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Title:Enhanced fertility estimation and novel ion channels in boar sperm
Author(s):Daigneault, Bradford W
Director of Research:Miller, David J.
Doctoral Committee Chair(s):Miller, David J.
Doctoral Committee Member(s):Krisher, Rebecca L.; Knox, Robert V.; Wheeler, Matthew B.; Bunick, David C
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):boar
sperm
in vitro fertilization (IVF)
cryopreserved
motility
oviduct
acrosome
fertilization
sperm function
transient receptor potential channel-2 (TRPP2)
polycystin 2 (PKD2)
capacitation
ion
Abstract:Cryopreserved boar sperm have many potential advantages when used for artificial insemination (AI), including increased time for pathogen testing and ease of semen transport. However, frozen boar sperm are seldom used for AI due to decreased pregnancy rates and litter sizes as a result of cryo-induced damage to sperm. Estimating the fertility of samples prior to AI would allow disposal of semen with poor predicted fertility. Unfortunately, conventional laboratory analyses of semen do not provide accurate estimates of fertility. Assessing sperm traits that more accurately predict fertility would improve commercial use of frozen boar sperm. Our objective was to identify traits of cryopreserved sperm that are related to boar fertility by including novel sperm assays with traditional laboratory analyses that may collectively provide a more accurate model for estimating fertility. Semen from 14 boars of several breeds was cooled to 15°C for overnight shipping prior to freezing. Semen was thawed and motility was estimated and confirmed using Computer Automated Sperm Assessment (CASA). Sperm viability and acrosome integrity were measured at 0, 30 and 60 min post-thaw. In addition to traditional analyses, each sperm sample was tested by IVF in two to three independent replicates and fertilization, cleavage and blastocyst development were recorded. As an assessment of sperm reservoir formation, a sperm-oviduct binding assay was used to compare the number of sperm bound to epithelial aggregates harvested from gilt isthmus. Additionally, a competitive zona binding assay using two distinct fluorophores for sperm identification was employed to measure the number of sperm from each boar bound to the zona. Frozen sperm from the same ejaculates subjected to laboratory analyses were used to determine boar fertility. Fertility was measured by AI of mature gilts using 4.0 x 106 sperm from one boar at 24 h and a second boar at 36 h after the onset of estrus and calculated as the percentage of the litter sired by each boar. AI order was reversed in consecutive replicates so that order of insemination was evenly distributed among boar comparisons. Reproductive tracts were harvested at ~ 32 d after AI and the number of fetuses were recorded and sampled for paternity identification using microsatellite markers. The least-squared means (LSM) of each laboratory evaluation were modeled by boar using multiple regression analyses to test their collective values in predicting fertility. The model generated was highly predictive of fertility (P < 0.001, r2 = 0.87) and included 5 traits; acrosome compromised sperm (0 and 30 min), percent live sperm (0 min), percent total motility (30 min) and the number of zona bound sperm. An additional model in which fertility was assessed by the number of piglets sired by boar also predicted fertility (P < 0.05, r2 = 0.57) and shared many of the same traits including percent live sperm, motility and the number of sperm bound to zona. These models indicate that the fertility of cryopreserved boar sperm can be predicted using both traditional and novel laboratory assays that consider multiple functions of sperm. This model provides a method to cull low fertility cryopreserved boar semen more accurately, thus enhancing the potential adoption of semen cryopreservation by the swine industry. Both models that successfully estimated boar fertility included sperm traits that evaluated binding ability to the zona pellucida. Further, binding to the oviduct epithelium was related to fertility. Impaired functions of sperm membrane proteins that are critical for normal sperm interactions within the female reproductive tract are not always measured during routine semen analyses and therefore likely contribute to idiopathic subfertility. Sperm must undergo important capacitation-like changes for normal fertilization such as a change in motility parameters and an increase in intracellular calcium. Transient receptor potential channel-2 (TRPP2) is a membrane protein with regulatory roles in ion homeostasis. TRPP2 is important for sperm motility, storage and male fertility in lower organisms (Drosophila melanogaster), but the protein has not been described in mammalian sperm. My objective was to identify, localize and begin to determine the functions of TRPP2 in porcine sperm. TRPP2 was detected in both capacitated and non-capacitated boar sperm and was localized to the head and principle piece. Antisera to TRPP2 inhibited sperm movement and increased tail cross-beat frequency. Sperm incubated with TRPP2 antibody had lower levels of free intracellular calcium and did not experience a rise in calcium over time when maintained in capacitating conditions. We conclude that porcine TRPP2 is a previously unreported mammalian sperm ion channel that regulates capacitation-like changes in boar sperm.
Issue Date:2015-07-08
Type:Thesis
URI:http://hdl.handle.net/2142/88160
Rights Information:Copyright 2015 Bradford Daigneault
Date Available in IDEALS:2015-09-29
Date Deposited:August 201


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