Improvement in Simple Artificial Insemination in Sheep Using Chilled Extended Semen and

108 Pages·2017·1.14 MB·English

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Graduate Theses and Dissertations
8-2017
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Mohan Acharya
University of Arkansas, Fayetteville
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Acharya, M. (2017). Improvement in Simple Artificial Insemination in Sheep Using Chilled Extended
Semen and Consideration of Fertility in Ewes. Graduate Theses and Dissertations Retrieved from
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Improvement in Simple Artificial Insemination in Sheep Using Chilled Extended Semen and
Consideration of Fertility in Ewes

A dissertation submitted in partial fulfillment
of the requirement for the degree of
Doctor of Philosophy in Animal Science

by

Mohan Acharya
Tribhuvan University
Bachelor of Veterinary Science and Animal Husbandry, 2010
University of Arkansas
Master of Science in Animal Science, 2014

August 2017
University of Arkansas

This dissertation is approved for recommendation to the Graduate Council.

Rick W. Rorie, Ph.D
Dissertation Director

Joan M. Burke, Ph.D. Kenneth Coffey, Ph.D.
Committee Member Committee Member

Dan. J. Donoghue, Ph.D.
Committee Member
Abstract
Three studies were conducted to improve simple artificial insemination in sheep using
chilled extended semen and evaluate methods to predict fertility in ewes. The first study
evaluated the effect of semen extenders and storage temperature on motility parameters of ram
semen stored for up to 72 h. Single semen collected from each ram was divided into four parts
and extended using four extenders: milk, TRIS, TEST, and CJ-2, diluted to 600 million sperm/ml
and loaded into 0.25 ml straws for storage at 4 and 15° C. Most of the CASA motility parameters
were higher after extension and storage at 4 compared with 15° C (P < 0.05). Ram semen stored
using milk or TRIS based extenders at 4° C maintained similar sperm motility parameters but
both were higher compared with TEST or CJ-2 extenders after storage for up to 3 d (P < 0.05).
The second study evaluated the effects of different combinations of extenders, ethylene glycol
(EG), egg yolk and penicillamine, hypotaurine and epinephrine (PHE) on ram sperm motility
parameters during storage. Semen electroejaculated from each ram was distributed across
treatment combinations consisting of either TRIS or milk extenders supplemented with 5 or 20%
(v/v) egg yolk, ± EG, and ± PHE. Milk extender supplemented with 1% EG, PHE and either 5 or
20% egg yolk can protect sperm cells better than other extender and supplement combination
after storage at 4° C for up to 72 h. The third study was conducted to correlate serum AMH and
lambs born with estimated breeding values determined through National Sheep Improvement
Program for reproductive performance (NLB or number of lambs born; NLW or number of
lambs weaned; MWWT or maternal weaning weight; and the Katahdin index) and WWT or
weaning weight. Additionally, correlation between AMH quartiles and lambs born after fall
versus summer breeding was determined. There was no correlation between serum AMH and
EBVs for NLB, NLW, MWWT, WWT and the Katahdin index in Katahdin ewes (P > 0.05).
Ewes in the lowest quartile (Q1) had lower number of offsprings born from summer breeding
compared with ewes in other AMH quartiles.
Acknowledgements
I would like to express my deepest gratitude and sincere appreciation to my research
advisor Dr. Joan M. Burke. Thank you for all course of studies, research, and training
opportunities you provided during 5 years (MS and PhD). You have thousands of qualities to be
a good advisor.
I would like to thank Dr. Rorie for accepting me as a PhD student from pile of applicants.
I will be missing every morning chats. You have good sense of humor behind quietness. Thank
you for providing opportunities on cattle research studies/practical experiences.
I would acknowledge Dr. Kenneth Coffey, and Dr. Dan J. Donoghue for serving in my
PhD thesis committee and providing valuable suggestions.
My dear parents, Chhabilal Acharya and Sabitra Acharya, my grandfather Dhananjaya
Acharya, a graduate from Banaras Sanskrit University, about a century ago, my big brother
Kamal Acharya (PhD graduate from Norway), my small brother Dinesh Acharya (MSc engineer
graduate from Switzerland), my sisters Manju Acharya and Ranjana Acharya and my beautiful
wife Roshani Sharma Acharya (PhD candidate at University of Arkansas). My achievement
today would not have been possible without the help of USDA-ARS Booneville Folks, Chad
Lee, Erin Wood, Jackie Cherry, Connie Cox, Samuel Tabler, Hannah Toland, Caleb Crelia,
Berlin Howell, and Keren Chapman. Graduate students at the University of Arkansas,
Fayetteville, AR, Saleh A. Alrashedi, Amanda Davis, Fanli Kong, Angella Boyer, Michelle
Thomas, Elizabeth Backes, Brandon Smith, Tony Ryan, Tom Devine, Cristina Turner, Eva wary,
Xheena, Christine Weingartz-Hernandez. Secretaries and staffs at the department, Chris Hansen,
Toby, Linda, Robert Story, Jeannie, Dianna, Connie, Palika, Tes, and TC thank you all help.
I cannot and will not forget Dr. Charles F. Rosenkrans name in here for all his love.
Dedication
I want to dedicate this dissertation, PhD degree and my all course work to my research
advisor Dr. Joan M. Burke. You accepted me as your first Master student as well as first PhD
student. You have thousands of qualities to be a good advisor. Thank you for all the help you did
in this important and productive 5 years.
Table of Contents Page number
Chapter I: Introduction………………………………………………………………………….1
Chapter II: Review of Literature……………………………………………………………….3
a. Common extenders and storage temperatures for extension and cold storage of ram
semen……………………………………………………………………………………...3

Early extension of bull semen…………………………………………………………......3
Common semen extenders used in different species……………………………………...3
Common buffers and bases used for storage of ram semen……………………………….4
Milk extender……………………………………………………………………………...6
Comparison of milk with other extender………………………………………………….8
Storage temperatures……………………………………..................................................10
Use of different percentage of egg yolk in the extender medium………………………..11
Low sodium choline based medium (CJ-2)……………………………………………...13
Ethylene glycol…………………………………………………………………………..14
Penicillamine, hypotaurine and epinephrine (PHE)……………………………………..16
b. Anti-Mullerian hormone as an endocrine marker to predict fertility………………18
Anti-Mullerian hormone…………………………………………………………………18
Sexual differentiation in males and females……………………………………………..18
Anti-Mullerian hormone and FSH……………………………………………………….21
Anti-Mullerian hormone studies in ewes………………………………………………...22
Summary and objectives…………………………………………………………………22
References…………………………………………………………………………..........24
Chapter III: Effect of semen extender and storage temperature on ram sperm motility
over time

Abstract………………………………………………………………………………….34
Introduction…………………………………………………………………………......35
Materials and Methods…………………………………………………………………36
Animal Management……………………………………………………………..36
Evaluation of fresh semen by computer assisted sperm analysis (CASA)………36
Preparation of semen extenders………………………………………………….37
Milk extender ……………………………………………………………37
TEST extender…………………………………………………………...37
TRIS extender……………………………………………………………38
CJ-2 extender ……………………………………………………………38
Preparation of semen for storage………………………………………………...38
Evaluation of extended semen using CASA …………………………………….39
Statistical analysis……………………………………………………………......40
Results…………………………………………………………………………………...40
Discussion……………………………………………………………………………….43
References……………………………………………………………………………….48
Tables
Table 1. CASA parameters………………………………………………………50
Table 2. Hourly change in temperature of semen filled straws kept inside
Koolatron cooler maintained at 15° C or laboratory refrigerator at 4° C………..51
Table 3. IVOS machine settings for ram semen motility analysis………………52
Table 4. Effect of temperature on ram sperm motility parameters over 72 h of
storage. Ram sperm motility parameters of extended and stored ram semen diluted
in CJ-2, milk, TEST, and TRIS extenders and stored at 4 or 15° C for 72 h…...53
Table 5. Effect of time of storage on ram sperm motility parameters, CJ-2, milk,
TEST or TRIS and stored at 4 and 15° C for 72 h……………………………….54
Chapter IV: Evaluation of semen extenders for short-term storage of ram semen at 4° C
Abstract………………………………………………………………………………….55
Introduction……………………………………………………………………………..56
Materials and Methods…………………………………………………………………57
Animal Management……………………………………………………………..57
Evaluation of fresh semen by computer assisted sperm analysis (CASA)………58
Experimental treatments…………………………………………………………58
Extenders………………………………………………………………………...59
Milk and TRIS extender…………………………………………………59
Determination of semen extension rate …………………………………………59
Semen extension using extenders ……………………………………………….59
Preparation of PHE ……………………………………………………………...60
Evaluation of extended semen …………………………………………………..61
Statistical analysis ……………………………………………………………….61
Results ………………………………………………………………………...………...62
Discussion……………………………………………………………………………….65
References…………………............................................................................................70
Tables
Table 1. IVOS machine settings for ram semen motility analysis ……………...72
Table 2. CASA parameters……………………………………………………....73
Table 3. Sperm motility parameters after extension using milk or TRIS and stored
at 4° C for 72 h. ………………………………………………………………….74
Table 4. Sperm motility parameters after extension using 5 or 20% (v/v) egg yolk
in milk or TRIS and stored at 4° C for 72 h. …………………………………….75
Table 5. Sperm motility parameters after extending with or without ethylene
glycol in milk or TRIS and stored at 4° C for 72 h. ……………………………. 76
Table 6. Effect of addition of penicillamine, hypotaurine, and epinephrine (PHE)
on sperm motility parameters before evaluation on cold stored ram semen, stored
at 4° C for 72 h.………………………………………………………..................77
Table 7. Sperm motility parameters after extension with or without ethylene
glycol in milk or TRIS with 5 or 20% egg yolk and stored at 4° C for 72 h. Only
significant ethylene glycol × hours interactions (P < 0.05) are expressed……....78
Table 8. Sperm motility parameters after extension using with or without
ethylene glycol (EG) in milk or TRIS extender. Only significant ethylene glycol ×
extender interactions (P < 0.05) are expressed…………………………………..79
Table 9. Sperm motility parameters after extension including 5 or 20% egg yolk
in milk or TRIS extender. Only significant egg yolk (EY) × extender interactions
(P < 0.05) are expressed…………………………………………………………80
Chapter V: Correlation between Anti-Mullerian hormone and estimated breeding value for
reproductive performance in ewes ……………………………………………………………81

Abstract………………...………………………………………………….……………81
Introduction….……………………….…………………………………….…………..82
Materials and Methods ………………………………….…………………………….83
Location …………………………………………………………………………83
Animal management …………………………………………………….………83