ABSTRACT:We evaluated the expression of proteins in the accessory sex gland fluid (AGF) and their relationships with fertility indexes of dairy bulls. Fertility was normalized as the percentage point deviation of their nonreturn rates (PD) from the average fertility of all bulls from a given artificial insemination center. Services associated with each sire ranged from 269 to 77 321 and PD values from ϩ7.7% to Ϫ18.1%. AGF, from 37 bulls, was obtained with an artificial vagina after cannulation of the vasa deferentia. Proteins from AGF were separated by 2-dimensional SDS-PAGE followed by staining with Coomassie blue and analysis of polypeptide maps using PDQuest software. Bulls were divided in groups based on PD values and the optical density of spots in the AGF gels used as independent variables to predict bull fertility. Proteins were identified by capillary liquid chromatography nanoelectrospray ionization tandem mass spectrometry (CapLC-MS/MS). An average of 52 Ϯ 5 spots was detected in the AGF gels, but there were no spots unique to groups of either high-(PD Ն 0) or low-(PD Ͻ 0) fertility sires. The former were neither less nor more homogeneous than the latter based on correlations of all matched spots between pairs of AGF maps. However, high fertility of dairy bulls was significantly associated with lower expression of 14-kDa spermadhesin Z13 isoforms and higher amounts of 55-kDa osteopontin and 58-kDa phospholipase A 2 (PLA 2 ) isoforms. The average intensity of 5 spots identified as BSP 30 kDa in the AGF gels had a quadratic association with fertility indexes (R 2 ϭ .18; P ϭ .03). PD values of bulls were related (R 2 ϭ .56) to the quantity of spermadhesin, osteopontin, and BSP 30 kDa in the AGF polypeptide maps. Bull fertility was also determined by another equation (R 2 ϭ .53) with spermadhesin, BSP 30 kDa, and PLA 2 as independent variables. We conclude that interactions among several proteins in accessory sex gland fluid explain a significant proportion of the variation in fertility scores of mature dairy sires.

No abstract

During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

No abstract

The objective of the present study was to describe the relationship of seminal plasma and total sperm cell proteins with the semen freezability parameters of Guzerat bulls. Thirteen bulls were subjected to breeding soundness evaluation. Semen samples were collected, cryopreserved, and then post-thawing sperm kinetics were assessed, where high ( = 7) and low ( = 6) freezability groups were defined. Seminal plasma and total sperm proteins from the 2 groups were separated by 2-dimensional SDS-PAGE, and spots were identified by mass spectrometry. Semen parameters post-cryopreservation were as follows in the high and low freezability groups, respectively: mean total motility, 52.4 ± 20.5 and 13.7 ± 3.9; percentage of normal sperm, 89.0 ± 2.6 and 64.7 ± 14.0; and reactivity of hypo-osmotic swelling test, 38.9 ± 4.7 and 13.6 ± 3.7. Three seminal plasma proteins (osteopontin-K, DNase γ precursor, and DNASE1L3) and 6 proteins from sperm cells (acrosome formation-associated factor isoform 2, annexin A1, disintegrin and metalloproteinase domain-containing protein 2, dihydrolipoyl dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase) were highly expressed ( < 0.05) in the high freezability group. Another 6 seminal plasma proteins (acrosin inhibitor 1, glutathione peroxidase 3, metalloproteinase inhibitor 2, ephrin-A1, annexin A1, and platelet-activating factor acetylhydrolase) were significantly higher ( < 0.05) in the low freezability group. We described the associations of seminal plasma and sperm cell proteins with post-thawing sperm viability of Guzerat bulls raised in a typical semiarid environment. Such associations indicate that specific seminal plasma proteins more abundant in bulls of low semen freezability may be a response to an early oxidative stress that is not detected by conventional prefreezing semen evaluation. Moreover, specific sperm proteins were more associated with good freezability. The results presented here can serve as guidelines for future research aiming to develop better extenders and/or to improve bull semen selection for cryopreservation.

We evaluated the relationships between proteins in cauda epididymis fluid (CEF) and fertility scores of dairy bulls. Fertility was expressed as the percentage point deviation (PD) of bull nonreturn rate from the average fertility of all bulls at an artificial insemination center. The number of services for each bull ranged from 1074 to 52 820, and PD values ranged from +7.7% to 26.6%. CEF from 20 bulls was obtained from vasa deferentia cannulae and was separated from sperm by centrifugation immediately after collection. Samples were evaluated by 2-dimensional (2-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis gels stained with Coomassie blue, and polypeptide maps were analyzed by PDQuest software. Protein quantities, defined as the total integrated optical density of the spots, were compared between groups of high-fertility sires (n 5 12; PD $ 0) and low-fertility sires (n 5 8; PD , 0) and were also used as independent variables in regression analysis. Proteins were identified by capillary liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry. An average of 118 spots was detected in 2-D maps of the CEF, but we were unable to distinguish any protein that was expressed only in high-fertility or in low-fertility bulls. However, the amount of a-L-fucosidase 2 and cathepsin D was 2.3-and 2.4-fold greater (P , .05) in high-fertility than in low-fertility bulls, respectively. Conversely, the intensities of 3 isoforms (24-27 kd; pI 6.3-5.8) of prostaglandin D-synthase (PGDS) were from 3.2-to 2.2-fold greater in low-fertility sires (P , .05). An empirical regression model established that a significant proportion (R 2 5 0.72; P , .0001) of the variation in fertility scores (PD values) was explained by the intensities of cathepsin D and 1 isoform of PGDS (24 kd; pI 6.3). Thus, multiple proteins present in the CEF are potential biomarkers of fertility in high-use, mature Holstein bulls.

No abstract

No abstract