Isolation, identification and quality assessment of bovine round spermatids

In mice and humans, it is possible to generate full-term individuals by round spermatids intracytoplasmic injection (ROSI). In cattle, ROSI is possible but with very low efficiency and no offspring have been born with this technique so far. One of the reasons for the low success rate is the ambiguous definition of a spermatid that hampers their selection. This work aims to set up a procedure for isolating a pure spermatids population from bull testes (n = 3 subjects of 2 years old), define morphological features for proper identification of spermatids, and analyse isolated cells for viability and quality immediately after their isolation (0 h) and after 24 h of culture at 4°C and 37°C. To this end, after enzymatic tissue dissociation, spermatids were isolated using discontinuous Percoll gradient, followed by the use of a 10-μm mesh cell strainer. Morphological analyses were run using haematoxylin and eosin and DAPI staining. Cell quality was assessed by viability (LIVE/DEAD staining), DNA integrity (acridine orange) and mitochondrial activity (red CMXRos and green FM mitoTrackers), at 0 and 24 h after their isolation and culture at 4°C in air atmosphere and 37°C in 5% CO2 incubators. Statistical analysis was run using one-way analysis of variance. The results show that 40% of cells collected from 35%–40% Percoll fractions were represented by round spermatids. After using a cell strainer, this value increased to 60% (P < 0.05). Morphological analysis revealed that round and elongated spermatids ranged between 7.0–12.0 μm in size. In round spermatids, nuclei were centrally located in the cell and had 1–3 nucleoli. In elongated spermatids, nuclei were darker and polarized and nucleoli were absent. At 0 h, all cells were viable and had an intact DNA. At 24 h after culture, the percentage of viable cells was 97.8 ± 1.2% at 4°C and 98.2 ± 2.0% at 37°C (P < 0.05). Interestingly, mitochondrial activity significantly increased in the spermatids cultured for 24 h at 4°C and 37°C compared with those analysed at 0 h (P < 0.05), suggesting the presence of oxidative stress. However, no difference in DNA integrity was found in the isolated spermatids after 24 h at both temperatures. Overall, our results indicate that distinctive morphological features were determined for identifying individual spermatids. Moreover, all the spermatids were viable and preserved their morphology after 24 h of culture at both 4°C and 37°C, suggesting that spermatids can be stored before ROSI. This work provides some preliminary data for developing an effective ROSI protocol in cattle. If successful and if it will be possible to generate competent spermatids from spermatogonia or even embryonic cells, this combined approach would provide a possible alternative for reducing the generational interval in cattle.