Sperm Selection for Assisted Reproduction in Nili Ravi Buffalo

Abstract

Sperm selection is of significance for cryopreservation procedures, in vitro fertilization and for production of desired sex of the offspring. Present study was planned to evaluate sperm separation methods (swim-up (SU), sephadex filtration (S-G15) and glass wool filtration (GWF)) for freezability (Objective 1) plus in vitro fertilization rate of buffalo semen (Objective 2); and to evaluate pre-freeze selection methods (modified swim up and sucrose density gradient techniques) for production of X and Y bearing sperm (SYBR green rt-PCR, Objective 3) confirmed through in-vivo fertility trials (objective 4). For this purpose semen was collected from five mąture Nilį Ravį buffąlo buļls kept at Semen Production Unit (SPU), Qadirabad District Sahiwal, Pakistan with artificial vagina (40 ˚C). For Objective 1, qualifying ejaculates from each bull were pooled and divided into aliquots and processed by swim-up, Sephadex filtration, glass wool filtration and routine procedure (control). After separation, semen was cryopreserved and evaluated in terms of post-thaw quality and in vitro fertility rate. The results indicate that total and motile sperm recovery rate was higher in samples processed through GWF, while post-thaw quality were improved (P < 0.05) in sperm selected by S-G15 and GWF (some parameters) compared to control. The sperm selected through S-G15 prior to cryopreservation showed highest (P < 0.05) embryo cleavage rate (%) compared to control. Methods of sperm preparation were evaluated for in vitro fertility rate (of cryopreserved semen (Objective 2). In vitro matured buffalo oocytes were fertilized by semen processed by routine procedure (control) or selected through swim-up, Sephadex filtration and glass wool filtration. Highest total and motile sperm recovery rate achieved through Glass wool filtration xxv while improved (P < 0.05) sperm quality (progressive motility, membrane integrity, viability, livability) was achieved by sephadex filtration. Sperm selection by S-G15 yielded higher in vitro fertility rate (in terms of cleavage rate) compared to glass wool filtration and swim-up (control). For objective 3, two separate experiments (modified Swim up and Sucrose density gradient) were conducted for sex sorting (X and Y bearing spermatozoa). In each experiment, semen was collected; qualifying ejaculates were pooled and processed by routine procedure (control) or sperm sexing techniques (modified swim-up and Sucrose density gradient). After separation, semen was diluted in trįs-cįtric acįd extendėr and cryopreserved usįng stąndąrd techniquės. Cryopreserved samples were analyzed for relative expression of presumptive X and Y bearing sperm in respective sexed doses by SYBR Green rt-PCR using SRY and PLP gene primers and post thaw semen quality. Using swim up technique, X chromosome bearing sperm fraction showed significantly higher sperm recovery rates, pre-freeze and post-thaw sperm quality than Y chromosome bearing fraction and control. X bearing sperm showed significantly higher (4–5 fold) mean fold relative expression in presumptive X bearing sperm fraction of supernatant than Y bearing fraction (0.06 fold), similarly Y bearing sperm was also showed significantly higher mean fold relative expression in Y bearing fraction (4 fold) of supernatant than X bearing fraction (0.15 fold) compared to control (1.00). For sucrose density gradient, X sorted sperm fractions showed significantly higher sperm recovery rates, pre-freeze and post-thaw sperm quality than Y sorted sperm fraction and control. X sorted fraction has comparable (1.6 fold) mean fold relative expression of X bearing sperm than Y sorted fractions (0.3 fold) whereas Y bearing sperm fraction showed significantly higher mean fold relative expression in Y xxvi sorted sperm (2.66 fold) fraction of supernatant than X sorted fractions (0.69 fold) compared to control (1.00). Sexed semen doses were evaluated for field fertility trials during peak breeding season to check the accuracy of method (Objective 4). From results it is concluded that sperm selection by sephadex filtration prior to and after cryopreservation showed improved quality and yielded better fertilization rates (in term of cleavage rate) of in vitro matured/ fertilized oocytes. Validation of modified swim up method by real time PCR and in vivo fertility trial (significantly higher production of female calf i.e. ~ 78%) proved that this method would be effective for Nili-Ravi buffalo sperm sexing for production of female calf. A sucrose density gradient method may be useful for production of male Nili-Ravi buffalo male calf.