Characteristics of chimpanzee (Pan troglodytes) ejaculates collected by rectal probe electrostimulation and by artificial vagina

This study compares characteristics of ejaculates collected from 16 adult male chimpanzees using rectal probe electrostimulation (RPE) and from 10 adult male chimpanzees trained to use an artificial vagina (AV). Ejaculate weight, semen volume, and sperm number were significantly lower (P < 0.01) and percentage liquefaction was significantly higher (P < 0.01) in ejaculates collected by RPE. Percentages of motile sperm and of live sperm in semen did not differ significantly between the two collection methods. Total amounts of protein and of α-glucosidase activity were significantly lower (P < 0.01) in seminal fluid from RPE samples. For ejaculates collected by RPE, semen volume correlated positively with protein (r = 0.8640, P < 0.001), fructose (r = 0.6976, P < 0.001), and citrate (r = 0.6976, P < 0.001); sperm number correlated positively with α-glucosidase activity (r = 0.6547, P < 0.001); and protein correlated positively with fructose (r = 0.5906, P < 0.002), citrate (r = 0.5926, P < 0.002) and α-glucosidase activity (r = 0.6006, P < 0.001). For ejaculates collected by AV, semen volume correlated positively with percentage liquefaction (r = 0.6058, P < 0.001), protein (r = 0.8055, P < 0.001), fructose (r = 0.6606, P < 0.001), and citrate (r = 0.8272, P < 0.001); sperm number correlated positively with percentage of motile sperm (r = 0.4196, P 0.004); percentage of motile sperm correlated positively with percentage of live sperm (r = 0.4388, P < 0.002); and, protein correlated positively with fructose (r = 0.6947, P < 0.002) and with citrate (r = 0.5926, P < 0.002). These data show that there is a significant difference in semen parameters and in biochemical parameters of ejaculates obtained by RPE and by AV. © 1995 Wiley-Liss, Inc.     

Motion characteristics of Murrah buffalo bull spermatozoa in various seasons and its relationship with functional integrity of the plasmallema

Semen was collected from six adult (3.5-7-year-old) Murrah buffalo bulls at weekly intervals for 1 year and evaluated for routine parameters, motion characteristics, reactivity in hypoosmotic solution, and acrosomal and other morphological abnormalities of the spermatozoa. The overall motility (MOT), straight line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), lateral head displacement (ALH) and average path velocity (VAP) were 66.85+/-2.79%, 26.58+/-0.24 and 107.07+/-1.47 microm/s, 26.91+/-0.01%, 11.19+/-0.09 and 61.78+/-2.79 microm/s, respectively. Significant seasonal variation was observed in sperm kinematics and hypoosmotic swelling (HOS) reactivity. Except for LIN, the mean values of sperm dynamics were higher during summer and rainy season and significantly lower in winter season. Sperm kinematics showed significant (P<0.01) positive correlation (r=0.25-0.60) with plasmallemal integrity. Ejaculates with less than 50% HOS-reactive spermatozoa had significantly lowered MOT, VSL, VCL and VAP as compared to the ejaculates with >50% HOS-positive spermatozoa. No significant difference was observed in sperm kinematics among the ejaculates having 50-70% and >70% HOS-reactive spermatozoa. The trend of motion dynamics of the spermatozoa with respect to HOS reactivity was similar in all the three seasons (summer, rainy and winter). The results indicate that ejaculates having more than 50% of HOS-reactive sperm show a higher magnitude of sperm kinematics compared to ejaculates having less than 50% HOS-positive spermatozoa.     

Semen characteristics of the adult male chimpanzee (Pan troglodytes)

The chimpanzee, because of its similarities to the human, is especially valuable in studies of reproductive function. However, relatively little is known about the physiology of reproduction in the adult male chimpanzee. This study provides, for five adult male chimpanzees, baseline values for testicular volume without and with pressure and for cellular and biochemical characteristics of ejaculates collected by artificial vagina (AV). There was no correlation between body weight and testicular volume measured without or with pressure. The ratios of mean testicular volume without and with pressure were not statistically different among animals. Statistical analysis of penetration of denuded hamster oocytes by ejaculated chimpanzee sperm revealed no correlations between sperm count and percentage of eggs penetrated. There was significant variability in concentrations of protein and fructose and in activities of alpha-glucosidase and acid phosphatase among samples from different animals. Computer-assisted motion analysis (CAMA) of sperm provided baseline data on motion parameters necessary for future evaluation of this technique for semen analysis in the chimpanzee. The level of demonstrated inter-animal variation mandates use of each animal as its own control for studies on normal and altered reproductive function. © 1993 Wiley-Liss, Inc.     

The effect of temperature and pH on the motility and viability of ostrich sperm

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.     

Prediction of human sperm penetrating ability using computerized motion parameters

The sperm penetration assay (SPA) is used to assess male fertilizing potential but it is tedious and costly. Computer analysis could replace the need for the SPA in some cases, if computerized sperm motility parameters are highly predictive of SPA performance. The objective of this study was to determine whether computerized motility parameters from fresh semen samples could be used to predict SPA performance. Computer automated semen analysis (CASA; CellSoft, Cryo Resources) was used to quantitate sperm concentration (CONC), percent motility (MOT), curvilinear velocity (VEL), linearity of swimming trajectory (LIN), mean amplitude of lateral head displacement (ALH), and beat/cross frequency (B/CF). The SPA was performed using either Biggers, Whitten, and Whittingham's medium (BWW) or TEST-yolk buffer (TYB). Patients were divided into three groups depending on SPA performance: group 1, BWW-treated, 0% versus greater than 0% penetration; group 2, TYB-treated, 0% versus greater than 0% penetration; and group 3, TYB-treated, less than 20% versus less than or equal to 20% penetration. SPA performance was highly correlated with CONC, MOT, VEL, and B/CF. CONC, MOT, VEL, and B/CF were significantly higher for patients who penetrated in the SPA than for those who failed to penetrate. Discriminant function analysis (DFA) successfully classified 76% of all patients treated with TYB (group 2) who penetrated and 86% of nonpenetrators based on their computerized motility parameters. For group 2 DFA predicted that 93 men would penetrate in the SPA with TYB. Of these, 90 (97%) successfully penetrated at least one egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Ile de France rams

The aims of this study were to identify different motile sperm subpopulations in fresh ejaculates from six Ile de France rams, by using a computer-assisted sperm motility analysis (CASA) system, and to evaluate the effects of individual ram and season on population distribution. Overall sperm motility and individual kinematic parameters of motile spermatozoa were evaluated for 125,312 spermatozoa, defined by curvilinear velocity (VCL), linear velocity (VSL), average path velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR), wobble coefficient (WOB), mean amplitude of lateral head displacement (ALH) and frequency of head displacement (BCF). A multivariate cluster analysis was carried out to classify these spermatozoa into a reduced number of subpopulations according to their movement patterns. The statistical analysis clustered the whole motile sperm population into five separate groups: subpopulation 1, constituted by rapid, progressive and non sinuous spermatozoa (VCL=126.41 μm/s, STR=92.87% and LIN=86.47%); subpopulation 2, characterized by progressive spermatozoa with moderate velocity (VCL=74.74 μm/s and STR=84.03%); subpopulation 3, represented by rapid, progressive and sinuous spermatozoa (VCL=130.45 μm/s, STR=76.02% and LIN=47.68%); subpopulation 4 represents rapid nonprogressive spermatozoa (VCL=128.69 μm/s and STR=44.09%); subpopulation 5 includes poorly motile, nonprogressive spermatozoa with a very irregular trajectory (VCL=36.81 μm/s and STR=47.04%). Our results show the existence of five subpopulations of motile spermatozoa in ram ejaculates. The frequency distribution of spermatozoa within subpopulations was quite similar for the six rams, and the five subpopulations turned out to be very stable along seasons.     

Computerized analysis of the motility parameters of hamster spermatozoa during maturation

A computer-aided semen analysis system was used for the objective assessment of hamster spermatozoa during epididymal maturation. The caput epididymal spermatozoa were extremely sluggish, achieved very little progression, and the three velocity parameters, namely curvilinear velocity (VCL), progressive velocity (VSL), and path velocity (VAP), were low. These spermatozoa during progressive movement alternated between the linear shape and “U” shape or attained an “S” shape prior to changing to the “U”; shape. The corpus epididymal spermatozoa were faster, displayed greater VSL, VAP, and VCL compared to caput epididymal spermatozoa, and, during forward motility, attained “U,” “C,”; and (or) “?” shape as in the wriggling motility pattern. The proximal cauda epididymal spermatozoa were actively motile and VSL, VAP, and VCL in these spermatozoa were more than 10 times greater compared to the caput epididymal spermatozoa. The proximal cauda epididymal spermatozoa predominantly moved in circles and with time became slower and more circular in their trajectories and exhibited a reduction in LIN (linearity). The distal cauda epididymal spermatozoa were very similar to the proximal cauda epididymal spermatozoa with respect to their fast motility (VSL, VAP, and VCL are similar) and beat cross frequency (BCF), but showed larger values for STR (straightness) and LIN and moved along curved trajectories. The amplitude of lateral head displacement (ALH) was also considerably lower in the distal cauda epididymal spermatozoa compared to the proximal cauda epididymal spermatozoa. Thus, this study provides for the first time data related to seven motility parameters for caput and corpus epididymal spermatozoa of hamster. It also provides additional data with respect to VCL, LIN, BCF, and ALH for proximal and distal cauda epididymal spermatozoa of hamster. © 1994 Wiley-Liss, Inc.     

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.     

Dynamics of motile-sperm subpopulation structure in boar ejaculates subjected to "in vitro" capacitation and further "in vitro" acrosome reaction

Incubation of diluted boar sperm from fresh ejaculates in a previously established "in vitro" capacitation medium induced a significant, time-dependent increase in several mean parameters of sperm motility, such as curvilinear velocity (VCL), linear velocity (VSL), mean velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR) and wobble coefficient (WOB). Furthermore, motile boar-sperm semen samples were structured in four definite subpopulations. Subpopulation 1 showed the lowest values of VCL, VSL and VAP and also low values of linearity. Subpopulation 2 showed the second lowest values of VCL and VAP and higher values of LIN and STR. Subpopulation 3 was characterized by high values of velocity and low values of linearity. Finally, Subpopulation 4 was characterized by high values of velocity and linearity. "In vitro" capacitation and further acrosome reaction induced changes in the motility characteristics of each subpopulation as well as in their percentage distribution, Subpopulations 3 and 4 being those that showed the most significant changes. However, despite these changes, the observed, overall four-subpopulation structure was firmly maintained during the entire "in vitro" capacitation and acrosome-reaction process. Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

Zwitterionic buffers preserve ram semen quality more efficiently than TRIS during storage at 15 °C

This study was designed to evaluate the effect of various buffers on the storage of ram semen at 15 °C. Second ejaculates from six adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15 °C and the sperm motility and viability (membrane integrity) variables assessed after 0, 24 and 48 h intervals. Significantly higher progressive sperm motility rates were recorded at 0 h of storage, and higher motile and progressive sperm motility at 24 and 48 h, when zwitterionic-based extenders (MOPS, TES and HEPES) were used, compared to citrate, TRIS, and phosphate-based extenders—with the last group showing a drastic reduction in sperm motility during storage. The zwitterionic groups were also superior to the other treatments in terms of sperm velocity (straight line velocity, VSL; curvilinear velocity, VCL; average path velocity, VAP) at 0 h of storage, although at 24 and 48 h the differences were minimal in the CITRATE group—regarding all velocity variables, and in the TRIS group, regarding the VCL parameter. Sperm diluted in the TRIS-based extender showed a marked increase in the proportion of circular sperm trajectories (lower sperm linearity, LIN, and straightness, STR), and a decrease in the VAP. The reduction in the vigour of the sperm in the TRIS extender (measured by VCL) was less pronounced than in the other groups. At the same time, VSL was reduced, as more sperm moved in circles, and the amplitude of lateral head displacement (ALH) was also dramatically increased. The CITRATE diluent recorded intermediate results—between that of TRIS and the other treatment groups, regarding the variables related to the quality of sperm movement at 0 h storage. However, following CITRATE dilution, semen underwent a clear improvement after a period of 24 and 48 h, so that differences with the zwitterionic groups were attenuated or disappeared. Similarly, the CITRATE group obtained similar or higher viable sperm values, compared to zwitterionic buffers during storage. The TRIS, and particularly the PHOSPHATE diluents, recorded poorer sperm viability after 24 and 48 h of storage. It was concluded that zwitterion-based buffers may be an acceptable alternative for inclusion in the composition of diluents for chilled ram semen storage. On the other hand, TRIS may be seen as having caused drastic modifications of certain sperm kinematic parameters during storage at 15 °C.     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.     

Trehalose enhanced the freezability of Poodle dog sperm collected by an artificial vagina (AV)

In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm.     

Collection method, season and individual variation on seminal characteristics in the llama (Lama glama)

The objective of the present study was to evaluate the effect of semen collection method (electroejaculation "EE" as compared with the artificial vagina "AV"), the season (summer versus winter) and the male used on macroscopic and microscopic characteristics of ejaculates in llamas. A total of 110 ejaculates were collected from six males and 92 of them were analyzed. Ejaculate volume, concentration, total sperm and the following sperm characteristics were studied: motility, membrane function (HOS test), membrane integrity (CFDA/PI fluorochromes) and morphology. A mixed linear model, that considered season and collection method as the fixed variables and the male as the random variable, was used for the statistical analysis. Variability was found between males (p相似文献   

Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters

Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at . That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish (Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300 frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was < or =10%, comparable to values obtained using commercial systems.     

Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes

The objective of this study was to determine the effective relationship between different motion characteristics of bull spermatozoa assessed by computer assisted semen analyzer (CASA) and in vitro fertilization percentage in zona-free hamster oocytes. A total of 64 frozen semen samples from 16 different crossbred bulls (Bos taurusxBos indicus) with four ejaculates from each bull were taken for analysis. Various motion characteristics of spermatozoa like progressive motility, path velocity, progressive velocity, beat cross frequency, straightness and linearity were recorded. Hypo-osmotic swelling test and sperm penetration bioassay were conducted to assess the membrane integrity and fertilization percentage of spermatozoa respectively. Significant positive correlation (P<0.01) was found between fertilization percentage and progressive motility (r=0.791) and between velocity parameters (VAP; r=0.612 and VSL; r=0.625) and fertilization percentage. Among different CASA variables, progressive motility alone contributed to 62.6% variation in the fertilization percentage. The velocity measurements (VAP and VSL) together with progressive motility and %HOS spermatozoa contributed to 66.1% of variation in fertilization percentage which was found to be significant (P<0.05).     

In vitro capacitation of stallion spermatozoa in calcium-free Tyrode's medium and penetration of zona-free hamster eggs

Capacitation of stallion spermatozoa in Tyrode's calcium-free (TCF) medium was assessed. Twelve gel-free ejaculates were collected. After removal from the seminal plasma, cells were washed three times with 0.85% saline containing 0.1% bovine serum albumin (BSA) and resuspended in TCF. Both washing and incubation media were adjusted to pH 8 and 300 to 310 mOsm. Final sperm concentration during incubation was 2 x 10(6) cells/ml. The diluted ejaculates were incubated for 2, 4, 6, 8 and 10 h at 37 degrees C in an atmosphere containing 5% CO(2). Acrosomes were stained with naphthol yellow and erythrocin B initially and after each incubation period and evaluated microscopically. Transmission electron microscopy was used to verify whether normal acrosome reaction was occurring or if cells were degenerating. Penetration of zona-free hamster oocytes was evaluated using 10(3) to 10(4) sperm/ml suspension and coincubating eggs for 3.5 to 4 h with sperm. Penetration tests were done for wash and incubation treatments and recorded positive when swollen sperm heads or male pronuclei were present. Incubation time affected acrosome integrity (P<0.001). Incubation for 8 to 10 h significantly improved acrosome reaction (P<0.001) and the percentage of reacted acrosomes increased sharply after 6 h of incubation (P<0.001). None of the washed sperm penetrated zona-free eggs at zero time, but sperm from all incubation treatments penetrated eggs. A peak penetration rate of 29.9% was observed at 8 h (P<0.001). Results indicate that under the conditions used, the requirement for Ca(++) in the medium for the process of capacitation and acrosome reaction can be substituted for by elevated pH.     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: effects of cryopreservation and between-bull variation

The aims of the present study were: (1) to determine the existence of sperm subpopulations with specific motility characteristics in fresh ejaculates from Holstein bulls, (2) to investigate the effects of semen cryopreservation and post-thaw incubation on the distribution of spermatozoa within the different subpopulations, and (3) to evaluate the existence of between-bull variation in the sperm subpopulations structure of fresh and frozen-thawed semen. Six ejaculates were collected from each of 9 Holstein bulls and cryopreserved following a standard protocol. Overall sperm motility and the individual kinematic parameters of motile spermatozoa, determined using a CASA system, were evaluated before freezing and after 0, 2 and 4h of post-thaw incubation at 37 degrees C. Data from 16,740 motile spermatozoa, defined by VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF, were analysed using a multivariate clustering procedure to identify and quantify specific subpopulations within the semen samples. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patters of movement: Subpopulation (Subp. 1) moderately slow but progressive spermatozoa (23.2%), (Subp. 2) highly active but non-progressive spermatozoa (16.0%), (Subp. 3) poorly motile non-progressive sperm (35.5%), and (Subp. 4) highly active and progressive sperm (25.3%). Subpopulations 2 and 4 significantly (P<0.01) decreased during cryopreservation and post-thaw incubation (Subp. 2: 21.1%, 18.1%, 8.7% and 5.9%; and Subp. 4: 34.1%, 20.6%, 15.2% and 7.3%, respectively, for fresh, 0, 2 and 4h post-thaw) whereas Subp. 3 significantly (P<0.01) increased (10.7%, 27.2%, 27.2% and 30.7%, respectively, for fresh, 0, 2 and 4h post-thaw). The frequency distribution of spermatozoa within subpopulations was quite similar for the 9 bulls, either in fresh or frozen-thawed semen, and differences among bulls were mainly due to differences in the Subp. 4. Significant correlations (P<0.01) were found between the proportions of spermatozoa assigned to Subp. 4 in the fresh ejaculates and those in frozen-thawed semen after 0 (r=0.473), 2 (r=0.513) and 4h post-thaw (r=0.450). This indicated that the ejaculates with the highest subpopulations of rapid and progressive sperm were also the most resistant to cryopreservation and showed the best post-thaw sperm longevity.     

Estimation of genetic parameters for semen quality traits and growth rate in a paternal rabbit line

Variance components of sperm quality traits were estimated in a paternal line of rabbits selected on the basis of daily weight gain (DG, g/day) between 28 and 63 days of age. Features of the marginal posterior distributions for the genetic variance ratios, variance due to non-additive plus environmental permanent male effects, and variance due to litter of birth effects with respect to phenotypic variance are reported. The correlation between sperm quality traits and the selection criteria were also estimated. Nine sets of two-trait analyses were performed involving 12 908 DG records, 2231 ejaculates corresponding to 412 males, and 14 700 animals in the pedigree file. Heritability values (h²) of sperm quality traits commonly evaluated in a classic spermiogram were 0.18, 0.19, and 0.12 for normal acrosome status (NAR) (%, percentage of sperm with intact acrosome), sperm abnormalities (ANR) (%, percentage of sperm abnormalities), and sperm motility (MOT) (%, percentage of total motile sperm cells), respectively. The h² of some motion computer-assisted sperm analysis (CASA) Parameters 0.09, 0.11, 0.10, 0.11, 0.11 and 0.11 for average path velocity (VAP) (μm/sec; average path velocity), straight-line velocity (VSL) (μm/sec; straight-line velocity), curvilinear velocity (VCL) (μm/sec; curvilinear velocity), linearity index (LIN) (%, linearity index), amplitude of lateral head displacement (ALH) (μm; amplitude of the lateral head displacement) and straightness (STR) (%, straightness) were also estimated. Permanent environmental effects were lower than the corresponding values of h² and varied between 0.04 and 0.14. Genetic correlations between DG and sperm traits showed a high interval of highest density of 95% (HPD)_95% (interval of highest density of 95%). However, there is some consistent evidence of the negativity of the genetic correlations of DG with NAR and MOT (−0.40 and −0.53, respectively). Permanent correlations were low, including the zero in the HPD_95%. Litter birth correlations between DG with LIN and STR showed that a favorable effect for growth could be detrimental for them (−0.47 and −0.53). Therefore, as the magnitude of the genetic correlations does not seem very high, it may be possible to define a selection index, including some sperm quality traits that allow improvement of DG without diminishing the semen quality.     

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4 h at 5 °C, and at 0 and 2 h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4 ± 17.8 μm/sec) and high progressiveness (LIN: 65.1 ± 14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7 ± 25.6 μm/sec) but a nonprogressive trajectory (LIN: 33.1 ± 10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3 ± 24.3 μm/sec) and nonprogressive sperm (LIN: 39.6 ± 18.3%); and Subpopulation 4 included very rapid (VCL: 152.8 ± 25.7 μm/sec) and highly progressive sperm (LIN: 70.9 ± 13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P < 0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.