Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes

In a group of normospermic donors exhibiting hamster oocyte penetration scores of 0-100%, multiple regression analysis indicated that only 20% of the variation in fertilizing potential could be explained by differences in the movement characteristics of the spermatozoa following incubation in vitro. When the movement characteristics of the spermatozoa in semen were considered this figure was reduced to 6.8% as a result of significant differences in the motility patterns exhibited by the seminal and post-incubation sperm populations. A much closer relationship was observed between the movement characteristics of human spermatozoa in semen and their ability to penetrate cervical mucus. When differences in motile sperm densities were taken into account, 76% of the variation in cervical mucus penetration could be accounted for by the existence of linear correlations with certain aspects of sperm movement (multiple R = 0.874). Of the various attributes of sperm motility measured (linear velocity of progression, frequency of rotation, amplitude of sperm head displacement, % rolling and % yawing), a failure to exhibit an adequate amplitude of lateral sperm head displacement was consistently found to be the most significant factor determining the success of sperm-cervical mucus interaction (R2 = 0.53).     

Rhesus monkey sperm penetration into zona-free hamster ova: Comparison of preparation and culture conditions

A major problem in development of nonhuman primate in vitro fertilization is the selection of donor males and repeated collection of consistent sperm samples. In practice, collection of a viable semen sample is highly dependent on operator technique and the type of animal restraint. We report an updated method for semen collection from the rhesus monkey (Macaca mulatta), use of TES-Tris (TEST) Yolk Buffer (TYB) for prolonged sperm storage and improved results of hamster ovum penetration assay. Semen was obtained from adult males restrained with 2.0 mg/kg IM ketamine hydrochloride prior to direct penile stimulation (Grass SD-9, frequency 150, delay 9, duration 7, volts 12–18, repeat mode, twin pulse). Liquified semen was washed and centrifuged twice at 100 × g for 5 min in BWW, Ham's F-10 and TALP and allowed to swim-up 60 min at 37° in 5% CO₂ and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4°C, centrifuged at 100 × g for 5 min, and the pellet resuspended in 1.0 ml of TALP or BWW prior to use. Hamster ova penetration was achieved with capacitated macaque sperm. Penetration was significantly improved (P < .001) with preincubation in TYB followed by resuspension in TALP (79%).     

Zona drilling increases the penetrability of rat oocytes matured in vitro

Immature rat follicular oocytes were cultured either with cumulus cells intact (CI) or cumulus-free (CF) in bovine serum albumin (BSA)- or serum-supplemented medium under conditions in which meiotic maturation occurs spontaneously. After 12 h of culture to permit in vitro maturation (IVM), the cumulus cells were stripped from the CI group. Control oocytes recovered 2-4 h after ovulation from oviducts of pregnant mare's serum gonadotropin (PMSG)-treated rats were similarly stripped of cumulus cells. Half the oocytes in each group had holes "drilled" in their zonae pellucidae by topical application of acid Tyrode's solution with a micropipette to enable bypass of the zona barrier to penetration. They were cultured for a further 14-16 h with epididymal sperm and then were assessed for sperm penetration and pronuclear formation. In a preliminary study using various concentration of sperm, 50,000 sperm/ml was identified as an appropriate concentration and was used in all subsequent experiments. For oocytes matured in serum-supplemented medium, penetration rates of non-drilled oocytes-expressed as a percentage of oocytes exposed to sperm for CF, CI, and ovulated oocytes were 10%, 34%, and 80%, respectively (p less than 0.01). Drilling significantly increased the penetration rates of both IVM groups (CF: 40%, CI: 77%) but not of ovulated oocytes (78%). Forty-one percent of non-drilled CF oocytes failed to form normal pronuclei after penetration. This was significantly higher than either the CI (0%) or ovulated (1%) groups (p less than 0.001). Drilling increased the incidence of failure to form normal pronuclei in penetrated oocytes of the CF group (64%) but not of the CI or ovulated groups.2z=     

Motile cells lacking hyaluronidase can penetrate the hamster oocyte cumulus complex

Sperm hyaluronidase is thought to assist in penetration of the extracellular matrix (ECM) between the cumulus and corona radiata cells surrounding mammalian oocytes. The question was asked: Can motile cells which lack hyaluronidase penetrate the hamster oocyte cumulus complex (OCC)? Sea urchin (Strongylocentrotus purpuratus) and frog (Rana catesbeiana) sperm and the unicellular, biflagellated, green alga Chlamydomonas reinhardtii were extracted and found to contain no hyaluronidase activity. Moreover, none of these cells was able to disperse the cumulus cells of hamster OCC, nor did they affect the ultrastructure of the ECM between cells. Fresh hamster OCC were challenged with suspensions of each cell type. Frog and sea urchin sperm penetrated to the zona pellucida surface in less than 5 min. A cell wall-less mutant of Chlamydomonas also penetrated to the zona surface but required longer than 5 min. Wild-type Chlamydomonas penetrated only halfway to the zona, perhaps because its cell wall adhered to the ECM between the cumulus cells and retarded its movement. The motility of the frog and sea urchin sperm was not affected by the ECM of the OCC. Frog sperm exhibited slow lethargic motility yet had no difficulty penetrating to the zona; this indicates that hyperactivated motility is not required for penetration of the ECM. None of the challenge cells penetrated the zona pellucida, although the frog sperm did compress the weave of the zona. These data show that motile cells which lack the enzyme hyaluronidase can readily penetrate the ECM of the hamster cumulus and corona radiata and suggest that the significance of hyaluronidase in fertilization should be reevaluated.     

Factors impacting equine sperm recovery rate and quality following cushioned centrifugation

Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P<0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P<0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400xg or 600xg were applied (90 and 90%, respectively; P>0.05), and no resulting differences in semen quality were detected between these treatment groups (P>0.05). The type of iodixanol cushion medium used (i.e., OptiPrep, Eqcellsire Component B, or Cushion Fluid did not impact post-centrifugation semen quality, based on the laboratory values measured (P>0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal.     

Functional parameters of chimpanzee (Pan troglodytes) sperm from ejaculates collected by rectal probe electrostimulation and by artificial vagina

This study compares functional parameters of sperm from ejaculates collected from 15 adult male chimpanzees using rectal probe electrostimulation (RPE) and from 10 adult male chimpanzees trained to use an artificial vagina (AV). Computer assisted motion analysis (CAMA) showed no significant differences in mean values for straight line velocity (VSL), linearity (LIN), curvilinear velocity (VCL), and lateral head movement (ALH) of sperm from ejaculates collected by RPE and by AV. There was, however, a significant difference (P < 0.01) in the population distribution for VSL and LIN, which indicates that sperm swim in a more convoluted manner in ejaculates collected by RPE than in ejaculates collected by AV. In the hamster zona-free ovum penetration assay (SPA), there were no significant differences in the percentages of hamster oocytes penetrated by sperm or in the number of sperm which penetrated each oocyte after 4 or 24 h incubation using sperm from ejaculates collected by RPE and by AV. Therefore, the lack of success using sperm from ejaculates collected by RPE to initiate pregnancy in the chimpanzee does not appear to result from abnormalities in sperm fertilizing capacity as measured in SPA. © 1996 Wiley-Liss, Inc.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing

The objective of this study was to evaluate the effects of two commercially available semen extenders on the motility of cryopreserved goat sperm and to simplify the cryopreservation protocol. Individual goat ejaculates were split and processed in parallel for freezing in either commercially available soy-based extender (Bioxcell®) or egg yolk-based extender (Irvine TYB). Sperm quality was assessed using total and progressive sperm motility, measured by computer-assisted sperm analysis (CASA). Total motility was higher for samples processed in soy-based extender, both at pre-freeze (P = 0.002) and at post-thaw (P < 0.0001). Progressive motility was higher for semen processed in soy extender at post-thaw (P < 0.0001). Approximately 10% of samples processed in egg yolk-based extender had a large (> 50%) reduction in total motility prior to freezing. However, this type of extreme reduction in pre-freeze motility did not occur in semen samples processed in soy extender. In addition, the use of soy-based extender eliminated the need for a time-consuming sperm washing protocol. We concluded that a commercially available soy-based extender was superior to an egg yolk-based extender in preserving motility of cryopreserved goat sperm, using a two-step method.     

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.     

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

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.     

Differences in sperm function in vitro but not in vivo between inbred and random-bred mice

Genetic variation in spermatozoa was used to examine mechanisms important for fertilization in the mouse. A significantly greater proportion of cauda epididymal sperm from C57BL/6 (inbred) males were motile than from random-bred (CFW) males. Random-bred sperm, however, were able to fertilize a significantly greater percentage of eggs in vitro than were inbred sperm. When sperm of these two genotypes were used for insemination in vivo, and the penetrated eggs cultured through the first cleavage, the levels of cleavage were similar, suggesting that neither levels of sperm motility nor sperm penetration in vitro accurately reflect the ability of the same sperm populations to penetrate eggs in vivo.     

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

Genetic correlations between yield traits or days open measured in cows and semen production traits measured in bulls

We used a bivariate animal model to investigate the genetic correlations between yield traits or days open (DO) as characters measured in cows and semen production traits as characters measured in bulls. Lactation records of 305-day milk, fat, and protein yields, and DO, from 386 809 first-lactation Holstein cows in Hokkaido, Japan, that calved between 2008 and 2014 were used. Semen production records were collected between 2005 and 2014 and included volume per ejaculate (VOL), sperm concentration (CON), number of sperm per ejaculate (NUM), progressive motility index of sperm (MOT), and MOT after freeze-thawing (A-MOT). Number of sperm per ejaculate was log-transformed into a NUM score (NUMS). A total of 30 373 semen production records from 1196 bulls were obtained. The pedigree file used for analysing the records was involving 885 345 animals. Heritability was estimated for VOL (0.42), CON (0.12), NUMS (0.37), MOT (0.08), and A-MOT (0.11). Weak and negative genetic correlations were recorded between yield traits measured in cows and VOL, CON or NUMS measured in bulls. Moderate and negative genetic correlations were obtained between DO and MOT (–0.42) or A-MOT (–0.43). Selection focused on MOT or A-MOT measured in bulls may therefore improve DO measured in cows.