Retrograde flow of spermatozoa into the urinary bladder of boars during collection of semen by electroejaculation

Boars that had a catheter implanted in the urinary bladder (n = 11) were used to determine the magnitude of retrograde flow of spermatozoa into the urinary bladder during electroejaculation. The overall mean (+/- SD) number of spermatozoa in the electroejaculate of boars was 22 +/- 20 x 10(9), with a mean range for individual boars of 3 +/- 3 to 48 +/- 13 x 10(9). The overall mean adjusted total number of spermatozoa in the post-electroejaculation urine was 1.038 +/- 2.656 x 10(9), and the mean percentage of retrograde flow of spermatozoa into the urinary bladder among boars ranged from 0% to 32.69%, with an overall mean percentage of retrograde flow of 7.51 +/- 17.82%. These findings indicate that in boars electroejaculation is associated with retrograde flow of spermatozoa into the bladder.     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

Retrograde ejaculation occurs in the dog, but can be prevented by pre-treatment with phenylpropanolamine: a urodynamic study

Retrograde ejaculation is partial or total propulsion of semen from the posterior urethra into the urinary bladder; it is well characterized (and relatively common) in humans, with only a few reports in animals. Our objectives were to determine whether retrograde flow of semen occurred during ejaculation in mature dogs with normal fertility, and to determine the effects of phenylpropanolamine on this phenomenon (dose-titration, switch-back study). Retrograde ejaculation and urethral pressure profile measurements were evaluated (double-blind) in six dogs after 5 days of oral treatment with phenylpropanolamine (0, 2, 4, or 8mg/kg); all dogs received all treatments (at 2-week intervals). The number of sperm in the urine was determined before and after each manual sperm collection. Urethral pressure profiles were obtained three times during each procedure. In the absence of phenylpropanolamine, sperm were present in the bladder after semen collection in all dogs (number varied significantly among individuals). The mean (+/-S.D.) number of sperm in the bladder was 17.0+/-5.0, 18.5+/-1.2, 5.1+/-5.0, and 4.8+/-0.1x10(6) sperm for 0, 2, 4, and 8mg/kg, respectively (no significant difference between dogs given 4 or 8mg/kg, but both were significantly lower than those given 0 or 2mg/kg). This reduction was significantly correlated to the increase in mean urethral pressure at the level of the sphincter (39cm versus 59cm H(2)O in placebo-treated dogs versus those given 8mg/kg). In conclusion, we confirmed that retrograde ejaculation occurred during the ejaculatory process in normal dogs, and we demonstrated that phenylpropanolamine (4 or 8mg/kg once daily for 5 days before collection) increased urethral pressure and reduced the number of sperm voided into the bladder during ejaculation.     

A preliminary technique for electro-ejaculation of agouti (Dasyprocta leporina)

This study focused on the effectiveness of electro-ejaculation of the agouti (Dasyprocta leporina) and analysis of the collected semen. Sexually mature male agouti were anesthetized with ketamine and electro-ejaculated. The means of ejaculation time was 5.48+/-0.31min, ejaculation voltage was 9.33+/-0.69V, ejaculation voltage range was 6-12V, amps was 0.5-1.5amps, ejaculation volume was 0.47+/-0.12ml and pH was 8.3+/-0.09. Mean spermatozoa concentration, motility and percentage abnormalities were (106.7+/-31.1)x10(6)spermatozoa/ml, 50.44+/-4.44%, and 35.14+/-2.76%, respectively. Thirty percent of the semen samples collected contained spermatozoa. The most significant result from these experiments was the inverse relationship between ejaculation time and ejaculate volume, which suggests that the maximum ejaculation time for electro-ejaculation of the agouti should not proceed beyond 6min. Secondly, the results indicate that the time of the off periods should be shortened to 3-4s.     

A modified technique for semen collection by electroejaculation in the domestic cat

A method was developed for collecting feline semen by electroejaculation combined with the use of a urethral catheter. The catheter facilitated handling the small volumes of semen for laboratory analysis. In 14 cats, semen volumes ranged from 0.019 to 0.284 ml (mean 0.076) and spermatozoa counts of ejaculates collected in the catheter ranged from 0.32 to 49.60 x 10(6) (mean 11.64 x 10(6)). Nine individuals were evaluated for retrograde ejaculation by quantitation of spermatozoa in pre-ejaculation and post-ejaculation urine samples. No spermatozoa were detected in pre-ejaculation samples but post-ejaculation urine samples contained from 0 to 11.88 x 10(6) (mean 4.55 x 10(6)) spermatozoa. The antegrade portion of the ejaculate contained from 6.3 to 100% (mean 59.1%) of the total number of spermatozoa ejaculated.     

Dietary supplementation with a source of omega-3 fatty acids increases sperm number and the duration of ejaculation in boars

Development of nutritional strategies to increase the production of fertile sperm would further enhance the distribution of superior genetic material by AI. The objective was to determine the effects of a dietary source of omega-3 fatty acids in boars on semen characteristics and sexual behavior. Boars were fed daily 2.2 kg of a diet top-dressed with 0.3 kg of corn (controls; n=12) or 0.3 kg of a supplement containing 31% omega-3 fatty acids (n=12) for 16 weeks. Semen was collected weekly and for boars that received the supplement containing omega-3 fatty acids, total sperm per ejaculate averaged 84.3+/-2.3 x 10(9) (mean+/-S.E.M.) during Weeks 0-7, and increased (P=0.02) to 95.6+/-2.3 x 10(9) during Weeks 8-15. Control boars averaged 86.3+/-2.3 x 10(9) sperm per ejaculate during Weeks 0-7 and 86.4+/-2.3 x 10(9) during Weeks 8-15. Other semen characteristics were similar (P>0.1) between groups. Duration of ejaculation was affected by treatment (343.9s for controls and 388.8s for boars fed omega-3 fatty acids; S.E.M.=15.7; P=0.05). In summary, semen characteristics and sexual behavior were altered in boars fed a supplement containing omega-3 fatty acids. Boar semen is typically diluted to create AI doses containing 3 x 10(9) sperm each; therefore, use of the supplement increased the number of potential AI doses by approximately three per ejaculate after the initial 7 week supplementation period.     

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).     

Capacity of boar spermatozoa to bind zona pellucida proteins in vitro in relation to fertilization rates in vivo

The purpose of this study was to determine variation among boars in the percentage of sperm in an ejaculate that express enhanced binding of zona pellucida proteins during treatment for capacitation in vitro, and to determine whether this relates to fertilizing ability in vivo. Ejaculates (n=35) were collected from 12 boars. A sample of each ejaculate was treated for capacitation in vitro. During incubation, the zona binding ability of spermatozoa was assessed at regular intervals with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP) and propidium iodide, using a flow cytometer. After incubation, a percentage of the sperm had enhanced FITC-sZP binding. The percentage of viable sperm with enhanced FITC-sZP binding, expressed as a percentage of the total sperm population, increased rapidly over the first 60 min and thereafter reached a plateau after 120-180 min. Averaged over all ejaculates, the percentage at 180 min was 46% (range 27-61%); this percentage was significantly different among boars. However, the variation between ejaculates within a boar was relatively small. There was no significant boar effect on the rate at which the percentage of viable cells with enhanced FITC-sZP binding reached the maximum. In ejaculates (n=14) from four boars (selected from the group of 12), we investigated the increase in the percentage of viable sperm with enhanced sZP binding during treatment for capacitation in vitro in relation to the ability to fertilize in vivo. Sows (n=44) were inseminated 4 h after ovulation with a suboptimal insemination dose (0.5x10(9) spermatozoa). Time of ovulation was determined using transrectal ultrasonography and sows were killed at 120 h after ovulation. The percentage of fertilized oocytes, embryo development, and numbers of accessory spermatozoa were determined. The percentage of spermatozoa that were viable and showed enhanced sZP binding after 180 min of incubation was 48 +/- 12% (range 28-56%). The percentage of fertilized oocytes was 85 +/- 27% and 64% of the sows had 100% fertilized oocytes. The percentage of sows with 100% fertilized oocytes correlated well (P< or =0.05, R2=0.98) with the percentage of viable spermatozoa with enhanced FITC-sZP binding after capacitation in vitro.     

Evidence for retrograde flow of spermatozoa into the urinary bladder of bulls during electroejaculation

Spermatozoa were not found in the urine collected from 12 bulls before electroejaculation, whereas spermatozoa were found in the urine collected after electroejaculation. The concentration of spermatozoa in four consecutive samples of urine collected during the first postelectroejaculation micturition did not differ (P > 0.80) within bulls, suggesting that the spermatozoa found in the urine were those that had flowed into the urinary bladder during electroejaculation. The mean percentage of retrograde flow was 21% and ranged from 1 to 50% for the 12 bulls. These findings demonstrate that there was a significant urinary loss of spermatozoa during electroejaculation.     

Effect of ejaculation frequency and season on donkey jack semen

Semen characteristics of first and second successive ejaculates from 6 jacks were evaluated weekly for 12 mo. The semen was collected at 4-h intervals, using an artificial vagina with a female in either natural or induced estrus. The statistical analysis was done by factorial delineation 2 x 2 in randomized blocks. Due to some ejaculation failures, the data had to be divided into 2 Groups (A and B) for statistical analysis: Group A - ejaculates preceded by 2 ejaculates in the previous week and Group B - ejaculates preceded by only 1 ejaculate in the previous week. If no statistical difference was observed between the groups in a given parameter, the data was grouped together. Semen characteristics for the first and second ejaculates, respectively, showed the following mean +/- SEM: gel-free semen volume 29.2 +/- 2.2 and 31.7 +/- 2.2 ml; progressive motility 71.0 +/- 1.6 and 72.9 +/- 1.6%; sperm vigor 3.8 +/- 0.1 and 4.1 +/- 0.1; live spermatozoa for Group A 82.6 +/- 2.1 and 82.3 +/- 2.1%, and for Group B 84.6 +/- 1.4 and 86.6 +/- 1.4%; total number of spermatozoa for Group A 10.6 +/- 0.8 x 10(9) and 5.8 +/- 0.8 x 10(9), and for Group B 13.3 +/- 1.2 x 10(9) and 9.6 +/- 1.2 x 10(9); head abnormalities for Group A 1.2 +/- 0.3 and 1.4 +/- 0.3%, and for Group B 1.6 +/- 0.3 and 1.9 +/- 0.3%; mid piece abnormalities 7.7 +/- 0.7 and 6.1 +/- 0.7%; tail abnormalities 7.3 +/- 0.7 and 6.8 +/- 0.7%; pH 7.6 +/- 0.0 and 7.6 +/- 0.0. Significant differences (P < 0.05) were observed between the animals for all sperm characteristics except for sperm vigor. The means for the first and second ejaculates were significantly different (P < 0.05) for the total number of spermatozoa in all the animals, while the percentage of mid piece abnormalities was significantly different in only 1 jack. Seasonal effects on sperm parameters were observed only for semen pH.     

Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa

The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were evaluated after thawing by computer automated sperm analyzer. The percentage of motile spermatozoa (faster than 30 microns/sec) was assessed. In addition, the velocity of the average path (VAP), the straight line velocity (VSL), the curvilinear velocity (VCL) and the amplitude of the lateral head displacement (ALH) were also measured. In Experiment 1, only glutamine (40 mM) significantly improved sperm motility (56.0% +/- 3.0 vs 49.7% +/- 1.6; P < 0.05) compared with the control extender, while velocities were unaffected at concentrations of 40 to 120 mM. However, at 160 mM, a significant decrease in motility and velocity was observed for all amino acids. In Experiment 2, motility in glutamine (range 41.1% +/- 3.8%; 42.4% +/- 3.6) and proline (43.0% +/- 3.7; 45.6% +/- 3.8) extenders compared with the control (34.7% +/- 1.6) was improved significantly (P < 0.05). Sperm velocity was improved at concentrations higher than 40 mM glutamine and 50 mM proline.     

Effects of prostaglandins and prostaglandin synthesis inhibitors on sexual behavior in boars

Experiments were conducted investigating the effects of prostaglandins and prostaglandin synthesis inhibitors on libido in boars. In Experiment 1, two prostaglandin products were compared with regard to expediting the training of boars for semen collection. On each of five consecutive days, boars received i.m. treatment with saline, dinoprost tromethamine or cloprostenol sodium (n=12/group). On each of day 1 (p=0.06), day 2 (p<0.05), and day 3 (p<0.05), but not on day 4 or 5 (p>0.1), the percentage of boars collected after dinoprost tromethamine, but not cloprostenol sodium, was greater than controls. In Experiments 2 and 3, libido in boars that were trained previously for semen collection was assessed after treatment with prostaglandin synthesis inhibitors, testing the hypothesis that endogenous release of prostaglandin is necessary for expression of sexual behaviors. In Experiment 2, boars treated with flunixin meglumine (n=12) had suppressed (p<0.01) levels of 15-ketodihydro-prostaglandin-F(2) (PGFM) in serum but characteristics of libido were similar (p>0.1) to controls (n=12). In Experiment 3, boars were administered indomethacin orally (n=12) or served as untreated controls (n=12). Indomethacin decreased (p<0.01) serum levels of PGFM, increased (p<0.05) the number of false mounts (mounting artificial sow but dismounting before an ejaculate was collected), and tended (p=0.09) to lengthen the interval between entering the collection pen and the start of ejaculation. These results suggest that prostaglandin synthesis and release is necessary for the complete display of normal sexual behaviors in boars.     

Seminal collection, seminal characteristics and pattern of ejaculation in llamas

Semen was collected from 10/10 llamas during 26/30 (87%) collection attempts using an artificial vagina mounted inside a surrogate female. For the 26 semen collections, the duration of copulation (mount to dismount) with the artificial vagina was 31.7 +/- 12.0 min (mean +/- SD). Seminal pH was 8.1 +/- 1.1, and seminal volume per collection was 3.0 +/- 1.9 ml. Sperm concentration per collection was 1.0 +/- 0.8 x 10(6) sperm/ml, total number of spermatozoa was 2.9 +/- 3.1 x 10(6), total sperm motility was 23.7 +/- 20.0%, and the percentage of morphologically normal spermatozoa was 39.7 +/- 18.5%. Morphologically abnormal spermatozoa were categorized according to abnormal heads (20.1 +/- 19.9%), tail-less heads (8.7 +/- 8.9%), abnormal acrosomes (12.9 +/- 12.4%), abnormal midpieces (1.0 +/- 3.7%), cytoplasmic droplets (11.1 +/- 12.4%), and abnormal tails (6.6 +/- 12.0%). There were 0.3 +/- 0.3 million motile, morphologically normal spermatozoa per collection: less than 1000 during the first 5 min of copulation, 0.01 +/- 0.01 x 10(6) between 5 and 10 min of copulation, 0.04 +/- 0.08 x 10(6) between 10 and 15 min of copulation, 0.09 +/- 0.21 x 10(6) between 15 and 20 min of copulation, and 0.15 +/- 0.28 x 10(6) between 20 min and the end of copulation.     

Cellular and biochemical characteristics of semen obtained from pubertal chimpanzees by masturbation

Semen characteristics were studied in 6 wild-born chimpanzees with dental ages ranging approximately from 6 to 12 years. The animals formed 2 groups, early pubertal (EP, N = 3, 6-9 years) and late pubertal (LP, N = 3, 11-12 years). Mean body weight, testicular volume and serum androgen concentration were significantly lower in Group EP (32.2 +/- 1.6 kg, 34.0 +/- 7.7 cm3, 2.1 +/- 0.1 ng/ml) than in Group LP (55.7 +/- 5.7 kg, P less than 0.01; 100.5 +/- 11.9 cm3, P less than 0.01; 3.6 +/- 0.7 ng/ml, P less than 0.05). Ejaculates were obtained by masturbation in all subjects. The mean ejaculate volume was lower in Group EP (0.56 +/- 0.20 ml) than in Group LP (3.77 +/- 0.73 ml, P less than 0.01). In Group EP, 2 animals were azoospermic while the third produced semen with means of 57.1 x 10(6) spermatozoa per ml, 20% motility and 40% vitality. These values were low when compared with the mean values of Group LP (376 x 10(6) spermatozoa per ml, 67% motility and 78% vitality). Mean total sperm count was correlated with testicular volume (r = 0.84) and serum androgen concentration (r = 0.96). The mean concentrations of L-carnitine, fructose, citrate and acid phosphatase for the two groups were not significantly different; but, related to the differences in ejaculate volumes, their total amounts in total ejaculate were lower in Group EP than in Group LP. These results suggest that, in chimpanzees, mechanisms of seminal plasma production and ejaculation are functional early in the reproductive life and that the emission of spermatozoa occurs later.     

Adjustments in IVF system for individual boars: value of additives and time of sperm-oocyte co-incubation

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.     

Thawing dog spermatozoa in just-boiled water: submersion time and effect on sperm quality compared to thawing in water at 70 degrees C

Dog spermatozoa have better quality after thawing in water at 70-75 degrees C instead of 35-38 degrees C. The aim of Experiment 1 was to determine the time needed to thaw 0.5 mL straws in just-boiled (98 degrees C) water and that of Experiment 2 to determine whether thawing frozen dog spermatozoa in just-boiled water will result in better quality than thawing in water at 70 degrees C. Prior to freezing the straws of Experiment 1, a Type J thermocouple with wire diameters of 0.08 mm (Osiris Technical Systems, Centurion, South Africa) was placed in the center of each of ninety-three 0.5 mL straws (IMV Technologies, L'Aigle, France) filled with extender (Biladyl* with 0.5%, v/v of Equex STM paste**) and 54 filled with extender plus 200 x 10(6)spermatozoa/mL (Minitüb, Germany (*) and Nova Chemical Sales, MA (**)). Thirty straws with extender were thawed in water at 70 degrees C and the others in just-boiled water. Temperatures inside straws were recorded 10 times/s during warming. Two ejaculates were then collected from each of eight dogs and one from each of three others. Extended ejaculates from the same dog were pooled, frozen 8 cm above liquid nitrogen, and 2 straws from each of the 11 batches thawed in water at 70 degrees C for 8s and 2 in just-boiled water for 6.5s. Sperm morphology and viability were assessed on eosin-nigrosin smears made after thawing and the percentage progressively motile spermatozoa was estimated immediately, 1, 2 and 3h after thawing. The optimal submersion time in just-boiled water was 6.5s for both sperm concentrations, resulting in average temperatures of 23.6+/-1.5 degrees C (+/-S.E.M.) and 24.9+/-1.6 degrees C inside straws with extender or extender plus spermatozoa (P=0.6). The temperature inside straws thawed in water at 70 degrees C was 13.6+/-1.7 degrees C after 8s. Apart from a 1.5% higher (P<0.05) mean percentage motile sperm 2h after thawing, thawing dog spermatozoa in just-boiled (98 degrees C) water holds no benefit over thawing in water at 70 degrees C, which is easier to do.     

Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser

Computer-assisted sperm morphometry has the potential to eliminate several drawbacks inherent to the current methods of sperm morphology evaluation, and allows for the identification of subtle sperm characteristics which cannot be detected by visual evaluation. In the present study, the Metrix Oval Head Morphology software implemented in the Hamilton-Thorne CEROS (version 12.1; HTR 12.1 Metrix) computer-aided semen analyser was evaluated for canine sperm morphometry and morphology analysis. Comparison of sperm morphometric measurements of 200 spermatozoa from pooled semen samples (n = 4) at 40x and 60x demonstrated a more accurate identification of the sperm head boundaries at a magnification level 60x. Dilution of pooled semen samples (n = 4) to a sperm concentration of 50 x 10(6) ml(-1) allowed for a correct evaluation of the sperm cell dimensions whereas 100 x 10(6) and 200 x 10(6) ml(-1) resulted in a higher percentage of rejected spermatozoa due to overlapping. No differences in morphometric dimensions were found when 100 or 200 spermatozoa were evaluated for each of 15 dogs. The mean morphometric parameters of canine spermatozoa, based on the fresh ejaculates of 23 dogs, were: major 6.65 +/- 0.20 microm; minor 3.88 +/- 0.14 microm; area 20.66 +/- 1.04 microm2; elongation 58.64 +/- 2.58 %; perimeter 17.57 +/- 0.43 microm and tail length 48.93 +/- 10.16 microm. Large variations in morphometric dimensions were detected among individual dogs. After cryopreservation, significantly lower morphometric dimensions were obtained for all the evaluated sperm samples (n = 12). Finally, a correlation of 0.82 (P < 0.05) was established for the percentage of normal spermatozoa assessed by subjective evaluation and by the HTR 12.1 Metrix (n = 39 semen samples). In conclusion, dilution of the semen samples to approximately 50 x 10(6) spermatozoa/ml and an objective lens magnification of 60x, analysing at least 100 spermatozoa, are the technical settings proposed to obtain reliable and objective sperm morphometric measurements by the HTR 12.1 Metrix in canine.     

Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.     

Platelet-activating factor content in boar spermatozoa correlates with fertility

The objective of this study was to evaluate the level of platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] content in spermatozoa between two groups of boars that differ in farrow rate percentages. The boar farrow rate was defined as High if it was > or = 70% and Low if it was < 70%. Fresh, extended semen was collected from sexually mature boars and used in the PAF extractions. Platelet-activating factor was detected in all semen samples assayed. The amount of PAF detected in spermatozoa obtained from the High group ranged from 1.90 to 11.30 pM/10(6) cells. The level of PAF in the Low group ranged from 0.92 to 4.96 pM/10(6) cells. Regression analysis revealed a positive (R2 = 0.369) and significant (P = 0.021) relationship between PAF content in boar spermatozoa and farrow rate. Spermatozoa-derived PAF levels (mean +/- SEM) were significantly higher (P = 0.015) in the High-farrow group (6.75 +/- 1.25 pM/10(6) cells) than in the Low-farrow group (2.45 +/- 0.51 pM/10(6) cells). The PAF content in spermatozoa was significantly higher (P = 0.035) in the High-average (> or = 10.5/litter) number of piglets born group (5.78 +/- 1.24 pM/10(6) cells) than in the Low-average (< 10.5/litter) number of piglets born group (3.34 +/- 1.19 pM/10(6) cells). Additionally, PAF content in spermatozoa was significantly higher (P = 0.034) in the High-average (> or = 9/litter) number of piglets born alive group (6.82 +/- 1.35 pM/10(6) cells) than the Low-average (< 9/litter) number of piglets born alive group (3.00 +/- 0.87 pM/10(6) cells). The data demonstrate that PAF is present in boar spermatozoa and that levels are significantly higher in individuals with a high-farrow rate status and high-number of piglets born and born-alive.     

Effect of administration of prostaglandin F2alpha or presence of an estrous teaser bitch on characteristics of the canine ejaculate

Semen was collected from eight dogs after SC administration of 0.1mg/kg PGF2alpha or 0.6 mL 0.9% NaCl solution 15 min prior to collection in the presence or absence of an estrous teaser bitch (switchback design; all dogs given all four treatments in random sequence). There were more spermatozoa (P=0.02) in ejaculates collected after administration of PGF2alpha in the presence of an estrous teaser bitch ((852+/-736)x10(6), mean+/-S.D.) than in ejaculates collected in saline-treated dogs in the absence of a teaser bitch ((371+/-620)x10(6)). However, the number of spermatozoa in the ejaculate of dogs given PGF2alpha in the absence of a teaser bitch and in dogs given saline in the presence of a teaser bitch ((556+/-494 and 600+/-622)x10(6), respectively) were not significantly different from each other or from the other two groups. The percentage of morphologically normal spermatozoa did not vary by treatment (P=0.51). In conclusion, treatment with PGF2alpha and presence of a teaser bitch had an additive effect on the number of spermatozoa. This, coupled with relatively minor side-effects, suggests this is a useful technique to increase number of spermatozoa in a single canine ejaculate.