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Introduction

Breeding of dairy cattle for high production and the reproductive management of herd is the biggest problem and it accounts for a large part on costs of production. A negative association has been observed between the level of livestock production and fertility. This is linked both to genetic factors (inbreeding and high production) and physiological factors (metabolic by high production)1. A lot of resources have been used for enhancement of cattle fertility but few studies and interventions are reported to control and to enhance the effect on the bull reproductive efficiency. As the patterns of selection and reproductive management of dairy cattle is based on the use of artificial insemination (AI) it is easy to understand the importance of assessing the level of fertility of bull breeder. One method of evaluating relative sire fertility currently used is the estimated relative conception rate (ERCR). ERCR is the difference in conception rate (nonreturn rate at 56 day) of a sire compared with other AI sires used in the same herd2. In this work the nonreturn rate was estimated at 56 d for first insemination of lactating cows (www.anafi.it). At present, validation of genomic markers that are able to predict with high confidence high or low fertility of a given sire it is very difficult using population estimates of sire fertility. The reason is because these methods do not measure the bull ‘true fertility’3. To unravel the biological display of the bull genome, proteomics, that focus at the protein level could lead to the development of novel biomarkers that may allow for detection of bull fertility levels4,5. The aim of this study is to evaluate, through the differential proteome analysis, changes in protein expression profiles of spermatozoa from bulls with high fertility (high ERCR score) and low fertility (low ERCR score) in order to identify possible protein markers to be used as indices of fertility.

Methods

Four classes of ERCR score were selected (from very low to very high fertility) for proteomic analysis. Sperm proteins were separated by 2-DE and digitized maps from each class subjected to image analysis with Progenesis SameSpot software. Differentially expressed spots (P<0.05) were excised, digested and tryptic peptides analyzed by MALDI-TOF/TOF mass spectrometry.

Results and discussion

Image analysis highlighted three significantly up and down regulated proteins in ERCR groups (Figure 1).

Figure 1
figure 1

(up) Progenesis SameSpot image analysis of differentially expressed bull sperm proteins, (bottom) normalized expression levels of differentially expressed proteins in the four classes analyzed. **=P<0.01; ***=P<0.001.

Full size image

Alpha-enolase was found to be strongly up-regulated in very high fertility (ERCR++) group. In human reproduction, elevated dimeric form of α-enolase (ENO-αα) characterizes abnormal immature spermatozoa, and elevated levels of ENO-S isoform (an isoform sperm-specific6) characterizes normally developed spermatozoa7. At present there are no data about elevated expression of α-enolase in bull sperm but only in fluid derived from cauda epididymal of mature Hollstein bull in association with a high fertility profile8,9. Other two proteins, isocitrate dehydrogenase subunit alpha (IDH-α) and triosephosphate isomerase (TPI) showed highest expression in ERCR−/− group which is associated with a very low score of fertility. Triosephosphate isomerase (TPI), an important glycolytic enzyme, is underexpressed in ERCR++ group respect to the others groups (P=0.048). In literature are not present data about this protein in bull sperm but similar profiles of expression are found in human sperm from asthenozoospermic (low motility sperm) patients10. In this work there is an overexpression of IDH-α in sperm samples with low ERCR score. The explanation of this phenomenon can be found either in a possible modulation of hypoxia-inducible factor-1 in sperm cell due to several type of metabolic problems11 or an increased necessity of NADPH in response to an increased oxidative stress. The latter possibility is largely supported by literature data. In effect defective human spermatozoa show intense redox activity and oxidative stress has been associated with impaired sperm motility12 and also sperm-oocyte fusion is inhibited by oxidative stress13.

Conclusion

In conclusion, the present study provides the first evidence for protein variations linked at the ERCR values in the bull sperm proteome and demonstrates that 2-D gel electrophoresis coupled to mass spectrometry and bioinformatics is useful for the identification of biomarkers for evaluation the level of fertility. The present data have indicated several possible candidate protein biomarkers for high and low ERCR. Further investigations will be necessary to evaluate possible use of these markers in fast screening of bull semen (by flow cytometry), and to clarify the causes of bull infertility.