Genetic Parameters for Semen Production Traits in Austrian Dual-Purpose Simmental Bulls

Genetic parameters were estimated for semen production traits collected in an Austrian AI centre in the years 2000-2004. In total, 12,746 ejaculates from 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls were examined considering different effects on ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. The model for genetic parameter estimation included the fixed effects age of bull, collection interval, number of collections on collection day, bull handler, semen collector, year and month of collection, a random additive genetic component and a permanent environmental effect. Correlations between estimated breeding values for semen traits and male fertility from the routine evaluation were calculated. The fertility trait considered in the routine evaluation is non-return rate 90 for the first insemination. All semen production traits were moderately heritable. Heritabilities for volume, concentration, percentage of viable spermatozoa, total number of spermatozoa and motility were 0.18, 0.14, 0.10, 0.22 and 0.04, respectively. Correlations between breeding values for semen quality traits and routinely estimated breeding values for male fertility were low and ranged from 0.08 to 0.17 indicating that semen production traits are rather poor predictors of male fertility.     

Fertility Results of Artificial Inseminations Performed with Liquid Boar Semen Stored in X-CellTM vs BTS Extender

The objective of the present field study was to compare the fertility results for boar semen diluted in X-cell stored up to 4-5 days before artificial insemination (AI) with semen diluted in Beltsville thawing solution (BTS) used for AI following 2-3 days of storage (where the first day being the collection day). A total number of 2601 double inseminations in Norwegian herds were included in this two-trial study. All the boars used in the study were mature cross-bred Norwegian Landrace x Duroc (LD), which were routinely used for AI in Norway. The inseminated gilts and sows were Norwegian Landrace x Yorkshire (LY). The AI doses contained 2.5 billion spermatozoa, and consisted of a mixture of semen from three, occasionally four, boars (i.e. heterospermic semen). Fertility was measured in terms of the likelihood of farrowing and subsequent litter size. The fertility of the semen in both of the extenders was satisfactory and no significant differences were found either in semen stored 4-5 days in X-cell compared with 2-3 days in BTS or in semen stored 2-3 days in X-cell compared with 2-3 days in BTS. The storage capability findings for the long-term extender X-cell could significantly simplify the practical issues of semen production and the distribution of AI doses containing 2.5 billion spermatozoa. However, in pig production systems where all semen is used within 2-3 days, the short-term extender BTS is as good as the more expensive extender X-cell.     

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.     

Comparison of acetamide and dimethylsulfoxide as cryoprotectants of European brown hare (Lepus europaeus) spermatozoa

The aim of the present study was to assess the effect of dimethylsulfoxide (DMSO) and acetamide on the post-thaw properties of hare semen and to perform an AI trial with frozen-thawed semen. Semen was collected under general anaesthesia by the electroejaculation method from 6 males. Immediately after collection, the semen was diluted with an extender containing the following components: 250 mM Tris, 80 mM citric acid, 70 mM glucose, 1.0 M DMSO, egg yolk (17% v/v) and kanamycin (80 mg/l); this extender was used for Protocol I (n=17). In Protocol II (n=15), the DMSO was replaced with 1.0 M acetamide. Immediately after thawing and after incubation for 90 and 180 min at 37 C, the properties of semen were evaluated by computer-assisted semen analysis, and the percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. During the 3-h incubation, the percentages of motile spermatozoa and spermatozoa with progressive motility were significantly higher in Protocol I (P<0.01). Immediately after thawing, path and straight velocity were significantly higher in Protocol I (P<0.01), as was the curvilinear velocity (P<0.05). The amplitude of lateral head displacement was higher after 3-h incubation in Protocol I (P<0.05), and no differences in beat cross frequency were found between Protocol I and II at any incubation time. The percentage of viable, acrosome intact spermatozoa determined with flow cytometry was higher in Protocol I (P<0.01) at all incubation times. As a result of artificial insemination with the semen frozen with DMSO as a cryoprotectant, two out of three inseminated females delivered two healthy young each. Following artificial insemination with the semen frozen with acetamide as a cryoprotectant, two out of three inseminated females delivered one healthy young each. On the basis of the results, it should be stated that DMSO ensures better post-thaw properties of hare spermatozoa than acetamide.     

Assessment of Sperm Viability by Measurement of ATP,Membrane Integrity and Motility in Frozen/Thawed Bull Semen

Control of sperm quality after commercial freezing/thawing of bull semen is still restricted to the subjective assessment of sperm motility, despite its low correlation to fertility (Söderquist et al. 1991, Kjaestad et al. 1993). Although no single in vitro method has yet been designed to predict the fertilizing ability of a given semen sample, the quantitation of viable spermatozoa (with intact plasma and acrosome membranes, and metabolically active) seems to be most promising (Woelders et al. 1991). The present report describes the use of a bioluminiscence technique to determine ATP-levels and a novel supravital stain (using fluorescent dyes) to assess the amount of viable spermatozoa in frozen/thawed semen from 3 A.I. dairy bulls with significantly different motility after thawing.     

Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987-1988).

Information pertaining to evaluation of single ejaculates of semen and records for 2 consecutive breeding seasons were obtained. In all, data for 99 individual breeding seasons (n = 43 Standardbreds and 56 Thoroughbreds) were evaluated. Included in each semen evaluation was examination of semen characteristics and computer-aided analysis of spermatozoal movement characteristics. On the basis of the analysis of breeding records for 4,175 mares (7,017 estrous cycles), a per-estrous cycle fertility rate was calculated from data for 96 of the breeding seasons. Stallions with lower fertility than the mean overall season fertility had significantly (P less than 0.01) lower mean values for subjective appraisal of the percentage of motile and progressively motile spermatozoa and for percentage of morphologically normal spermatozoa. Lower mean values were obtained for computer-aided movement analysis of the percentage of motile and progressively motile spermatozoa, and for mean velocity of motile spermatozoa. Semen characteristics, including spermatozoal movement characteristics, and fertility were significantly (P less than 0.05) correlated for Thoroughbred and Standardbred stallions when analyzed individually and when data for both breeds were combined. Characteristics most highly correlated (P less than 0.01) with fertility data for both breeds combined were: subjective appraisal of the percentage of motile (r = 0.40) and progressively motile (r = 0.46) spermatozoa; percentage of morphologically normal spermatozoa (r = 0.36); and computer-aided analysis of percentage of motile spermatozoa (r = 0.34). However, on the basis of evaluation of a single ejaculate for each stallion, the variation in these characteristics only accounted for approximately 20% of the observed variation in fertility rate.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Distribution of Spermatozoa and Embryos in the Female Reproductive Tract after Unilateral Deep Intra Uterine Insemination in the Pig

The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6-8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 x 10(6) motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 x 10(6) motile spermatozoa. The sows were anesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero-tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 x 10(6) motile spermatozoa. The sows were anesthetized 61.1 +/- 12 h after insemination (48-72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI-group, and from unilateral side of the reproductive tract in the DIUI-group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI- than the DIUI-group (p < 0.001). In experiment II, fertilization occurred in five of eight sows (62.5%) after DIUI. The number of ova that ovulated were 16.4 +/- 2.6 per sow and the embryos numbering 11.4 +/- 2.3 per sow were recovered from both sides of the reproductive tract. In conclusion, the spermatozoa given by DIUI could be recovered from only one side of the reproductive tract of sows at approximately 24 h after DIUI via the flushing technique. However, embryos were found in both sides of the oviducts and the proximal part of the uterine horns 48-72 h after insemination, indicating that the fertilization occurred in both sides of the oviducts.     

Computerized and subjective assessments of post-thaw motility of semen from Finnish Ayrshire AI bulls in relation to non-return rates.

A semen analyser (Lazymot), was used to evaluate post-thaw motilities in 296 batches of semen from 74 Ayrshire bulls used for artificial insemination (AI). Motility was also assessed subjectively. A significant correlation was observed between assessments of motility using the Lazymot analyser and the subjective assessments. There was no correlation between post-thaw motility assessments and non-return rates in relation to the batches examined, which met Finnish criteria for use in AI. This suggests that criteria for post-thaw semen motility should not be increased beyond the present requirement for 40% motile spermatozoa.     

Estimation of genetic parameters and genome scan for 15 semen characteristics traits of Holstein bulls

A QTL detection experiment was performed in French dairy cattle to search for QTL related to male fertility. Ten families, involving a total of 515 bulls, were phenotyped for ejaculated volume and sperm concentration, number of spermatozoa, motility, velocity, percentage of motile spermatozoa after thawing and abnormal spermatozoa. A set of 148 microsatellite markers were used to realize a genome scan. First, genetic parameters were estimated for all traits. Semen production traits were found to have moderate heritabilities (from 0.15 to 0.30) while some of the semen quality traits such as motility had high heritabilities (close to 0.60). Genetic correlations among traits showed negative relationships between volume and concentration and between volume and most quality traits such as motility or abnormal sperm while correlations between concentration and these traits were rather favourable. Percentages of abnormal sperm were negatively related to quality traits, especially with motility and velocity of spermatozoa. Three QTL related to abnormal sperm frequencies were significant at p < 0.01. In total, 11 QTL (p < 0.05) were detected. However, the number of QTL detected was within the range of expected false positives. Because of the lack of power to find QTL in this design further analyses are required to confirm these QTL.     

Sperm distribution in the reproductive tract of sows after intrauterine insemination

The purpose of the present study was to compare the number of spermatozoa obtained from different parts of the oviducts and the uterine horns of sows after intrauterine insemination (IUI) and conventional artificial insemination (AI), 24 h after insemination. Twelve crossbred (Landrace x Yorkshire) multiparous sows were used in the experiment. The sows were examined for standing oestrus using a back pressure test and were examined every 4 h after standing oestrus by real-time B-mode ultrasonography to estimate the time of ovulation. The sows were allocated to two groups, group I sows (n = 6) were inseminated by a conventional AI technique with 3 x 10(9) motile spermatozoa in 100 ml of extended semen, and group II sows (n = 6) were inseminated by an IUI technique using 1 x 10(9) motile spermatozoa in 50 ml of extended semen. A single dose of AI or IUI was given using the same boar, 8-10 h before the expected time of ovulation during the second oestrus after weaning. Twenty four hours after insemination, the sows were ovario-hysterectomized. The oviducts and the uterine horns were removed and divided into seven parts, the cranial, middle and caudal uterine horns, the utero-tubal junction (UTJ), the cranial and caudal isthmus, and the ampulla. All parts of the reproductive tract were flushed and the spermatozoa were counted using a haemocytometer. The results revealed that the spermatozoa were found in both the oviducts and the uterine horns in all animals. The number of flushed spermatozoa in the UTJ of groups I and II, was 142,500 and 131,167 (p > 0.05), and in the caudal isthmus was 1411 and 1280 (p > 0.05), respectively. The proportion of spermatozoa in different parts of the reproductive tract in relation to the total number of spermatozoa within the tract was not significantly different between groups I and II (p > 0.05). It could be concluded that IUI, with a three-time reduction in the number of spermatozoa used resulted in the same number of spermatozoa to be deposited in the sperm reservoir around ovulation time.     

Qualitative changes and the fertilizing capacity of sperm after 6 years of preservation of boar semen

Sperm motility, acrosome morphology, changes determined by the vital-lethal test and aspartate aminotransferase (AST) concentration in semen plasma were evaluated in the semen of four boars; the semen was stored for six years. No statistically significant changes in the percentage of motile spermatozoa were indicated when sperm motility was evaluated after four and six years of semen storage in liquid nitrogen. Neither did the fluctuation of the changes found on the basis of the vital-lethal test go beyond statistically insignificant values. After semen sample thawing in the BTS medium, the motility of spermatozoa was found to be somewhat higher than after thawing in the INRA-ITP medium, but after the termination of the thermoresistance test both media appeared to be equally effective. The AST level of the semen samples stored for four years was just slightly up on the initial values. After thawing in the BTS medium, AST level increased by 0.03 microcatal per litre of semen plasma, and in the INTRA-ITP medium by 0.06 microcatal per litre of semen plasma. The insemination of five sows with the semen stored for six years results in conception of two sows, i. e. 40%, and the average litter size was 7.5 piglets. It can be derived from the results that six years of boar semen storage in liquid nitrogen cause no further substantial changes in the structural and functional characteristics of spermatozoa.     

Laparoscopic artificial insemination in sheep

The goal of any AI program is to create improved offspring, and the achievement of this objective will depend on the breeding value of the ram and ewe selected. Laparoscopic AI is being utilized in the sheep industry to extend the use of superior rams, and it offers the producer the opportunity to maximize the reproductive potential of superior sheep. Rapid genetic trait infusion of known superior stud rams into the flock is the primary economic benefit of laparoscopic AI. The success of laparoscopic AI depends on events and factors that interrelate in a complex way. Once the selection and preparation of the ewe have been accomplished, one of the more important steps in the program is the successful synchronization of the ewe to deliver the necessary ova to the site of fertilization at a specific time. One of the best methods of synchronization for laparoscopic AI is the use of a progesterone product for a controlled time period and the administration of PMSG upon its removal. Detecting the onset of estrus is critical, and the addition of sterile (e.g., vasectomized) males is helpful, even essential, to accurately determine when each ewe begins her estrus. The ram effect has been shown to stimulate ovulation and estrus. Ewes must be inseminated within a narrow window of time after the synchronization product is removed. Ewes should be inseminated in the order in which they begin to exhibit signs of behavioral estrus, but age, stage of lactation, duration of behavioral estrus, and breed must be taken into account when this order is established. Fresh-extended semen works well throughout this preferred time frame established for laparoscopic AI, but frozen semen gives best results when used near the end. Advancement in manufacturing technology today removes equipment as a variable factor. It is important, therefore, that the inseminator develop a level of expertise in laparoscopy to ensure maximum fertilization rates. If available, fresh-extended semen is preferred over frozen semen, using at least the minimal number of spermatozoa necessary for fertilization. Evaluation of the post-thaw frozen or fresh semen is necessary to determine motility, morphology, and concentration, all of which help determine the volume of the insemination dose. The minimum necessary for laparoscopic AI in fine-wooled breeds is 20 X 10(6) normal motile spermatozoa; however, the more seasonal and less fertile American sheep need approximately 40 to 50 X 10(6) normal motile sperm to achieve acceptable fertility rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca²⁺-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca²⁺]_i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca²⁺-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.     

Relationship of the number of spermatozoa inseminated to fertility of turkey semen stored 6 h at 5 degrees C

The relationship was investigated between turkey hen fertility and the total number and the number of viable spermatozoa inseminated, after semen storage for 6 h at 5 degrees C. The minimum numbers of total and viable spermatozoa necessary for high fertility during the first 15 weeks of production were respectively 175 and 150 X 10(6) weekly. The number of viable spermatozoa produced by breeding males decreased with age. Adjusting the insemination dose of viable spermatozoa weekly over 15 weeks resulted in a consistent high rate of fertility.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

Electroejaculation and semen cryopreservation of free-ranging Japanese black bears (Ursus thibetanus japonicus)

The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and cryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) x 10(6), 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by freezing the thus collected electroejaculates.     

Relationship between ATP content and motility in bovine spermatozoa with reference to the effects of the bull and the A.I. centre.

Deep-frozen semen from 28 bulls belonging to 6 different A.I. centres was studied after thawing and the ATP content in the spermatozoa was assayed using a bioluminescence technique. The sperm motility was subjectively estimated under a phase contrast microscope and the sperm concentration of each ejaculate was calculated in a haemocytometer. The overall mean ATP content was 16.6 nmoles ATP/spermatozoa x 10(8). There was a significant variation in ATP content between A.I. centres. Significant differences between bulls in ATP content were found as well as a significant correlation between ATP concentration and the number of motile spermatozoa. This may indicate that ATP assessment may be useful as an additional, objective laboratory test.     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.