High-resolution melt analysis does not reveal mutagenic risk in sexed sperm and in vitro-derived bovine embryos.

The objectives of the present work were to verify whether simultaneous exposure to Hoechst 33342 and UV irradiation during sorting by flow cytometry may induce gene point mutations in bovine sperm and to assess whether the dye incorporated in the sperm may imply a mutagenic effect during the embryonic development. To this aim, high-resolution melt analysis (HRMA) was used to discriminate variations of single nucleotides in sexed vs. non-sexed control samples. Three batches of sorted and non-sorted commercial semen of seven bulls (42 samples) were subjected to HRMA. A set of 139 genes located on all the chromosomes was selected, and 407 regions of the genome covering a total of 83 907 bases were analyzed. Thereafter, sperm of one sexed and one non-sexed batch of each bull was used in in vitro fertilization, and the derived embryos were analyzed (n = 560). One hundred and thirty-three regions of the bovine genome, located in 40 genes, were screened for a total coverage of 23 397 bases. The comparison between the frequencies of variations, with respect to the sequences deposited, observed in the sexed and non-sexed sperm (843 vs. 770) and embryos (246 vs. 212) showed no significant differences (P > 0.05), as measured by chi-square tests. It can be concluded that staining with Hoechst 33342 and exposure to UV during sorting does not lead to significant changes in the frequencies of variants in the commercial sexed semen and in embryos produced in vitro with the same treated sperm.

Minimum number of spermatozoa per dose in Mediterranean Italian buffalo (Bubalus bubalis) using sexed frozen semen and conventional artificial insemination.

In buffaloes, AI with sexed semen is not fully optimized, and the procedure has only been performed using the approach currently in use for cattle. The objective of the present work was to compare the pregnancy rates in Mediterranean Italian buffalo cows inseminated with sexed frozen-thawed semen at 2, 4, 6, and 8 million sperm per dose, using the Ovsynch protocol and conventional AI at a fixed time. Fresh ejaculates from three buffalo bulls were processed according to Beltsville sperm sorting technology, and packaged in 0.25-mL straws with two total concentrations of 2 and 4 million live sorted sperm per straw. After thawing, semen was evaluated for total motility, forward motility, average path velocity, membrane and DNA integrity, and membrane fluidity. Sorting efficiency was estimated using a real time polymerase chain reaction method developed and validated in our laboratory. The artificial inseminations were conducted during the breeding season on 849 Italian Mediterranean buffalo heifers and cows distributed in 13 farms in northern and central Italy. No significant difference in quality parameters was reported between nonsexed and sexed straws produced with 2 and 4 million sperm. Lower pregnancy rate (P < 0.001) was reported when inseminating doses of sexed semen at 2 million were used (53/170; 31.2%), with respect to conventional nonsexed (78/142; 54.9%), and sexed doses at 4, 6, and 8 million spermatozoa (102/205, 49.8%; 84/175, 48.0%; and 74/157, 47.1%, respectively). No differences were evident using conventional doses and sexed semen with sperm numbers equal or higher than 4 million per dose. Pregnancies were not affected by the sire; 39/82 (47.6%), 120/270 (44.4%), and 151/355 (42.5%), respectively, for the three bulls. Variability in pregnancy rates observed in different herds was not significant. Furthermore, no significant difference was reported between pregnancies obtained with sexed semen in heifers and multiparous, respectively, 179/407 (44.0%) and 131/300 (43.7%). The results of the present work indicate that in Mediterranean Italian buffalo the dose of 4 million represents an optimal compromise when using sexed semen with conventional technologies of insemination, together with estrus synchronization, and the minimum number of spermatozoa per dose. In addition, the real time polymerase chain reaction method was optimized and is now available for estimating sorting efficiency in buffalo.