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.     

Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa.

Approximately 1 week was required to stabilize the extragonadal sperm reserves in stallions ejaculated daily for 10 weeks. The true daily sperm output of a stallion was equal to the mean daily sperm output of seven ejaculates +/- 1-35 X 10(9) spermatozoa. Mean concentrations of spermatozoa/ml and number of spermatozoa/ejaculate were higher (P less than 0-01) for X1 and X3/week ejaculation frequencies than for a X6/week frequency. Sperm output/week was nearly identical for a X6/week frequency. Sperm output/week was nearly identical for the X3 and X6 frequencies and higher (P less than 0-01) than the X1 frequency. Increase of ejaculation frequency from one to two ejaculates/day twice weekly significantly (P less than 0-01) raised the output of spermatozoa/week. Gel-free semen volume, spermatozoa/ml, and number of spermatozoa/ejaculate were higher (P less than 0-01) in the first, than in the second, ejaculate. Collection of semen on alternate days would be a practical ejaculation frequency for inseminating mares. Two ejaculates collected twice a week would be a practical ejaculation frequency for long-term storage of stallion semen.     

Relationship between semen characteristics and fertility in electroejaculated mice

Ejaculates were obtained from C57BL mice by applying two successive series of electrical stimuli which were delivered via a bipolar rectal probe. The ejaculates thus collected contained fertile spermatozoa as indicated by results from in-vitro fertilization. Once separated from the seminal plasma, ejaculated spermatozoa possessed the same in-vitro fertilization rate as epididymal sperm. Ejaculates were analysed for coagulum weight, ejaculate volume, sperm count, sperm motility, acid phosphatase content and fructose content. Significant differences were present between several of these values for fertile and infertile mice, and values were therefore empirically assigned to represent minimal amounts for 'normal' fertility (1.5 microliters ejaculate volume; 10.2 mg coagulum weight; 2.5 x 10(6) spermatozoa/ml; 2.3 x 10(3) motile spermatozoa/ejaculate). One half of the fertile animals had no deficiencies in any of the characteristics measured, whereas 97% of the infertile animals had at least one deficiency. No fertile male had more than 2 deficiencies. These data show that the characteristics of mouse semen obtained by the present method of electroejaculation are related to the fertility status of the animal.     

Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years.

OBJECTIVE: To determine whether the quality of semen has changed in a group of over 500 Scottish men born between 1951 and 1973. DESIGN: Retrospective review of data on semen quality collected in a single laboratory over 11 years and according to World Health Organisation guidelines. SETTING: Programme of gamete biology research funded by Medical Research Council. SUBJECTS: 577 volunteer semen donors. Of these, 171 were born before 1959, 120 were born in 1960-4, 171 in 1965-9, and 115 in 1970-4. MAIN OUTCOME MEASURES: Conventional criteria of semen quality including semen volume (ml), sperm concentration (10(6)/ml), overall motility (% motile), total number of sperm in the ejaculate (10(6)), and total number of motile sperm in the ejaculate (10(6)). RESULTS: When the four birth cohort groups were compared a later year of birth was associated with a lower sperm concentration, a lower total number of sperm in the ejaculate, and a lower number of motile sperm in the ejaculate. The median sperm concentration fell from 98x10(6)/ml among donors born before 1959 to 78x10(6)/ml among donors born after 1970 (P=0.002). The total number of sperm in the ejaculate fell from 301x10(6) to 214x10(6) (P=0.0005), and the total number of motile sperm in the ejaculate fell from 169.7x10(6) to 129.0x10(6) (P=0.0065). CONCLUSION: This study provides direct evidence that semen quality is deteriorating, with a later year of birth being significantly associated with a reduced number of sperm in adult life.     

Seasonal emission of seminal coagulum and in vivo sperm dynamics in the black-handed spider monkey (Ateles geoffroyi)

The ejaculate of diverse primate species consists of two portions, liquid and solid; the latter, known as the seminal coagulum, is thought to sequester large numbers of sperm. In the black-handed spider monkey (Ateles geoffroyi), ejaculates collected by electroejaculation did not always contain seminal coagulum. The objective of the present study was to determine seasonal emission of seminal coagulum and in vivo sperm dynamics in the black-handed spider monkey. Seminal coagulum emission was related to season; it was more frequent in the dry season, coincident with maximal female fertility. Sperm concentration was higher (P = 0.02) in the dry season (dry vs. rainy season: 137.9 +/- 15.7 sperm/mL vs. 82.56 +/- 14.7 x1 0(6) sperm/mL; mean +/- S.E.M.) but also in ejaculates (collected during the rainy season) that had seminal coagulum (coagulum vs. no coagulum: 140.0 +/- 29.3 sperm/mL vs. 31.2+/-0.1 x 10(6) sperm/mL, P<0.001). In semen samples collected from the uterus after AI, the percentage of linearly motile sperm was higher during the dry season (dry vs. rainy: 9.1+/-2.1% vs. 5.9+/-2.5%), as well as whenever coagulum was present (coagulum vs. no coagulum: 13.0+/-3.2% vs. 2.0+/-0.9%, P<0.001).     

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.     

Semen characteristics of the guinea fowl Numida meleagris meleagris

Eighteen adult exotic Golden Sovereign guinea fowl (Numida meleagris meleagris) males identified by leg bands and housed individually in cages were ejaculated two times a week at 4- and 3-d intervals (Mondays and Thursdays) for 6 wk. Semen was obtained manually by gently massaging the dorso-lateral lumbo-sacral region. Semen collection and evaluation were done between 1400 and 1800 h each day of test. Mean semen volume was 0.032 +/- 0.001 ml, sperm motility was 37.1 +/- 0.1% and sperm concentration/ml was 2.62 +/- 0.01 x 10(9). Percentage live sperm averaged 91.6 +/- 0.1, while the mean percentage morphologically normal spermatozoa was 76.9 +/- 0.5. Primary and secondary sperm abnormalities were 11.9 +/- 0.2% and 11.3 +/- 0.2%, respectively. Birds differed significantly in all ejaculate characteristics studied except percent secondary abnormalities, indicating that considerable variation exists for improvement of these semen traits. There were significant bird x collection interval (P<0.05) and bird x week (P<0.01) interactions for sperm concentration/ml and bird x week interaction for sperm motility (P<0.05). The results generally are within acceptable levels and show that the semen is suitable for use in artificial insemination.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Comparison of seminal quality in Holstein bulls as yearlings and as mature sires

Semen quality was compared in 5 Holstein bulls from samples collected as young sires (yearlings) and again as mature bulls after a mean interval of 1,265 d. At both sampling periods, the semen was examined for ejaculate volume, sperm numbers, post-thaw progressive motility and sperm viability. Sperm viability was assessed on cryopreserved samples with fluorescent SYBR-14 to stain living spermatozoa and propidium iodide (PI) to identify dead spermatozoa. The fluorescent populations of stained spermatozoa were quantified by flow cytometry. The percentages of living spermatozoa for the individual bulls, as determined by green fluorescence of SYBR-14, ranged from 44 +/- 3.1 to 54 +/- 0.3 for yearlings, and from 38 +/- 1.5 to 55 +/- 1.0 for mature sires. No differences in sperm viability were found between samples taken from yearling bulls and those of mature bulls. The percentage of spermatozoa stained with SYBR-14 was negatively correlated (r = -0.97; P = 0.0001) with the percentage of dead spermatozoa as indicated by PI staining. Comparisons of identical samples run on 2 different flow cytometers indicated that either flow instrument could be used to assess sperm viability. Although the individual bulls differed (P < 0.05) in ejaculate volume and sperm numbers as yearlings, they did not differ in these parameters as mature bulls. The average number of spermatozoa per ejaculate changed as a result of maturation, increasing from 6.2 +/- 1.0 to 10.7 +/- 1.1 x 10(9). Aging was significantly correlated with ejaculate volume (r = 0.76; P = 0.01) but not with the total number of spermatozoa per ejaculate (r = 0.51; P = 0.13). The maturational changes that occurred in the 5 bulls were minimal with the exception of the increased volume of the ejaculate and the number of spermatozoa per ejaculate.     

Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa

Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 x 10(6)/ml) were incubated for 30 min at 38 C with neutrophils (0,0.5 x 10(6),1 x 10(6), 5 x 10(6) and 10 x 10(6)/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H2O2 was measured at T30 with the Amplex Red assay kit. At T30, there was a significant (P < 0.01) increase in H2O2 with the addition of 5 x 10 and 10 x 10(6) neutrophils/ml activated by FMLP (0.76 +/- 0.3 and 0.99 +/- 0.4 microM, respectively, versus 0.0024 +/- 0.002 microM in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 +/- 5.1 and 48 +/- 6.0%, respectively, versus 80 +/- 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H2O2 with the addition of 5 x 10(6) and 10 x 10(6) neutrophils/ml activated by PMA (1.88 +/- 0.2 and 2.07 +/- 0.3 microM, respectively, versus 0.0009 +/- 0.0006 microM in sperm alone). The results of this study demonstrate that 5 x 10(6) activated neutrophils/ml are sufficient to impair equine sperm motility in vitro.     

Electroejaculation, semen characteristics and serum testosterone concentrations of free-ranging african elephants (Loxodonta africana)

A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anaesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93.3 ml, 2408.6 X 10(6) spermatozoa/ml, 70%, 3.9 and 7.4, respectively. A high percentage (mean 77.5%) of spermatozoa within each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic droplet defect. When sperm viability was tested in vitro at 37 degrees C, sperm motility rating declined by at least half of the initial assessment within 3.5 h of semen collection. Generally, spermatozoa maintained motility in vitro for less than 6 h. Serum testosterone ranged from 1.4 to 8.2 ng/ml in 4 males evaluated in the morning (07:30-08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00-18:00 h), testosterone levels were less than 0.9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25.6 ng/ml) and a preputial discharge potentially indicative of 'musth'. The present study demonstrates that high quality semen can be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.     

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 +/- 0.4 vs 5.08 +/- 0.44; width: 3.08 +/- 0.34 vs 2.71 +/- 0.28 mum, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.     

Frequent semen collection and sperm reserves of the male Angora goat (Capra hircus).

Semen was collected from six mature and sexually rested Angora bucks at one-hour intervals five times a day on each of 5 consecutive days in the breeding season. There was a marked decline in semen volume (P less than 0.001), sperm concentration (P less than 0.05) and number of spermatozoa (P less than 0.001) on consecutive days. Successive ejaculates within days differed only in number of spermatozoa (P less than 0.001). The following year at the beginning of the breeding season, the weights of testes and epididymides and the reserves of spermatozoa in these parts were examined after slaughter of the six bucks. The mean number of spermatozoa in the paired testes, capita, corpora and caudae of the epididymides were (22.8 +/- 1.24) x 10(9), (9.4 +/- 1.19) x 10(9), (3.4 +/- 0.22) x 10(9) and (35.0 +/- 2.21) x 10(9), respectively. Epididymal reserves of spermatozoa were correlated with testicular weight (r = 0.50, P = 0.01) and number of spermatozoa in the testes (r = 0.42, P = 0.07), but not with epididymal weight. The daily production of spermatozoa per animal in the breeding season was estimated to be 4.0-6.4 x 10(9).     

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.     

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).     

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.     

Semen quality and sperm output of Yankasa rams at different ages

Three groups of three Yankasa rams aged 1.4, 2.4, and 3.4 yr, weighing 32.8 +/- 0.8, 47.9 +/- 1.4, and 48.8 +/- 1.3 kg, and with scrotal circumferences measuring 26.8 +/- 0.9, 28.9 +/- 0.3, and 30.3 +/- 1.3 cm (mean +/- SEM), respectively, were used for this study. The rams were ejaculated once per day for 14 d using an artificial vagina and their semen quality and sperm output determined. For the three groups, ejaculate volume, sperm concentration, sperm motility, percentage of morphologically normal sperm, and total sperm output per ejaculate averaged 0.72 +/- 0.03, 0.90 +/- 0.04, and 0.88 +/- 0.03 ml; 3.421 +/- 0.133, 4.025 +/- 0.179, and 4.673 +/- 0.184 x 10(9)/ml; 83.2 +/- 2.3, 84.6 +/- 1.5, and 74.0 +/- 2.4%; 96.2 +/- 0.3, 96.0 +/- 0.4, and 95.4 +/- 0.4%; and 2.469 +/- 0.141, 3.663 +/- 0.237, and 4.163 +/- 0.247 x 10(9) sperm, respectively. Differences between groups were significant for all semen traits except for percentage of morphologically normal sperm. Total sperm output per ejaculate (Y; x 10(9)) was significantly correlated with scrotal circumference (X; cm) by the geometric regression equation Y = 0.000128 X(3.03) (r = 0.71; P < 0.05). Ejaculate characteristics were not influenced by day of collection except for percentage of morphologically normal sperm, which was significantly (P < 0.01) lower on the first day of collection.     

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.