Semen quality and fertility effectiveness of cloned buffalo (Bubalus bubalis) bulls

In India, due to a severe shortage of elite bulls, semen available from progeny-tested bulls covers a limited breedable population of buffalo. Animal cloning has proved beneficial in making identical genetic copies in the shortest possible time to meet the target of providing elite breeding bulls. ICAR-National Dairy Research Institute, Karnal, has produced several cloned breeding bulls, of which two breeding bulls (named Swarn and Rajat) were used to demonstrate the fertility of cloned bulls. The semen parameters such as volume (2.83±0.33 vs. 2.44±0.23 ml), sperm concentration (1607.56±161.90 vs. 1589.44±211.76 million/ml), mass motility (3.17±0.08 vs. 3.11±0.20), progressive motility (87.78±0.88% vs 85.00±1.44%) and acrosome integrity (85.39±1.66% vs 87.61±1.80%), respectively in cloned and non-cloned bull’s semen and had no significant (p<0.05) difference. Post-thaw motility was also similar to non-cloned bulls or their parents. When these spermatozoa were subjected to a transcriptomics study, 27,481 transcripts were identified, out of which 18,703 transcripts were expressed commonly in both cloned and somatic cell donor bull’s spermatozoa. Only 566 transcripts were up- regulated, and 410 transcripts were down-regulated significantly (FC≥2; P<0.05) in clone bulls spermatozoa relative to somatic cell donor bulls spermatozoa. KEGG analysis revealed that the Up- and down-regulated transcripts affect excision repair, autophagy, lipolysis, AMPK, and insulin signaling pathways. A total of 278 miRNAs were found in both cloned and parent bull spermatozoa, of which 239 were common and 28 were unique to cloned bulls spermatozoa. Out of 62 miRNAs (at FC ≥2; p<0.05) found by differential expression analysis, 31 were up-regulated, and 31 were down-regulated in cloned bull spermatozoa compared to donor bulls. The expression pattern of some necessary transcripts and miRNAs associated with spermatogenesis, sperm motility, sperm capacitation, bull fertility, and early embryonic development was almost similar in cloned bulls spermatozoa and parent bulls spermatozoa. To determine the fertility of cloned bulls, 20 female buffaloes were inseminated with the semen of two above-mentioned cloned bulls. A 65% conception rate was achieved following insemination, which is typical in buffaloes. Thirteen healthy calves (seven males and six females) have been produced, and further attempts are ongoing to produce more calves. These calves are physiologically normal, growing well and healthy. In conclusion, with this limited study, we can say that the semen produced by cloned bulls is similar to that of non-cloned bulls and parent bulls, which can be further used in various assisted reproductive technologies and sustainable milk production.