Sub-fertility in crossbred bulls: Identification of proteomic alterations in spermatogenic cells using high throughput comparative proteomics approach.

The present study was carried out to compare the proteomic profiles of spermatogenic cells of crossbred and zebu cattle in an effort to understand the possible reasons for a higher incidence of sub-fertility in crossbred bulls. The spermatogenic cells collected from the testes of pre-pubertal (6 mo) and adult (24 mo) crossbred and zebu males through fine needle aspiration were proliferated in vitro, and proteomic profiling was done using a shotgun proteomics approach. The age- and species-specific variations in the expression level of proteins were identified in spermatogenic cells. The number of differentially expressed proteins (DEPs) identified in pre-pubertal zebu and crossbred was 546, while 579 DEPs were identified between adult zebu and crossbred bulls. Out of these, 194 DEPS were common to these groups and 40 DEPs displayed a fold change ≥2. However, only 20 proteins exhibited similar expression variation trends (upregulated or downregulated) among pre-pubertal as well as adult zebu and crossbred bulls. Out of these 20 DEPs, 13 proteins were upregulated, and 7 proteins were downregulated in spermatogenic cells of zebu compared to crossbred bulls. Among the upregulated proteins were RPLP2, PAXIP1, calumenin, prosaposin, GTF2F1, TMP2, ubiquitin conjugation factor E4A, COL1A2, vimentin, protein FAM13A, peripherin, GFPT2, and GRP78. Seven proteins that were downregulated in zebu bulls compared to crossbred included APOA1, G patch domain-containing protein 1, NAD P transhydrogenase mitochondrial, glutamyl aminopeptidase, synaptojanin 1 fragment, Arf GAP with SH3 domain ANK repeat and PH domain-containing protein 1, and protein transport protein sec16B. It was inferred that the proteins associated with sperm function and fertilization processes, such as calumenin, prosaposin, vimentin, GRP78, and APOA1 could be studied further to understand the precise cause of subfertility in crossbred bulls.

Testicular cytology indicates differences in Sertoli cell counts between "good freezer" and "poor freezer" bulls.

In artificial insemination, poor quality of semen unsuitable for cryopreservation and susceptibility of spermatozoa to cryodamage in crossbred bulls have been a matter of concern. Present study was designed to identify the testicular cytology indices that might be used to predict the semen quality and cryotolerance of spermatozoa in bulls. Based on the ejaculate rejection rate and sperm cryotolerance, bulls (Holstein Friesian X Tharparkar crossbred) were classified into either good (producing good quality semen with spermatozoa having good cryotolerance; n = 4) or poor (producing poor quality semen with spermatozoa having poor cryotolerance; n = 4). Testicular cytology was studied in all the 8 bulls using fine needle aspiration technique. Testicular cytology of good bulls and poor bulls differed significantly. The proportion of Sertoli cells was significantly higher in good bulls (25.3 ± 1.6) compared to poor bulls (11.0 ± 0.8). The Sertoli cell index was 46.1 ± 5.0 in good bulls while it was only 13.8 ± 1.3 in poor bulls. The cut off values, as determined using Receiver Operating Characteristics analysis, indicate that the bulls having testicular cytogram comprising of < 15.5% Sertoli cells, < 24.3 Sertoli cell index and > 4.0 spermatogenic cells to Sertoli cell ratio might be a poor bull in terms of semen quality and cryotolerance of spermatozoa. The proportion of Sertoli cells in the testicular cytology had positive (P < 0.05) relationship with semen quality and cryotolerance of spermatozoa.

Comparative proteomic analysis of Taurine, Indicine, and crossbred (Bos taurus × Bos indicus) bull spermatozoa for identification of proteins related to sperm malfunctions and subfertility in crossbred bulls.

Subfertility is one of the most common problems observed among Taurine × Indicine crossbred bulls in tropical countries; however, the etiology remain unknown in most of the cases. In present study, we compared the proteomic profile of spermatozoa from crossbred bulls (Bos taurus × Bos indicus) against their purebred parent lines (Holstein Friesian [Taurine] and Tharparkar [Indicine]) to find out alteration in expressions of proteins, if any. The proteomic profiles of freshly ejaculated spermatozoa from these breeds were compared by two-dimensional difference gel electrophoresis, and differentially expressed proteins were identified through mass spectrometry. It was observed that compared to Holstein Friesian, nine proteins were underexpressed and eight proteins were overexpressed (P < 0.05) in the spermatozoa of crossbred bulls. Similarly, four proteins were overexpressed and four proteins were underexpressed (P < 0.05) in the spermatozoa of crossbred bulls compared to Tharparkar bulls. In concurrent three breed comparison, 14 proteins were found to be differentially expressed (P < 0.05) between these breeds. From the findings of the study, it is apparent that the expression levels of several functionally significant proteins are either upregulated or downregulated in spermatozoa of crossbred bulls, which might be related to high incidence of subfertility in these bulls.