higher (P&amp;lt;0.05) than those obtained by density gradient (VCL 98.3µm/s; VSL 45.01µm/s). However, the total and progressive motility by density gradient method was slightly higher (89% and 57%) compared with that assessed by swim-up (64% and 43%) or microfluidic technique (74% and 54%) respectively. Microfluidic sorting (11.3%) showed lower (P&amp;lt;0.05) DNA fragmentation levels compared with density gradient method (16.6%), whereas the swim-up technique (12.5%) was similar between both groups. No significant difference was detected between the 3 groups for sperm morphometric and vitality parameters. Moreover, cleavage rates were similar (P&amp;gt;0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P&amp;lt;0.05) using sperm selected by density gradient (28.1%) and swim-up (21.9%) compared with microfluidic sorting (15.3%). In conclusion, sperm selection using microfluidic and swim-up techniques improved kinetic parameters with lower levels of DNA fragmentation, without affecting sperm morphometry. However, both the density gradient and swim-up techniques are efficient systems for producing invitro bovine embryo.

Frecuencia de fotogramas óptima para evaluar la cinética espermática de verracos con un sistema CASA-Mot

Introducción. La movilidad es el indicador de calidad seminal más utilizado, por tanto, es necesario determinar una adecuada frecuencia de fotogramas (frame rate, FR) para estandarizar los análisis cinéticos con los equipos de análisis computadorizado de semen (CASA-Mot) y disminuir la variabilidad entre laboratorios, lo que optimizará el número de dosis seminales y su calidad para la industria porcina basada en inseminación artificial (IA). Objetivo. Determinar la frecuencia óptima de fotogramas sobre la cinética espermática de verraco mediante un sistema comercial CASA-Mot y analizar la distribución de subpoblaciones espermáticas con el FR óptimo. Materiales y métodos. Se utilizaron veinte dosis seminales de diez verracos Pietrain (Sus scrofa domestica). El periodo experimental fue entre febrero y julio del año 2017. Se utilizó el sistema CASA-Mot (ISAS®v1) con cámaras de recuento ISAS®D4C20 precalentadas a 37 °C. El tiempo de captura fue de dos segundos. Resultados. La frecuencia de fotogramas afectó los parámetros de cinética espermática, exceptuando la velocidad rectilínea (VSL), debido a que no representó un comportamiento dependiente del FR. Las demás variables presentaron alteraciones significativas al cambiar su FR, y la velocidad curvilínea (VCL) y velocidad promedio (VAP) fueron las variables más sensibles al cambio. Se obtuvieron tres componentes principales (PC) definidos por patrones de movilidad, progresividad y oscilación, en donde el PC1 (velocidad-ondulación) fue el más influyente, debido a que representó el 51,34 % y 55,08 % de la varianza total explicada para 25 y 200 fps (fotogramas por segundo), respectivamente. Conclusión. La frecuencia óptima de fotogramas para analizar variables de cinética espermática en verraco con un sistema CASA-Mot fue de 200 fps. Una tasa de fotogramas inferior provoca errores en la estimación de la trayectoria de los espermatozoides. La distribución de subpoblaciones espermáticas varía según la tasa de fotogramas utilizada. Es necesario establecer un protocolo de análisis para homogenizar los resultados.

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration.

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5mM caffeine-treated groups). Compared to that in the control, the addition of 5mM caffeine induced an increase in sperm motility (38.9±2.8 versus 32.6±3.4%), and a decrease in nitrite concentration (11.4±2.1 versus 12.8±2.9µM/µg protein, p<.05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.

Butaphosphan and Cyanocobalamin Supplementation in Semen Extender on Chilled Boar Sperm Quality and Life Span.

The objective of the present study was to determine the effect of butaphosphan and cyanocobalamin supplementation in semen extender on chilled boar sperm quality and life span. A total of 35 ejaculates of boar semen were included. The semen was diluted with Beltsville thawing solution extender supplemented with different concentrations of butaphosphan and cyanocobalamin [0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5%] in the diluted semen. The semen samples were evaluated using a computer-assisted sperm analysis system to determine sperm motility and sperm kinetic parameters (i.e., the curvilinear velocity, VCL; straight line velocity, VSL; average path velocity, VAP; linearity, LIN; straightness, STR; amplitude of lateral head, ALH; wobble, WOB; and beat cross frequency, BCF). Additionally, sperm viability, acrosome integrity, mitochondrial activity, and plasma membrane integrity were evaluated after 4 (day 0), 72 (day 3), 120 (day 5), and 168 (day 7) h of storage using SYBR-14–ethidium homodimer-1 (EthD-1), EthD-1, JC-1, and the short hypo-osmotic swelling test, respectively. The analyses were carried out by using the general linear mixed model (MIXED) procedure of SAS. The statistical models for each data set included group, day after storage, and interaction between group and day after storage. The boar was included as a random effect. On day 0 after storage, progressive motility, VCL, VSL, VAP, and plasma membrane integrity of boar sperm in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the 0.4 and 0.5% groups (P < 0.05). On day 3 after storage, total motility and progressive motility, VCL, VSL, VAP, LIN, WOB, BCF, and plasma membrane integrity in 0.3% of butaphosphan and cyanocobalamin supplementation were significantly greater than those in the control group (P < 0.05). The total motility and progressive motility, VAP, and WOB in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 5 after storage (P < 0.05). No effects of butaphosphan and cyanocobalamin supplementation on acrosome integrity and mitochondria activity were found on days 3, 5, and 7 after storage. However, the motility and progressive motility and the values for all sperm kinetic parameters except ALH in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 7 after storage (P < 0.05). In conclusion, 0.3% of butaphosphan and cyanocobalamin supplementation in semen extender improved sperm motility, sperm activity, morphology, and life span in chilled boar sperm.

Parámetros cinéticos de espermatozoides en semen fresco y crioconservado de alpaca (Vicugna pacosL.)

Cryopreservation of alpaca sperm is important for genetic improvement. The purpose of this work was to evaluate the sperm kinetic parameters in fresh semen and cryopreserved in dilutors, using the CASA team. 3 male alpacas were used, which were trained for a month in the semen collection with the artificial vagina. The sperm was frozen using two diluents (commercial AndroMed) and (commercial AndroMed, citric acid, gentamicin and glycerol). The semen had the following characteristics, volume 1.5±0.7 mL, viscosity 5.9±1.5, pH 7.1, with an alkalinity tendency and the color that predominated was the translucent white. The averages of kinetic parameters found in fresh semen were VCL (25.8±4.2 μm/s), VSL (10.3±2.5 μm/s) and VAP (18.9±3.2 μm/s). Kinetic parameters in cryopreserved semen showed that dilutor 2 VCL (8.4±1.4 μm/s), dilutor 1 (7.2±1.2 μm/s), VSL dilutor 2 (3.4±0.8 μm/s), dilutor 1 (2.9±0.7μm/s), VAP dilutor 2 (5.8±1.0 μm/s), dilutor 1 (4.8±0.8 μm/s). It is concluded that dilutor 2 is another alternative for cryo-preservation of alpaca semen.

D-aspartate treatment in vitro improves mouse sperm fertility in young B6N mice

We previously reported that the administration of d-aspartate (D-Asp) in drinking water over a 2-4-week period to 7-week-old mice resulted in higher sperm quality and increased in vitro fertilisation (IVF) rates associated with a systemic increase of luteinizing hormone and testosterone levels in the serum. The goal of this study was to investigate the effects of in vitro treatment with D-Asp on the IVF rate, embryo transfer, and sperm parameters of cryopreserved-thawed C57BL/6NTacCnrm (B6N) spermatozoa derived from young and adult mice. In this study, cryopreserved-thawed B6N spermatozoa from males aging 9, 11, 13, and 16 weeks were treated for 1 h with 4 mM D-Asp during capacitation. Thereafter, the in vitro fertilisation ability and the embryo transfer efficiency were analysed. Also, the kinetic activity of the treated spermatozoa and the acrosome reaction were measured after 1 h, 2 h, and 5 h of incubation. The capacitation rate of spermatozoa was determined after 1 h of pre-incubation. Spermatozoa from 9- and 11-week-old mice, which were treated with D-Asp, led to significantly increased IVF rates. However, spermatozoa derived from 13- and 16-week-old mice did not lead to a significant improvement in the fertilisation rate. At all ages examined, no differences were observed in the birth rate and sperm kinetic parameters. After 1 h incubation under the same conditions as the IVF was performed, the capacitation rate and the acrosome reaction were significantly higher with the D-Asp-treated spermatozoa from 9-week-old (67.5% vs. 41% and 14.5% vs. 10.5%, respectively) and 11-week-old mice (78.5% vs. 41.1% and 21.0% vs. 3.8%, respectively), corresponding to the improved IVF results. Therefore, the present results demonstrate, for the first time, a direct role of D-Asp in the capacitation process and acrosome reaction.

Effect of Tripterygium Glycosides Tablets on reproductive toxicity in male rats with Ⅱ type collagen induced arthritis

The aim of this paper was to observe the toxic effect of Tripterygium Glycosides Tablets on the reproductive system of Ⅱ type collagen induced arthritis(CIA) male rats, and to explore the toxic mechanism preliminarily. Fifty SD rats were randomly divided into normal control group(Con), model group(CIA), Tripterygium Glycosides Tablets clinical equivalent dose groups of 1, 2, 4 times(9, 18, 36 mg·kg~(-1)), 10 rats in each group, and were given by gavage once a day for 42 days after the first immunization. The organ index of testis and epididymis were calculated on days 21 and 42. Histopathological and morphological changes of testis and epididymis were observed under optical microscope. Sperm count, sperm malformation rate and sperm kinetic parameters in epididymal tissues were observed by computer assisted sperm analysis(CASA). The concentration of testosterone(T), nitric oxide synthase(NOS) and aromatase(CYP19 A1) in serum were detected by ELISA. Immunohistochemistry was used to observe the expression of Bax and Bcl-2 related proteins in the apoptosis pathway of testis and epididymis. The results showed that, compared with Con group, CIA group significantly increased the rate of testicular spermatogenic tubule lesion and sperm malformation, decreased the average path speed, and no significant changes were observed in other groups. Tripterygium Glycosides Tablets at 4 times clinical equivalent dose can significantly reduce the testis index(P&lt;0.01), each dose group can reduce the epididymis index(P&lt;0.05). Each dose group of Tripterygium Glycosides Tablets could cause different degrees of damage to the testis and epididymis, the proportion of testicular histopathology lesions increased, the number of spermatogenic cells in the seminiferous tubules decreased, and so on. It could reduce the number of sperm, increase the rate of sperm deformity, make the parameters of sperm dynamics abnormal, and so on. Tripterygium Glycosides Tablets at 4 times dose could significantly reduce the content of serum sex hormone T and key enzyme of androgen synthesis(P&lt;0.05 or P&lt;0.01), but had no effect on CYP19 A1. The expression of Bax and Bcl-2 in testis and epididymis were increased by 2 and 4 times doses of Tripterygium Glycosides Tablets(P&lt;0.05, P&lt;0.01 or P&lt;0.01). The results showed that 21 d administration of Tripterygium Glycosides Tablets at equal or higher doses could induce obvious toxic effect to the reproductive organs of CIA male rats, and lower the level of serum sex hormone T and the key enzyme of androgen synthesis, NOS. The mechanism of abnormal changes of Bax and Bcl-2 in Testis and epididymis is still to be elucidated.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca)

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.