Effect of age on spermiogram of Holstein Friesian × Sahiwal crossbred bulls

This study was conducted on 94 Frieswal (5/8 Holstein Friesian 3/8 Sahiwal) crossbred bulls of three different grades, categorized based on their semen freezability visualising Group 1 (consistently freezable semen producer bulls, N = 11), Group 2 (inconsistent freezable, N = 16) and Group 3 (Non freezable, N = 67). Each group was further divided into two classes that is young (up to 30 months) and adult (31 to 70 months) bulls depending upon their age. Sperm morphology was studied by using the eosin-nigrosin staining technique. Bulls age significantly (P < 0.01) affected semen quality and sperm morphology. In adult bulls, semen volume, mass activity and sperm concentration were 36%, 17.56% and 19.6%, respectively, higher than young. Initial progressive motility (%) and livability showed significant (P < 0.01) improvement with the advancement of age (43.37 ± 1.21 and 67.71 ± 1.11, respectively, in young; 53.02 ± 1.11 and 74.17 ± 1.03, respectively, in adult). In young bulls, sperm head, mid piece, tail abnormality and total abnormal sperm percent (12.38 ± 0.92, 4.87 ± 0.24, 11.01 ± 0.60 and 28.26 ± 1.34, respectively) were 1.85, 1.27, 1.20 and 1.44 folds higher than that of their mature stage (6.69 ± 0.64, 3.82 ± 0.32, 9.14 ± 0.64 and 19.66 ± 1.31, respectively). Significant reduction (P < 0.01) in micro cephalic sperm, free heads, bent mid piece, looped mid piece and proximal protoplasmic droplets were observed at mature age as compared with their younger stage. In bulls of consistent freezing category, abnormal sperm heads significantly decreased from 4.40 ± 0.31% to 3.28 ± 0.02% on maturity. Similarly, in inconsistent freezing grade bulls sperm head abnormality (9.28 ± 0.75% to 5.13 ± 1.20%) and total abnormal sperm percent (24.89 ± 1.43 to 18.73 ± 3.40) was decreased over the age. On the contrary, in non-freezing category bulls' sperm morphology did not show significant (P > 0.05) improvement with age advancement, rather some abnormalities like long slender head, under developed/deformed head, abaxial implantation of mid piece, double mid piece, stump tail and distal protoplasmic droplets tend to increased significantly (P < 0.05) with age of bulls. Results indicated that in potential Frieswal bulls semen quality and sperm morphology were improved from young to mature stage, where as, in poor quality (non-freezing) semen producer bulls neither the morphology nor the semen quality showed any improvement with maturity. It was recommended that crossbred bulls producing more than 25% morphologically abnormal sperms in young age (below 30 months) along with poor progressive motility (<50%) and low sperm concentration (<1000 million/ml) need immediate culling with out any expectation of further improvement in semen quality with age advancement.     

Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility.

The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%-20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.     

Successful pregnancies in cows following double freezing of a large volume of semen

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.     

Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation

Keeping in view the poor freezability of bubaline semen in conventionally used extenders, this study was conducted on three Murrah bulls to improve semen cryopreservation with the incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ml(-1)) in routinely used egg yolk tris-glycerol (EYTG) extender. Bradykinin (2.0 ng ml(-1)) had significant (P<0.05) beneficial effect on live sperm % (81.6+/-1.8) and hypo osmotic swelling (HOS) % (63.0+/-1.3) as compared to their respective control values of 73.4+/-2.1 and 56.3+/-2.0 at 0 h post freezing. The post-thaw progressive sperm motility in semen samples diluted with EYTG containing 2.0 ng ml(-1) Bradykinin (65.5+/-1.4) was also significantly (P<0.01) higher than control (60.3+/-1.9) at 0 h post freezing. Thus incorporation of 2 ng ml(-1) Bradykinin in buffalo semen diluted in EYTG extender may be useful in improving the quality of cryopreserved bubaline semen.     

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni)

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p < 0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p < 0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20-39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p < 0.01, p < 0.03, p < 0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p < 0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.     

Effect of season on fertility of frozen buffalo semen

Twenty double ejaculates from each of ten water-buffalo bulls were collected in June (non-breeding season) and again in November (breeding season). Fresh semen was screened for sperm quantity, motility, eosin uptake, and sperm morphology and was frozen using lactose, skim-milk, and Tris extenders. Thawed semen was checked for motility and Sephadex filtration. Half of each semen batch was used for artificial insemination in the breeding season and the other half during the non-breeding season.Laboratory screening revealed that June semen had a significantly lower Sephadex filtration rate and a higher percentage of abnormal sperm cells, and three June ejaculates were excluded from further processing due to poor sperm motility. In the remaining ejaculates the motility before freezing and the sperm cell quantity were higher in June semen than in November semen. Eosin uptake, mass motility, and post-freeze-motility did not vary with season. November semen produced significantly higher pregnancy rates than June semen over a total of 3220 inseminations in both seasons. Forty percent of the observed seasonality of buffalo fertility was attributable to the male. No fertility differences appeared between extenders used. When November semen was used, the fertility in adult buffaloes in both seasons was higher than in heifers.     

Post-thaw viability of bull AI-doses with low-sperm numbers

Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 x 10(6) total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/PI), acrosome integrity (Carboxy-SNARF-1/PI/FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 degrees C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols.     

SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing.     

Comparison of electroejaculation and transrectal massage for semen collection in range and yearling feedlot beef bulls

Two experiments were conducted to compare electroejaculation (EE) and transrectal massage (RM) of the ampullary region for semen collection from beef bulls, and to determine the effect of semen collection method on semen traits. In experiment 1, semen was collected either by EE or RM randomly assigned on an alternate basis in 137 range beef bulls unaccustomed to being handled. The maximum time allowed for RM was 4 min and if no semen was obtained, EE was used. In experiment 2, semen was collected from 39 yearling feedlot beef bulls that were accustomed to being handled, by RM followed immediately by EE. The maximum time allowed for semen collection by both methods was 4 min. In both experiments, sperm concentration, percent of progressively motile sperm, percent of sperm staining alive, and sperm morphology were determined. In experiment 1, RM resulted in fewer (P<0.001) successful semen collections and fewer bulls with penile protrusion than EE (80.9% versus 100% and 54.4% versus 91.5%, respectively). The success of RM was not influenced by bull age or breed, or by the veterinarian performing the massage. Transrectal massage required more time (30s, P<0.001) for obtaining a semen sample and resulted in samples with lower sperm concentration (P<0.001), percent motile sperm (P<0.05) and percent live sperm (P<0.001) when compared to EE. In experiment 2, EE and RM were equally effective for obtaining a semen sample (97.4 and 94.9%, respectively), but the proportion of bulls exhibiting penile protrusion during semen collection was lower (P<0.0001) with RM compared to EE. Percent of sperm staining alive was also lower (P<0.01) in samples collected by RM. Sperm morphology (normal sperm, head defects, midpiece defects, proximal cytoplasmic droplets, and detached sperm heads) did not differ between samples collected by EE and RM. In conclusion, semen could be collected by transrectal massage from approximately 80% of range beef bulls and from 95% of yearling beef bulls accustomed to handling. Sperm morphology was not affected by the method of semen collection, but percent of motile sperm and live sperm were lower in samples collected by RM. A reduced ability to stimulate penile protrusion with RM precluded examination of the penis in a large proportion of bulls.     

Effects of Trypanosoma vivax and Trypanosoma congolense infections on the reaction time and semen characteristics of Zebu (Bunaji) x Friesian crossbred bulls

The effect of trypanosomosis on reaction time and semen characteristics of 12 Zebu (Bunaji) x Friesian crossbred bulls aged between 3 and 5 years was studied for a duration of 12 weeks. Four of the bulls were infected with Trypanosoma vivax, another four with Trypanosoma congolense and the remaining four bulls served as controls. Rectal temperatures and haematological parameters were monitored twice weekly. The pre-infection mean value of the rectal temperature was 38.3 degrees C, and this rose to a mean of between 40.5 and 41.1 degrees C in the infected animals. Concurrently, the infected animals exhibited signs of anaemia shown by pale mucous membranes and decreased packed cell volume (PCV), weight loss, lethargy, weakness and dullness.The reaction time (ejaculation time) of semen collection significantly increased from a pre-infection mean value of 20.46-25.14 s to a mean of 290.33-301.15 s within 12 weeks post-infection. Semen characteristics deteriorated progressively within the same period in the infected bulls. There were highly significant and drastic decreases in sperm concentration and volume of semen and increases in sperm morphological defects. By the third week, all the infected bulls were unfit for breeding because of very poor semen characteristics. Deterioration, also characterized by oligospermia at 6 weeks post-infection in all bulls which later culminated in azoospermia in two bulls infected with T. vivax and two bulls infected with T. congolense continued to the end of the investigation. The present results indicate that trypanosomosis due to T. vivax and T. congolense infections is very pathogenic and devastating in its effect on the reaction time (ejaculation time) and semen characteristics which resulted in very poor semen quality. The practical implication is infertility and sterility in Zebu x Friesian crossbred bulls in trypanosome endemic areas.     

Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at -5 and -15 degrees C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a 'normal apical ridge' (NAR) were 72.5+/-3.8 and 75.8+/-2.0 versus 46.3+/-4.8 and 36.0+/-3.7 (means+/-S.E.M., n=4). In ejaculates of 15 unselected AI boars, after seeding at -5 degrees C, the post-thaw % live and % NAR were 66.3+/-10.4 and 74.8+/-7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 x 10(9) cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8+/-1.9% versus 40.7+/-1.7% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3+/-3.0% versus 59.3+/-2.9% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.     

Post-thawing survival of motile ram sperm after isolation by layering on protein columns

Post-thawing survival of ram sperm was examined after semen which had been layered on top of isolation columns containing solutions of bovine serum albumin (BSA) in Tris diluent was processed for freezing by the pellet method. Sperm isolated from the bottom of the BSA column had better post-thawing survival than sperm from the top of the column. The efficiency of sperm isolation was affected by the concentration of BSA in the column, the holding time of semen on the column and the concentration of sperm in the layered semen. The best post-thawing survival of sperm occurred when semen diluted to a concentration of 200 x 10(6) sperm/ml was layered on a column of 6% BSA in Tris diluent and the bottom layer of the column was isolated for freezing after two hours' holding time.     

Embryo quality and andrological study of two subfertile bulls versus five control bulls with normal fertility

Andrological studies and embryo morphology evaluation of superovulated cows were performed on 2 randomly selected subfertile dairy bulls whose semen was used for artificial insemination and on 5 control bulls with normal fertility. Neither sperm motility studies, nor sperm morphology or testicular measurements differed between the subfertile and the control bulls. Altogether 315 ova were recovered from 41 superovulated cows inseminated with semen collected from either the subfertile or the normal control bulls. The spermatozoa of one of the 2 subfertile bulls was shown to have a decreased ability to fertilize superovulated ova, while the other subfertile animal, the bull with the lowest noreturn rate, was found by chromosome analysis to have a reciprocal translocation (60, XY, rcp 20:24), causing embryonic death. We suggest that subfertile bulls should not be used in commercial embryo transfer programs nor in artificial insemination and that andrological studies on subfertile bulls with good sperm motility should include evaluation of 6- to 7-day-old ova from superovulated cows to determine if the fertilization rate is normal or impaired. A chromosome analysis should also be performed when a subjertile bull has a normal fertilization rate of ova.     

Cryopreservation of semen in the dog: use of ultra-freezers of -152 degrees C as a viable alternative to liquid nitrogen

This experimental work was carried out to validate the use of a -152 degrees C ultra-low temperature freezer to freeze and store canine semen. The semen of three dogs was pooled and processed to obtain a final dilution with a concentration of 100 x 10(6) spermatozoa/mL, glycerol at 5% and Equex at 0.5%. Then, four freezing protocols were tested to evaluate the cryosurvival of sperm at 1, 7, 30, 60 and 120 days after freezing: (I) semen was frozen and stored in liquid nitrogen; (II) semen was frozen in liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (III) semen was frozen in the vapour of liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (IV) semen was frozen and stored in the ultra-low freezer at -152 degrees C. Data were statistically analyzed by repeated measures analysis of variance to determine the effect of the freezing protocol and time on the sperm characteristics assessed. The percentages of sperm motility and of dead/live spermatozoa were similar throughout the experimental period, with no significant differences (P < 0.05) to be observed between four different freezing techniques tested. At 120 days after freezing, the percentage of abnormal cells and the percentage of sperm cells with abnormal acrosome were not significantly different between the freezing techniques. Although the number of dogs used was slightly low, in vitro results of this preliminary study showed that the use of ultra-freezers at -152 degrees C to freeze and store canine semen could be a viable alternative to liquid nitrogen.     

Sperm chromatin alterations in fertile and subfertile bulls

Alterations in sperm chromatin have been related with subfertility in several mammals. In this study, chromatin alteration types (Base, Basal half, Central axis, Dispersed, and Whole) were assessed by toluidine blue (TB) staining, 6-diamidino-2-fenilindole (DAPI) and anti-protamine 1 antibody (anti-PR1) labeling in sperm samples of fertile and subfertile bulls. Semen samples were obtained from bulls kept in Artificial Insemination Center (fertile bulls) or from bulls subjected to scrotal insulation (subfertile bulls). The percentage of chromatin alterations identified by TB was similar (P?>?0.05) in semen samples of fertile and subfertile bulls. In contrast, a greater (P?<?0.01) chromatin decondensation and heterogeneity were recorded in semen samples of subfertile bulls. In DAPI and anti-PR1 methods, the subfertile bulls samples had a higher (P?<?0.05) percentage of alteration in the base as well as overall chromatin alterations (P?<?0.05). Moreover, the chromatin alterations recorded with TB, DAPI, and anti-PR1 were compared in semen samples of fertile and subfertile bulls. In fertile bulls, the overall chromatin alterations were similar (P?>?0.05) among the methods In contrast, semen samples of subfertile bulls had a higher (P?<?0.05) percentage of overall chromatin alterations when labeled with DAPI. In conclusion, our findings shown that all dye tested had specific sperm stainability and can be feasible to monitor subfertility condition in bulls. Also, different chromatin alteration types in sperm samples of fertile and suberftile bulls were recorded.     

Seasonal influence on semen traits and freezability from locally adapted Curraleiro bulls

Studies were conducted to characterize the effect of season of the year on testicular morphology, fresh and frozen/thawed semen quality from Curraleiro (Pé-duro) bulls in the Brazilian Central west region. Five adult, healthy bulls underwent an andrological examination and semen collection using an electroejaculator, once a month for a year. Fresh and thawed semen were evaluated for progressive sperm motility and sperm vigor, sperm morphology and acrosomal integrity. Testicular length and volume were less (P<0.05) in April than in the other months of the year. For fresh semen, the ejaculate in April had less volume and sperm concentration (P<0.05), while sperm vigor was less (P<0.05) in June, increasing in January and February. With the frozen/thawed semen, the proportion of sperm was greater (P<0.05) in April to July, decreasing from October to December. Semen collected in December had the greatest (P<0.05) proportion of major defects while that collected in February/March had the highest proportion of minor defects. The proportion of live intact sperm reduced progressively from December to April/May. The marginal influence of the time of the year on testicular biometry and fresh semen in Curraleiro bulls shows the adaptation of this breed to the environmental conditions in the region. Thus, reproduction with natural mating should be successful at any time of year. For frozen semen collection for conservation programs, the best time of year is from June to September.     

DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay

During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P < 0.017) than the sorted samples and that there was no difference between the samples in tail length (TL) (P = 0.36). The SCSA showed that the DNA fragmentation index (DFI) was higher for conventional than the sorted samples (P = 0.011), but the standard deviation of DFI (SD-DFI) was higher for the sorted samples (P < 0.001). We conclude that the NCA and SCSA can be used in assessing DNA integrity in bovine sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.     

Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.