Sperm chromatin structure assay of bulls qualified for artificial insemination

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility

The DNA methylation pattern in spermatozoa of buffalo bulls of different fertility status was investigated. Spermatozoa isolated DNA from two groups of buffalo bulls (n = 5), selected based on their artificial insemination–generated conception rate data followed by IVF efficiency, were studied for global methylation changes using a custom-designed 180 K buffalo (Bubalus bubalis) CpG island/promoter microarray. A total of 96 individual genes with another 55 genes covered under CpG islands were found differentially methylated in sperm of high-fertile and subfertile buffalo bulls. Important genes associated with biological processes, cellular components, and functions were identified to be differentially methylated in buffalo bulls with differential fertility status. The identified differentially methylated genes were found to be involved in germ cell development, spermatogenesis, capacitation, and embryonic development. The observations hint that methylation defects of sperm DNA may play a crucial role in determining the fertility of breeding bulls. This growing field of sperm epigenetics will be of great benefit in understanding the graded fertility conditions of breeding bulls in commercial livestock production system.     

The effect of scrotal insulation on spermatozoal morphology and the rates of spermatogenesis and epididymal passage of spermatozoa in the bull

In five beef bulls spermatogenic cells labelled with (3)H-thymidine were used to estimate the duration of one cycle of the seminiferous epithelium (S.E.) and epididymal passage (E.P.) of spermatozoa after 10 or 20 hr of scrotal insulation designed to increase testicular temperature. From six autoradiographic estimates in three bulls, the mean value for 1 S.E. cycle was 13.4 days (range 12.9 to 13.6 days). Two estimates of E.P. were both 13.5 days. Scrotal insulation altered both semen quality (lowered percentage live and motile spermatozoa, raised percentage abnormal spermatozoa and lowered concentration per ml spermatozoa) and the histological appearance of the seminiferous epithelium. However, the rate of spermatogenesis and epididymal transport was unchanged.     

Hair whorl patterns on the bovine forehead may be related to breeding soundness measures

The objective of this study was to determine if there is an association between facial hair whorl patterns on the bovine forehead and sperm morphology. Breeding soundness exams were conducted on 219 yearling bulls at three Colorado State University facilities. There were 150 Angus bulls and the remaining bulls were of several different breeds. Hair whorl patterns on the forehead were classified as round or nonround epicenters. Angus bulls with round epicenters had a higher percentage of morphologically normal spermatozoa compared to nonround epicenters (P < 0.05). Hair whorls on Angus bulls were sorted into two extreme groups of round spirals, with rotation or long lines that were longer than the width of the eyes. Bulls with round spirals had a greater percentage of normal spermatozoa compared to bulls with long lines (P < 0.05). Hair whorl pattern on Angus bulls also had an effect on the percentage of bulls that had the minimum threshold value of 70% normal spermatozoa. Eighty percent of Angus bulls with round epicenters had > 70% or more normal spermatozoa compared to 59% with a nonround epicenter (P < 0.01). Fifty percent of Angus bulls with long lines failed because they had < 70% normal spermatozoa. There were no significant differences in the remaining non-Angus bulls. Hair whorl patterns may be useful for making early culling decisions.     

Individual differences in capacitation of bull spermatozoa by heparin in vitro: relationship to fertility

Several parameters of motility and integrity of frozenthawed spermatozoa are compared with the ability of selected motile, intact spermatozoa to acrosome reaction induced by 0.005% hyamin. Between semen donors there exist distinct individual differences; however only the induced acrosome reaction after heparin treatment showed a significant correlation with fertility. For 12 bulls the nonreturn rates from 334 to 559 services correlated significantly with induced acrosome reaction (r = 0.607). The in vitro fertilization of 53 to 93 tubal bovine oocytes with frozen-thawed spermatozoa from five bulls yielded a correlation of r = 0.621 with the rate of induced acrosome reaction. The different capacity levels of heparin-treated spermatozoa to undergo acrosome reaction appears to correspond to the varying intensity and kinetics of heparinmediated head-to-head aggregation of motile cells. The applied functional parameters could be used for the selection of bulls with low fertility in artificial insemination programs, and for spermatozoa donors for in vitro fertilization.     

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.     

Effects of sperm preparation and bull fertility on in vitro penetration of zona-free bovine oocytes

The objectives of this study were to develop and validate a zona-free bovine oocyte penetration assay for detecting relative differences in bovine sperm fertility and to determine the effect of different sperm preparation methods on oocyte penetration. Oocytes were incubated with heparin-capacitated spermatozoa which either were or were not induced to acrosome-react with lysophosphatidylcholine. Heparin-capacitated spermatozoa treated with lysophosphatidyl-choline penetrated more oocytes and had more penetrations per oocyte than spermatozoa capacitated in heparin but not induced to acrosome-react with lysophosphatidylcholine. Spermatozoa stained with Hoechst 33342, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, alone or in combination, penetrated similar numbers and percentages of zona-free bovine oocytes as the similar to non-stained spermatozoa. When spermatozoa from the same ejaculate were stained with either fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate and competed in penetrating the same oocytes, the number of penetrations generated by the 2 differently stained spermatozoa was similar. Spermatozoa from bulls of differing in vivo fertilities were labeled with different fluorescent dyes, and their relative abilities to penetrate the same oocytes were assessed. Comparisons between spermatozoa from high and low fertility bulls demonstrated that high fertility spermatozoa had a significant oocyte penetrating advantage over low fertility spermatozoa in 13 of 16 paired competitions. We concluded that the results of the competitive penetration of zona-free bovine oocytes by fluorochrome-labeled spermatozoa from bulls of different fertilities were indicative of their relative in vivo fertility.     

Assessment of the fertilizing potential of frozen bovine spermatozoa by in vitro induction of acrosome reactions with calcium ionophore (A23187)

The relationship between nonreturn rates of bulls in a commercial artificial insemination program and in vitro induction of acrosome reactions in frozen-thawed spermatozoa by the calcium ionophore, A23187, was investigated. Washed spermatozoa from 3 to 5 ejaculates, collected from each of 23 Holstein bulls, were incubated for 1 h with 1 microM A23187. Acrosome reactions were determined by fluorescence microscopy. The percentage of increase in acrosome reaction in the ionophore-treated compared with control samples was significantly correlated to the 90-d nonreturn rate of the bulls (r = 0.86; P < 0.001). In a second experiment, a significant correlation was obtained between the fertility of bulls predicted on the basis of induced acrosome reaction and achieved 90-d nonreturn rate (r = 0.84; P < 0.005). No other assessments of semen quality (post-freezing motility, percentage of morphologically normal spermatozoa) was significantly correlated with fertility. Finally, the regression between acrosome reaction induction obtained from young bulls was used to predict the fertility of mature bulls whose semen was in widespread use (actual versus predicted nonreturn rate, r = 0.88; P < 0.0001).     

Up-regulation of glucose metabolism during male pronucleus formation determines the early onset of the s phase in bovine zygotes

After in vitro fertilization with spermatozoa from bulls with high in vitro fertility, a beneficial paternal effect is manifested during the G1 phase of the first cell cycle. This benefit determines an earlier onset of the first S phase, and then a successful morula-blastocyst transition 7 days later. We hypothesized that the origin of the paternal effect could be a shift of the metabolism of the fertilized oocyte, because in mice, sperm decondensation is responsible for a dramatic increase in glucose metabolism. In this study we investigated the interaction between both pronuclei and compared glycolysis and pentose phosphate pathway (PPP) activities in bovine oocytes fertilized with spermatozoa from bulls of high or low fertility. Here we demonstrate that male pronucleus formation is necessary for the onset of the S phase in the female pronucleus, and that the component promoting an early S phase in both pronuclei is metabolic and linked to an up-regulation of the PPP during the male pronucleus formation. This long-lasting paternal effect is more evidence of the important role of epigenetic control during early embryo development.     

Spermatogenesis, sperm output and seminal quality of Holstein bulls electroejaculated after administration of oxytocin

The 12- to 24-month-old Holstein bulls were electroejaculated twice on each of 3 days per week throughout the study. After a 2-week stabilization period and subsequent 2-week pre-treatment period, 7 bulls were given 50 i.u. oxytocin via the jugular vein 10 min before each first ejaculate for 10 weeks. The 7 control bulls were handled identically but did not receive oxytocin. All bulls were castrated at the end of the study. Oxytocin was without effect on spermatogenesis (P greater than 0.10). Oxytocin did not alter the total number of spermatozoa harvested per collection day (P greater than 0.10), but increased the number of spermatozoa in first ejaculates by an average of 34.2% (P less than 0.025). Oxytocin did not affect sperm quality (P greater than 0.10) as judged by the motility of spermatozoa in fresh semen or by the motility or percentage of spermatozoa with intact acrosomes in thawed semen. It is concluded that 50 i.u. oxytocin enhanced sperm output in first ejaculates of electroejaculated bulls without altering daily sperm production or seminal quality.     

Relationship between bovine fertility and the number of spermatozoa penetrating the cervical mucus within straws

In this study, by using a recently developed test technique, the relationship between the total spermatozoa number penetrating determined sites of bovine cervical mucus in straws and potential fertility of bulls, and other spermatological characteristics were investigated. Furthermore, we aimed to determine the effect on the test results, of two different incubation temperatures (37 and 41 degrees C) and two sperm penetration distance ranges (PDRs). Frozen semen samples of six Holstein bulls were used in the study. The bulls were divided into two fertility groups (high and low fertility) according to the "non-return rates" (NRR). For the penetration test, cervical mucus was drawn into transparent plastic straws and incubated with semen at 37 and 41 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, concentrations of spermatozoa penetrated to the PDRs, each of which was 2.5 mm, between 32.5 and 35 mm (first penetration distance range, PDR1), and 50 and 52.5 mm (second penetration distance range, PDR2) distance in the straws from the open end, were measured. When compared with the low fertility group, bulls from the high fertility group showed a higher number of spermatozoa at the determined PDRs, and a significant positive correlation was found between the total number of spermatozoa at the penetration distances and the NRR scores of the bulls.