Sex ratio after insemination of bovine spermatozoa isolated using a bovine serum albumin gradient

This experiment was undertaken to determine if a method reported to successfully enrich the proportion of Y-chromosome-bearing spermatozoa in human semen could be adapted for separation of bovine spermatozoa. Semen was collected from four Angus bulls and aliquots were either separated on discontinuous gradients of bovine serum albumin (BSA) or untreated before processing for cryopreservation. Two hundred seventy-one cows or heifers were assigned randomly to be artificially inseminated (20 X 10(6) sperm/insemination) with separated or unseparated spermatozoa. The proportions of male offspring were 45 and 54% after inseminations with separated or unseparated spermatozoa, respectively. In a second phase of the experiment, pooled semen from three Holstein bulls was either extended and frozen without separation or frozen after separation using the discontinuous BSA gradient. Separated and unseparated spermatozoa were analyzed by flow cytometry to determine the ratio of X- and Y-chromosome-bearing spermatozoa based on differences in DNA content. The ratios of X- and Y-bearing spermatozoa in separated or unseparated samples were indistinguishable. We concluded that the separation method did not enrich the proportion of Y-bearing bovine spermatozoa.     

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.     

Characteristics of bovine spermatozoa after migration through a bovine serum albumin gradient

Experiments were conducted to determine the efficiency with which viable, morphologically normal bovine spermatozoa could be isolated using a discontinuous bovine serum albumin (BSA) gradient. In the first experiment, extended semen was layered on top of a BSA gradient (4% BSA over 10% BSA) contained in a 500-ml separatory funnel. When comparing 1, 3, 5, 7, 14 or 21 X 10(9) spermatozoa applied to the gradient, the percentage of spermatozoa recovered from the lower third of the 10% BSA ranged from 2.9 to 18.5%. The greatest recovery was achieved when 1 X 10(9) sperm cells were applied. Increasing the number of spermatozoa applied to the gradient increased the percentage of spermatozoa remaining in the upper portions of the gradient. Motility of spermatozoa immediately after collection from the 10% BSA layer of the gradient was greater than 90%, regardless of the number of spermatozoa applied. In a second experiment with freeze-thawed separated or unseparated spermatozoa, post-thaw motility (greater than 60%) and acrosomal integrity (greater than 85%) of separated spermatozoa (4 or 10% BSA layer) was greater (P less than .05) than that of unseparated spermatozoa (38 and 66%, respectively). The discontinuous gradient excluded decapitated spermatozoa and spermatozoa with mid-piece and principal piece abnormalities from entering the lower layers. Sperm cells with head abnormalities were not separated. These data indicate that a population of spermatozoa with a high frequency of viable, motile, morphologically-normal bovine spermatozoa can be isolated using a discontinuous BSA gradient.     

Biochemical properties of bull spermatozoa separated in iodixanol density solution

Bull spermatozoa samples contain variable portion of motile and normal morphology spermatozoa along with spermatozoa incapable of fertilization due to their pathologic changes. As semen quality is influenced by biochemical and morphological characteristics of all spermatozoa, the aim of the study was to separate spermatozoa in discontinuous iodixanol density gradient solution and to determine their cholesterol, phospholipid, triacylglycerol and lipid peroxide concentrations and creatine kinase activity. The study was performed in winter and included seven Simmental bulls aged 1.5-3.5 years. Semen samples were collected by use of artificial vagina. Upon evaluation of semen quality (volume, concentration and progressive sperm motility), the samples were centrifuged in iodixanol density solution to obtain two sperm fractions. The two fractions included sperms with progressive motility greater than 90% and less than 20%, respectively. A statistically significantly higher lipid peroxide concentration was determined in sperm fraction with <20% progressive motility. Different sperm subpopulations can be obtained by separating bull spermatozoa in different iodixanol density gradient solutions, while monitoring their biochemical properties can help assess the sperm quality.     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Kjxstad, H., E. Ropstad and K. Andersen Berg: Evaluation of spermatological parameters used to predict the fertility of frozen bull semen. Acta vet. scand. 1993,34,299-303.– Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.     

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.     

Fertility of stallion spermatozoa isolated on albumin gradients

Fertility of stallion semen extended with bovine serum albumin (BSA) sucrose extender or cream-gelatin extender was compared. Pregnancy rates were 95% of 47 mares and 86% of 46 mares inseminated with BSA-sucrose and creamgelatin extended semen, respectively. Foaling rates and cycles per conception were not significantly different between treatment groups.Semen from 5 mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. Immediately following collection, samples were evaluated for motility and forward movement. Seven to 10 ml of semen, extended 1:1 with BSA-sucrose extender, were pipetted onto BSA medium separation columns. After 1 hour incubation at 37°C, the top, middle and bottom layers were separately withdrawn from each separation column and pooled, respectively. A marked decrease (P<.001) was noted in the mean motility of spermatozoa in the top layer (35%) as compared to the mean pre-incubation motility (59%). Spermatozoa from middle and bottom layers were significantly (P<.001) more motile (70.6 and 87%) than those from the top layer and pre-incubation samples. Rate of forward movement (RFM) of spermatozoa in lower fractions was higher (P<.001) than RFM of spermatozoa in the top layer. Concentration of spermatozoa decreased (P<.001) as the concentration of BSA in the medium increased.     

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.     

Sexing sperm of domestic animals

The ability to preselect or predetermine the sex of offspring prior to conception is a highly desired technological tool for assisted female breeding programs specifically for milk production, and in males, for meat production and increasing livestock numbers. The current technology is based on the well-known differences in X- and Y-sperm in the amount of DNA. The technology uses modified flow cytometric instrumentation for sorting X- and Y-bearing sperm. The method can be validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content, and embryo biopsy for sex determination. Currently, the sex of animals has been predetermined with 90 % accuracy by sexing spermatozoa. In the bovine breeding industry, flow cytometric sperm sexing has not fulfilled its original promise. Sexed sperm doses are too expensive for widespread application while the fertility of sexed sperm doses is lower than unsexed ones. Essentially all bovine sexed semen is frozen and then applied through artificial insemination (AI) or in vitro fertilization. There is still a need in the animal breeding industry to develop a technique for sperm sexing that provides sufficient spermatozoa for AI doses, does not compromise sperm fertility, and is widely applicable to a range of species. In this review, we will summarize the current state-of-the-art in sex preselection in domestic animals and some wildlife species using flow cytometric sperm-sorting of X from Y sperm based on DNA differences.     

Einflüsse von Prostatasekret und Verdünner auf die Spermienmotilität und ATP-Konzentration sowie die Aktivität der sauren und alkalischen Phosphatase von Beagle-Samen*

Influences of seminal plasma and extender on sperm motility, ATP-concen-tration, and the activity of acid and alkaline phosphatases of beagle dog semen. The percentage of progressively motile spermatozoa, the time of sperm survival, ATP-concentration, and the activity of the acid and alkaline phosphatases were determined in the semen of five healthy beagle dogs immediately after collection and after storage for 24 hours at +5°C. The sperm rich fraction was examined in the native state as well as after the addition of prostatic secretion and a Tris-eggyolk-medium, resp. The percentage of progressively motile spermatozoa was 64.4% in the fresh undiluted semen, 68.4% after the addition of prostatic secretion, and 74.8% after dilution with Tris-eggyolk-medium. It decreased within 24 hours to 31.6%, 20.4%, and 59.2%, resp. After 0 und 24 hours, resp., the sperm survival time in a coverslide preparation was     

Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa

Two experiments were conducted to test whether stallionand/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, 15 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/ml) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment 1, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.     

Effects of sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.     

Effect of caffeine on metabolic activity of ejaculated and epididymal spermatozoa of buffalo (Bubalus bubalis)

Split fractions of 25 ejaculated semen samples and spermatozoa from 5 caudae epididymides were used to study the effect of different levels of caffeine on the motility and fructolytic activity. During the first hour of incubation at 37°C, addition of caffeine to unwashed ejaculated buffalo sperm significantly increased the percentage of motility and amount of fructose utilized. In presence of 2–8 mM caffeine, sperm maintained their initial motility at least 2 hours at 37°C. Maximal stimulation of fructolytic activity was obtained with 2 mM whereas the minimal stimulation was found with higher concentration of 8–10 mM caffeine. Compared to ejaculated spermatozoa, epididymal sperm appeared to be more influenced by caffeine. Fructolytic activity was stimulated at least 1.5 times in the presence of 2 mM caffeine. Nearly during all incubation periods, the amounts of fructose utilized by caffeine-treated sperm were greater than in control samples. Data on epididymal sperm motility demonstrated that caffeine (2–10 mM) significantly stimulated and maintained the initial motility at least 3 hours at 37°C .     

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.     

Relationship Between Motility and Membrane Integrity of Boar Spermatozoa in Media Varying in Osmolality

A study was conducted to evaluate the relationship between boar sperm motility and membrane integrity following exposure to media with 150–1120 mOsm. Total sperm motility was defined as the percentage of spermatozoa that had any form of motility was subjectively assessed under a light microscope. Sperm cell damage was expressed as a loss of membrane integrity as measured by a combination of fluorescent stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), and Hoechst 33258 (H33258). There were no significant differences between sperm motility and membrane-intact spermatozoa, as measured by CFDA-PI and H33258, in media with 250 and 300 mOsm. In anisosmotic conditions, a higher amount of membrane-intact spermatozoa than motile spermatozoa was observed. In hypo-osmotic conditions (150 mOsm), a high proportion of spermatozoa had curled or coiled tails and most of them retained their entire membrane integrity, as detected by CFDA-PI. In media with 350–1120 mOsm, some spermatozoa accumulated PI in the head region and CFDA in the mid-piece. These spermatozoa fluoresced blue at the lower region of the head, as detected by H33258. The ATP content in spermatozoa exposed to hypo- and hyperosmotic conditions was markedly reduced. There was no recovery of sperm motility on returning the spermatozoa to isosmotic conditions after 10 min incubation in anisosmotic conditions, indicating that the spermatozoa suffered an almost complete and irreversible loss of motility. This irreversible loss of motility may be a consequence of reduced ATP production in spermatozoa subjected to anisosmotic conditions. The results of this study demonstrate that plasma membrane integrity assessment in combination with sperm motility, using a range of media varying in osmolality, can give valuable information about the status and function of different sperm membranes, which might be relevant for semen preservation.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Retrospective Analysis of Canine Semen Evaluations with Special Emphasis on the use of the Hypoosmotic Swelling (HOS) Test and Acrosomal Evaluation Using Spermac®

Routine semen evaluation includes volume, motility, vital staining for live‐dead ratio and pathomorphology including Spermac^® staining for evaluation of the acrosome. In recent years, depending on the species, also the hypoosmotic swelling (HOS) test has been applied routinely for evaluation of semen quality. In this respect, a significant correlation between the ability of spermatozoa to swell in HOS test and the fertilizing ability has been reported. Also for evaluation of dog semen, reference has been made to the HOS test; however, its correlation to conventional semen parameters so far is discussed controversially. In the present study, the results of 400 semen examinations from stud dogs presented at our clinic were evaluated for their correlations between conventional semen parameters (motility, live/dead ratio, pathomorphology), conventional semen parameters and age, Spermac^® staining and HOS test, respectively. We found a significant correlation of age and sperm concentration (p < 0.01), total sperm count (p < 0.0001), percentage of progressively motile sperm (p < 0.01) and live spermatozoa (p = 0.012). Furthermore, several correlations between conventional semen parameters were identified. Percentage of sperm with normal acrosome identified by Spermac ^® staining correlated significantly with live spermatozoa (p < 0.0001) and percentage of progressively motile sperm (p < 0.01). A significant correlation was proven between curled tails in HOS test and age (p < 0.001), motility (p < 0.0001), live sperm (p < 0.0001), acrosomal status (p < 0.05), pathomorphology (p < 0.0001) and sperm concentration (p = 0.011). These results indicate that Spermac^® staining and the HOS test are useful in improving canine semen analysis.     

Long-term liquid storage of porcine spermatozoa separated using a discontinuous bovine serum albumin gradient

Two experiments were conducted to determine efficacy of a discontinuous bovine serum albumin (BSA) gradient for isolating viable porcine spermatozoa more tolerant to 5-d liquid storage in Beltsville Thawing Solution (BTS) at 15 degrees C. The gradient, contained in a 500-ml separatory funnel, consisted of 4% BSA (60 ml) over 10% BSA (60 ml). Spermatozoa were extended in 26 ml of BTS, layered on top of the gradient, and allowed to migrate through the BSA. The quality of spermatozoa separated by the gradient varied among boars. However, populations of spermatozoa isolated from the bottom 30 ml of the gradient (Fraction 4) consistently contained a high percentage of spermatozoa with acrosomes possessing normal apical ridges (NAR; 89.6%) and progressively motile spermatozoa (MOT; 84.0%), as well as spermatozoa with high velocity (VEL; 336.5 mu/s). Increasing sperm migration time, but not gradient temperature, increased the number of spermatozoa recovered in Fraction 4, but it did not reduce quality of the separated spermatozoa. Spermatozoa isolated in Fraction 4 had greater NAR, MOT and VEL after 5-d storage in BTS than did unseparated spermatozoa. Boar spermatozoa isolated on a discontinuous BSA gradient were more tolerant to storage at 15 degrees C than were unseparated spermatozoa. Such a population may be desirable for use in artificial insemination programs.     

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.