Improved bovine in vitro embryo production with sexed and unsexed sperm selected by chemotaxis

Abstract

Assisted reproductive techniques (ART) have been widely used in farm animals in the last decades. Sexed cryopreserved spermatozoa, ovum pick up, in vitro embryo production and transfer constitute the ART that have revolutionized the dairy industry. However, the efficiency of some of these techniques is still low due in part to sperm quality, which influences fertilization, embryo development and implantation. The Sperm Selection Assay (SSA), based on sperm chemotaxis towards progesterone, provides a sperm subpopulation enriched with spermatozoa that are capacitated, with intact DNA and low level of oxidative stress. Since the SSA selects a sperm subpopulation at optimum physiological state, the application of the SSA may improve the efficiency of the current ART. The aim of this study was to adapt the SSA for unsexed and sexed bovine frozen-thawed semen samples, and then to test whether sperm selection by the SSA improves the cleavage rate of bovine embryos in vitro. The optimal SSA conditions to obtain the higher sperm accumulation percentage given by chemotaxis were the same for both unsexed and sexed semen samples. Thus, sperm accumulation in W2 was significantly higher when: 2 million sperm per mL were placed in W1 (unsexed samples: 12 ± 1%, p = 0.002; sexed samples: 14 ± 3%, p = 0.02); 1 pM progesterone was placed in W2 (unsexed sample: 9 ± 1%, p = 0.009; sexed samples: 11 ± 2%, p = 0.02); and to incubate the SSA device for 10 min (unsexed samples: 17 ± 2%, p = 0.007; sexed samples: 10 ± 1%, p = 0.004). We found that the quality of spermatozoa recovered from W2 in unsexed and sexed semen was enhanced. Thus, the capacitation index was significantly increased (unsexed samples: 1.75 ± 0.1, p = 0.0001; sexed samples: 1.76 ± 0.2, p = 0.004), while DNA fragmentation index was significantly decreased (unsexed samples: 0.33 ± 0.07, p = 0.0003; sexed samples: 0.32 ± 0.04, p = 0.002). Moreover, the cleavage index of oocytes fertilized with either unsexed or sexed SSA-selected sperm was significantly improved (unsexed samples: 3.2 ± 0.4, p = 0.0001; sexed samples: 2.3 ± 0.33, p = 0.03). Thus, we show that the SSA can be used to recruit a bovine sperm subpopulation at optimal functional state regardless of whether the sample is previously sexed, and that this optimal state improves bovine embryo cleavage rate.