Colorado State University
Gonzalez-Castro, Raul A.
Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection.
Degree: PhD, Biomedical Sciences, 2018, Colorado State University
When performing intracytoplasmic sperm injection (ICSI), many in vivo mechanisms for sperm selection are bypassed; however, sperm must still be capable of activating the oocyte for successful fertilization. Limited information is available for horses on the effect of sperm preparation method and sperm characteristics that affect ICSI outcome. The overall objectives of this dissertation were to: 1) study the association between sperm sorting methods, sperm population characteristics, and equine ICSI outcome, and 2) characterize sperm oocyte activating factors in stallion sperm, such as phospholipase C zeta (PLCz) and postacrosomal WW binding protein (PAWP). In Experiment 1, a microfluidic device was used to sort frozen-thawed sperm from stallions (n=19), which resulted in a sperm subpopulation with improved motility, morphology, viability and DNA integrity (P<0.05) compared to the original sample. Then, microfluidic sorting was compared with the swim-up procedure and density gradient centrifugation. Swim-up was the least effective method to separate equine sperm. Microfluidic sorting and density gradient centrifugation sorted a sperm subpopulation with similar parameters, improving motility, viability and DNA integrity. After ICSI (n=45), no differences (P>0.3) were observed for cleavage and embryo development among sorting methods. In Experiment 2, sperm population parameters from which individual sperm were selected for injection were analyzed immediately after ICSI and correlated with the outcome. Sperm morphology, viability, membrane integrity measurement of hypoosmotic swelling and DNA integrity were evaluated in frozen-thawed sperm (n=114) used for ICSI in a program. Among sperm parameters, viability correlated positively with normal morphology and membrane integrity (P<0.05). Normal sperm morphology and DNA integrity were not predictive of ICSI outcome. Viability was predictive of cleavage and blastocyst formation, and membrane integrity was predictive of early pregnancy (P<0.05). In Experiment 3, PLCz and PAWP were identified, localized and quantified in stallion sperm, and the relationship with other sperm parameters was investigated. PLCz was identified as a 71 kDa protein and located in the acrosomal and postacrosomal region, midpiece and principal piece of the tail. PAWP was identified by two bands of ~28 and ~32 kDa, located in the postacrosomal region, midpiece and principal piece of the tail. The expression of PLCz and PAWP correlated positively (P=0.04) when analyzed for sperm of 14 stallions. Flow cytometric assessment was feasible for PLCz, but not for PAWP. Expression and percentages of positive labeled sperm for PLCz varied among stallions (n=21). Expression of PLCz was higher in live than dead sperm (P<0.005), and DNA fragmentation correlated negatively with PLCz expression (P<0.04). In conclusion, microfluidic sorting and density gradient centrifugation resulted in a subpopulation of sperm with high quality parameters for ICSI. The probability of sperm-injected oocytes to develop into an embryo…
Advisors/Committee Members: Carnevale, Elaine (advisor), Grahman, James (advisor), Seidel, George (committee member), Gerrit, Bouma (committee member), Ann, Hess (committee member).
Subjects/Keywords: microfluidics; PLC zeta; stallion; PAWP; ICSI; sperm
to Zotero / EndNote / Reference
APA (6th Edition):
Gonzalez-Castro, R. A. (2018). Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/191446
Chicago Manual of Style (16th Edition):
Gonzalez-Castro, Raul A. “Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection.” 2018. Doctoral Dissertation, Colorado State University. Accessed April 16, 2021.
MLA Handbook (7th Edition):
Gonzalez-Castro, Raul A. “Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection.” 2018. Web. 16 Apr 2021.
Gonzalez-Castro RA. Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. [Internet] [Doctoral dissertation]. Colorado State University; 2018. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/10217/191446.
Council of Science Editors:
Gonzalez-Castro RA. Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. [Doctoral Dissertation]. Colorado State University; 2018. Available from: http://hdl.handle.net/10217/191446