improvement in simple artificial insemination in sheep

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Theses and Dissertations

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Improvement in Simple Artificial Insemination inSheep Using Chilled Extended Semen andConsideration of Fertility in EwesMohan AcharyaUniversity of Arkansas, Fayetteville

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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

Blood sampling and serum collection …………………………………………...83

Anti-Mullerian hormone analysis ………………………………......…………...84

Description of animals and categories…………………………………………...85

Statistical analysis ……………………………………………………………….85

Results …………………………………...……………………………………………...85

Discussion ……………………………………………………...……………………….87

References ………………………...…………………………………………………....92

Table 1. Relationship between AMH quartiles and mean number of lambs born

from different summer and fall breeding. Data expressed in mean ± standard

error (SE)………………………………………………………………………...94

Chapter VI: Conclusions ………………………………… …………………………………...95

Appendix. University of Arkansas Animal Use Protocol (IACUC 15019)…….…...…….…97

1

Chapter I. Introduction

There are many advantages to using artificial insemination (AI) in sheep. In a

conventional breeding program, a ram can mate 50 to 100 ewes per year. However, through the

use of cervical AI, semen collected from a single ram can be used to inseminate over 1000 ewes

during 2 to 3 weeks (Evans and Maxwell, 1987). Faster genetic gain for desirable traits in the

flock is possible through the use of AI technique (Evans and Maxwell, 1987). Artificial

insemination is the cheapest means to spread superior genetic traits of elite rams across long

distances and beyond the limits of live animals (Foote et al., 2002). Artificial insemination

eliminates the need for small producers to keep a breeding ram in a herd and also enables them to

access semen from superior rams (Evans and Maxwell, 1987). For AI, semen quality is examined

on all rams before semen extension and storage, thus eliminating sub-fertile males from the flock

(Foote et al., 2002). When using AI, there is no direct contact between males and females, which

helps to minimize disease transmission during breeding (Evans and Maxwell, 1987). Semen can

be collected from superior rams which may not be able to breed naturally due to injury (Evans

and Maxwell, 1987). Ram-ewe preference during breeding is eliminated in AI (Evans and

Maxwell, 1987). Accurate record keeping is possible through AI, and thus, making it easier

to select desirable traits and minimize undesirable traits from the flock (Evans and Maxwell,

1987). During off-season (sheep are seasonal breeders), ram semen quality is low (Mickelsen et

al., 1981). Artificial insemination enables the collection of high quality semen during the

breeding season and use it to inseminate synchronized ewes during off-season (Evans and

Maxwell, 1987). It is possible to produce only ewe lambs through AI using sex sorted semen

(Foote et al., 2002).

Artificial insemination with frozen thawed semen results in low conception rates because

of poor survival of sperm (Salamon and Maxwell, 2000). A higher conception rate occurs when

2

extended semen is cool stored instead of cryopreserved. Cervical AI conception rate was

compared after extension and storage among various temperatures. Conception rate was higher

with extended semen stored at 4° C for 72 h (70%) or 23° C for 24 h (76%) compared with

cryopreservation (-196° C, 50%; Wusiman et al., 2012). Milk or tris[Hydroxymethyl]

aminomethane (TRIS) based extenders are the most common extenders used for liquid storage of

ram semen (Salamon and Maxwell, 2000). The TEST (N-Tris(hydroxymethyl) methyl-2-

aminoethane sulfonic acid, TES titrated with TRIS) was used in other species but not commonly

used for extension of ram semen (Bolanos et al., 1983; Pope et al., 1991). Very limited studies

are available on choline based (CJ-2) semen extender.

There was evidence of better motility of ram semen after storage at 5° C (O’ Hara et al.,

2010) while others reported better motility after storage at 15° C (Mata-Campuzano et al., 2014).

Conclusions cannot be generalized as those studies differed in types of extenders and storage

duration. The current study was designed to find best semen extenders, storage temperature, and

supplements for liquid storage of ram semen for 3 d.

Anti-Mullerian hormone (AMH) in circulation may reflect the total pool of follicles

within the ovaries, and may be positively correlated with fertility in ewes. An additional

objective of the study includes determining the correlation between serum concentrations of

AMH and EBV for NLB, NLW, MWWT, WWT, and the hair sheep index.

3

Chapter II: Review of literature

a. Common extenders and storage temperatures for extension and cold storage of ram

semen

Early extension of bull semen

Yolk-phosphate was the first most extensively-used semen extender for AI in the US

(Phillips and Lardy, 1940). Salisbury et al. (1941) reported superiority of sodium citrate extender

due to better buffering capacity, and maintaining of sperm motility for a longer period compared

with egg yolk-phosphate extender. During the early 1940’s, bull semen extended with sodium

citrate and stored at 5° C for up to 3 d was used for AI in cattle (Foot et al., 2002). A few years

later, the necessity of semen preservation for long periods developed, especially for dairy cattle;

therefore, a use of a glycerol based semen extender for cryopreservation was initiated (Polge et.

al., 1949). During the 1940’s and 1950’s, AI conception rate in cattle was very low (15%; Foote

et al., 2002). Several studies were conducted in the 1950’s to protect sperm cells from cold shock

and used antibiotics to control disease transmission in order to increase overall AI conception

rate in cattle (Foot and Bratton, 1950). Studies on milk glycerol semen extender for

cryopreservation of bull semen began in the 1950’s (Michajilov, 1950). Caproic acid and

catalase, with 5% egg yolk as antioxidants, were used to preserve bull sperm in New Zealand in

the early 1980’s (Shannon et al., 1984). Use of sugar for successful cryoprotection was initiated

by Polge in 1968 (Polge, 1968). The TRIS egg yolk extender for cryopreservation provided

excellent sperm protection for both cold and frozen storage (Davis et al., 1963), and thereafter,

this medium was used worldwide for semen storage of bull semen (Rehmana, et al., 2013).

Common semen extenders used in different species

Sodium potassium tartrate, sodium sulfate, and peptone were commonly used to extend

and store boar semen between 1920 - 1960 (Foote et al., 2002). After discovery of egg yolk-

4

citrate, and egg yolk-phosphate extenders in the bull, the same extenders were used in the boar

with little modification (Polge, 1956). Frozen storage of boar semen had little success; therefore,

fresh or liquid extended semen is commonly used (Foote et al., 2002).

The most common semen extenders used to extend buck and ram semen is described in

detail (Evans and Maxwell, 1987). The TRIS and citrate are common buffers, fructose or glucose

are used as energy sources, and egg yolk is added to the extender to protect sperm cell

membranes against cold shock (Evans and Maxwell, 1987). Egg yolk causes a negative

interaction with seminal plasma in buck semen; therefore, low egg yolk is preferred for

cryopreservation for goats (Evans and Maxwell, 1987). Sperm extension, extenders, storage

temperature, and AI techniques in sheep and goats has been extensively reviewed by Australian

scientists (Salamon, 1976; Salamon and Maxwell 1995; and Salamon and Maxwell 2000).

Common semen extenders used in poultry are a mixture of phosphates, citrates and/or N,

N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (BES), and TES (Donoghue and Wisharttris,

2000). Turkey semen requires oxygen during storage but chicken sperm can survive both in an

aerobic and non-aerobic environment (Donoghue and Wishart, 2000). Artificial insemination in

poultry is mostly from fresh collection. Cryopreservation of poultry semen is limited (Gill et al.,

1999).

Common buffers and bases used for storage of ram semen

Seminal plasma maintains semen pH, but semen pH may fluctuate after extension (Purdy,

2006). In order to maintain sperm cell viability or avoid damage to cells in extended semen and

storage, pH needs to be constant (Purdy, 2006). Buffers are used to minimize pH fluctuation after

extension. Buffering solution for semen storage should have a) pKa between 6 and 8, b)

maximum water solubility, c) minimum passage through cell membranes, d) little interaction

5

with salts, e) dissociation of buffer that is minimally affected by buffer concentration,

temperature, and ionic concentration of medium, f) resists enzymatic and non-enzymatic

digestion, and g) should be prepared from inexpensive materials and easily purified (Good et al.,

1966; Graham et al., 1972). Milk, TRIS, TES, and choline-based extenders meet most of the

above criteria. Appropriate concentrations of these buffers in semen extenders for maintenance

of pH and best survival of sperm was examined. Concentration of TRIS (150 to 550 mM in 50

mM increments) was tested in a medium to compare best post thaw survival of frozen ram semen

(Salamon and Visser, 1972). Spermatozoa tolerated a wide range of TRIS concentrations (250 to

400 mM), although the highest post thaw motility was obtained using 300 mM TRIS.

Raffinose-based extender was the extender of choice before in vitro semen motility was

tested with TRIS based extenders. Visser and Salamon (1973) compared AI conception rate

following cervical AI with frozen-thawed ram semen extended in either TRIS (300 mM TRIS,

27.75 mM glucose, 15% v/v egg yolk, and 5% v/v glycerol) or raffinose (166.5 mM raffinose, 68

mM sodium citrate, 15% v/v egg yolk, and 5% v/v glycerol) based extender. Lambing rates were

similar (40.0 versus 44.8%) for TRIS and raffinose-citrate extender, respectively, but freeze-

thaw sperm motility of frozen ram semen pellets without using thawing solution (pelleted

raffinose in inositol-sodium-citrate solution and pelleted TRIS in TRIS-fructose solution) was

lower in raffinose-citrate extenders compared with TRIS based extender. Thus, TRIS based

extender was recommended by Salamon and Visser (1972) as the extender of choice. A

concentration of 300 mM is still used as a concentration of choice for liquid storage of ram

semen in TRIS extender (Quan et al., 2016).

Studies have also been conducted to determine the best buffer and base combinations for

pH maintenance and sperm cell survival after freeze-thaw of extended semen. Seven different

6

zwitter ion buffers, 3-(Morpholin-4-ium-4-yl)-1-propanesulfonate (MES), 2,2’-(1,4-

Piperazinediyl)diethanesulfonic acid (PIPES), 3-(Morpholin-4-ium-4-yl)-1-propanesulfonate

(MOPS); 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol (BES), and N-

Tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid (TES), 2-[4-Hydroxyethyl)-1-

piperazinyl]ethanesulfonic acid (HEPES), and N-[1,3-Dihydroxy-2-(hydroxymethyl)-2-

propanyl]glycine (TRICINE) were used to titrate separately with bases (TRIS, NaOH, and

combination of NaOH and KOH) to pH 7.0 for extension and cryo-storage of bovine semen in a

glycerol-egg yolk medium (Graham et al., 1972). The TES titrated with TRIS better maintained

pH than other buffer-base combinations and maintained semen motility after freezing and

thawing of bovine semen (Graham et al., 1972). Comparison of TRIS (300 mM TRIS, 27.8 mM

glucose, 94.7 mM citric acid, 150 mg/l penicillin, 250 mg/l streptomycin, 0.3% bovine serum

albumin, and 10% (v/v) egg yolk) and TES-TRIS (176 mM TES, 80 mM TRIS, 9 mM glucose,

10 µg/ml gentamicin sulphate, and 10% egg yolk) was done for extension and cold storage (4º C)

of ram semen for 7 d (Quan et al., 2016). However, there were no significant differences in

sperm motility and viability parameters between TRIS and TES-TRIS based extenders. Since

TES is expensive and its inclusion in TRIS based buffer has no advantage for liquid stored ram

semen, its inclusion in TRIS based extender was not justified.

Milk extender

Milk contains lipids, lactose, casein, albumin, globulin, lactoferrins, and several other

compounds. Heating milk to 95° C for 10 minute inactivates lactenin in protein, a compound

toxic to sperm cells (Salamon and Maxwell, 2000). Several studies were done in the past to find

component(s) in milk that protect sperm cells after extension and storage of semen. No

difference in motility or field fertility was found when bovine semen was extended and stored in

7

skim milk (lipid free) or whole milk, suggesting the lipid fraction in milk is not a major factor

that contributes to protection of sperm cells (Almquist et al., 1954). Semen extended in lactose

(not fructose) based extender had higher frozen thaw motility when used with the dialyzable

portion of milk. However, fructose based extender (144 mM fructose, 3% v/v casein, 10 mM K,

2 mM Mg, 0.5 mM Ca, 20 mM mono and disodium phosphate) had higher sperm motility

compared with lactose (equal concentration of fructose replaced by lactose) based extender in

synthetic media (Choong and Wales, 1963). Addition of extra fructose to a 144 mM initial

concentration improved frozen thawed bovine semen motility; thus lactose can be replaced with

fructose without any deleterious effect (Choong and Wales, 1963).

Casein proteins are found in milk; making up to 80% of the total protein in cow and goat

milk. Casein exists in milk as casein micelles (Dalgleish, 1998). The effect of albumin, globulin,

or casein (all 3% w/v) on motility of cold stored bovine semen was compared by adding these

proteins in phosphate buffer solution (mono- and disodium phosphates and 22 mM fructose).

Motility measured after storage of extended bull semen at 5° C for 24 h showed increased

motility in casein but not in albumin or globulin (Choong and Wales, 1963). Based on their

research it was concluded that casein (not albumin or globulin) could help improve semen

motility after cold storage of bovine semen. The mechanism by which casein protein protects

sperm after extension was reviewed by Manjunath et al. (2002). The binder of sperm (BSP)

proteins are present in seminal plasma which deteriorates sperm cells after extension (Manjunath

et al., 2002). In ram semen, casein micelles bound to BSP proteins to decrease cholesterol and

phospholipid loss from the sperm membrane, thus protecting sperm cells after extension (Plante

et al., 2015). Therefore, it could be that casein protein in milk plays a major role in protection of

sperm cells when semen is extended and stored in milk based extenders.

8

Comparison of milk with other extender

Research was conducted TRIS and milk based extenders for better maintenance of ram

sperm cell motility after extension and storage. Motility of ram sperm was higher when extended

and stored at 5 or 20° C for 30 h in a commercial TRIS extender (Minitub, Tiefenbach,

Germany) with 20% v/v egg yolk or TRIS citrate (250 mM TRIS, 69.4 mM fructose, 88.5 g

citric acid, 20% v/v egg yolk) compared with milk (11% non-fat milk powder, 5% v/v egg yolk)

or sodium citrate (112 mM % trisodium citrate, 20% v/v egg yolk) based extenders (Paulenz et

al., 2002). Superiority of TRIS over milk based extender was also reported by Quan et al. (2016)

when milk (10 g skim milk powder, 0.2 g glucose in 80 ml distilled water, and 0.5 g of penicillin

and streptomycin), was compared with TRIS (300 mM TRIS, 27.8 mM glucose, 94.7 mM citric

acid, 150 mg/l penicillin, 250 mg/l streptomycin, 0.3% bovine serum albumin, and 10% (v/v)

egg yolk), and TES-TRIS (176 mM TES, 80 mM TRIS, 9 mM glucose, 10 µg/ml gentamicin

sulphate, and 10% egg yolk) extenders for extension and storage of ram semen at 4º C for 7 d.

Total motility, membrane integrity, and acrosomal status was better when extended and stored

TRIS or TES-TRIS compared with milk extender (Quan et al., 2016). In another study, TRIS

(300 mM TRIS, 27.7 mM fructose, 103 mM citric acid, and 15% v/v egg yolk was dissolved in

100 ml distilled water), sodium citrate (2.9% aqueous solution of trisodium citrate in 20% v/v

egg yolk), milk (11% w/v distilled water with 5% v/v egg yolk), glucose phosphate (108 mM

Na2HPO4, 24 mM KH2PO4, 34 mM glucose, and 2% v/v egg yolk), and commercial proprietary

extender Andromed® (Minitub, Tiefenbach, Germany) were examined for extension and storage

of ram semen at 4º C until no motile spermatozoa were observed. No difference in sperm

motility was observed until the third day between TRIS, sodium citrate and milk based

extenders, but sperm motility was higher than that extended in glucose phosphate, and

9

Andromed® extenders (Gündoğan, 2009). For longer storage (up to 14 d), semen extended in

TRIS maintained better sperm motility compared with milk and other extenders (Gündoğan,

2009). López et al. (1999) compared sodium citrate (112 mM aqueous solution of tri-sodium

citrate mixed in 20% of egg yolk), TRIS (250 mM Tris, 71 mM fructose, 88 mM g citric acid per

1000 ml extender plus 20% egg yolk), or skim milk extender (88 g non-fatty milk powder

dissolved in 800 ml distilled water, 220 ml of mixed solution was dissolved with 12 ml of egg

yolk) for extension and storage of ram semen at 5º C for 7 d. There was no difference in sperm

motility until 24 h of storage, but after 24 h, milk based extender had lower sperm motility

compared with TRIS or sodium citrate based extender (López et al., 1999).

Not all studies reported superiority of TRIS over milk based extender for preservation of

cool stored ram semen. Hollinshead et al. (2004) compared ultra-heat treated milk, TRIS with

10% v/v egg yolk, a proprietary AndroHep® (Minitub, GmbH, Landshut, Germany), and TES-

TRIS 10% v/v egg yolk. Semen stored in ultra-heat treated milk had higher motility, acrosomal

integrity, and greater migration through artificial mucus and at on all times compared with other

extenders (TRIS, AndroHep®, and TES-TRIS; Hollinshead et al., 2004).

Superiority of milk as an extender was also reflected in in vivo studies. There was a

higher vaginal insemination conception rate obtained with semen extended and stored in milk

compared with TRIS based extenders (Paulenz et al., 2003). It could be possible that short term

(< 30 h) storage of semen in milk based extenders will have a higher motility and AI conception

rate compared with TRIS based extenders, and TRIS based extenders may be beneficial for long

term storage (> 24 h). Nevertheless, this is hard to generalize as milk based extenders had low

motility when stored for less than 30 and 24 h in the study reported by Paulenz et al. (2002) and

López et al. (1999), respectively.

10

Storage temperatures

Ram semen extended in sodium citrate (124 mM sodium citrate, 177 mM glucose, 20%

v/v egg yolk, with 6% v/v glycerol added in cryopreserved medium) was stored at 4, 23 or -196°

C (Wusiman et al., 2012). Cervical insemination of ewes with semen stored at 4° C for 72 h

resulted in a similar conception rate compared with semen stored at 23° C for 24 h (69.6% vs

76%), but both were higher than the 50% conception rate for semen cryopreserved and stored at -

196° C (Wusiman et al., 2012). Ram semen quality characteristics were also compared using

milk extenders for storage at 4 or -196° C for 48 h and 4 wk, respectively (Kulaksiz, et al.,

2012). Total motility of sperm cells in milk extender after storage at 4° C for 48 h had a higher

than compared with frozen-thawed ram semen stored at -196° C for 4 wk (47.5 versus 21.2%;

Kulaksiz, et al., 2012). From the two studies above, it can be concluded that ram semen motility

can be better maintained and have a higher AI conception when stored at a low temperature (4°

C) for a short period of time (2 to 3 d) compared with freeze-storage at -196° C.

For cool storage of extended ram semen, different low temperatures have been compared.

Ram semen was extended using milk, TRIS, commercial TRIS or sodium citrate and stored at 5

or 20° C for 30 h (Paulenz et al., 2002). Sperm motility was higher when stored at 5 compared

with 20° C (Paulenz et al., 2002). However, Salamon and Maxwell (2000) stated that 5 or 15° C

was the most suitable temperatures for liquid storage of extended ram semen. To find the best

temperature between 5 or 15° C, ram semen was extended using standard skim milk-egg yolk,

OviPro®, AndroMed®, or INRA 96® for storage at 5 or 15° C. Sperm motility score (0 = non-

motile to 5 = rapid motility) of extended and stored semen at 5° C for 72 h ranged from 2.3 for

skim milk extender, to 3.2 for AndroMed®, whereas those stored at 15° C for a similar time

period ranged from 0 for skim milk based extender to 1.16 for AndroMed® (O’Hara, et al., 2010).

11

In this study, ram semen extended and stored at 5° C had higher motility than that storaged at 15°

C when evaluated at 72 h of storage (O’Hara, et al., 2010). However, the results in this study

were not consistent with other studies in which ram semen was evaluated at 48 h after storage at

5 or 15° C (Mata-Campuzano et al., 2014) using TES-TRIS or commercial INRA96 extenders. It

could be possible that for short time storage (< 48 h) 15° C could be better than 5° C storage.

Consistent with better motility when stored for < 48 h, storage at 15 was better than 5° C with

semen extended in ultra-heat treated milk. Another study compared TRIS 10% v/v egg yolk,

AndroHep®, and TES-TRIS 10% v/v egg yolk (Hollinshead et al., 2004). Spermatozoa stored at

15° C for 6 h migrated a greater distance in artificial cervical mucus than spermatozoa stored at

5° C for 3 d (Hollinshead et al., 2004). Even though semen storage at 5° C was better than at 15°

C for 3 d (O’Hara, et al., 2010), shorter storage (6 h or 48 h) at 15 °C was better than at 5° C.

However, it is hard to make conclusion from these studies, as they differed in type of extenders,

use of supplements, and storage periods.

Use of different percentages of egg yolk in the extender medium

Sperm plasma membrane protects sperm cells from its external environment. Cooling

semen during freezing or cold storage causes massive rearrangements and leakage in sperm

plasma membrane leading to permanent damage to the sperm cell (Holt and North, 1984).

Studies were conducted to find the component of egg yolk and its protective action on sperm

cells during storage of extended semen. Studies between the 1970’s and early 1980’s concluded

that low density lipoprotein in egg yolk protects sperm cells during freezing by making a

spermatozoa-lipoprotein complex (Pace and Graham, 1974; MacDonald and Foulkes, 1981).

Ricker et al. (2006) used purified phosphatidylcholine (PC; soy-PC, or egg-PC) to replace

egg yolk in INRA 82, a commercial semen extender to freeze donkey semen. Total motility,

12

progressive motility, and viability of frozen-thawed semen showed that egg yolk can be replaced

with soy or egg-PC for frozen storage of semen without affecting semen quality. Inclusion of

external lipids (phosphatidylcholine in this case) does not incorporate in sperm membrane but

associates strongly, and this strong attachment remains even after the freeze-thaw process

(Ricker et al., 2006). Thus, low density lipoprotein can be replaced with phosphatidylcholine

without negative effects to maintain sperm motility and viability (Ricker et al., 2006). Thus, the

spermatozoa-lipoprotein complex formation may not be the only mechanism for sperm cell

protection. Purification of phosphatidylcholine either from soy or egg yolk is a very expensive

process, making it less practical.

According to a third hypothesis, bovine seminal plasma contains BSP proteins which are

secreted by seminal vesicles during ejaculation (Manjunath et al., 1988; Desnoyers and

Manjunath, 1992). The BSP proteins modify the sperm membrane and cause cholesterol efflux,

and continuous exposure leads to detrimental effects on the sperm plasma membrane (Manjunath

et al., 2002). When semen extender is supplemented with egg yolk, the low-density fraction

(LDF) of egg yolk binds BSP protein preventing detrimental effect on sperm membranes

(Manjunath et al., 2002).

Ram semen supplemented with egg yolk, or combination of egg yolk and glycerol had

higher sperm subjective motility and membrane integrity compared with no egg yolk (Gil et al.,

2011). Extender supplemented with glycerol but without egg yolk had no protective effect on

cool storage of ram semen (Gil et al., 2011). This shows the importance of egg yolk to protect

extended, cooled sperm cells.

López et al. (1999) used three different extenders: sodium citrate (112 mM aqueous

solution of tri-sodium citrate) with 20% of egg yolk, TRIS with 20% egg yolk, or skim milk with

13

5.5% egg yolk to store ram semen at 5 °C for 7 d. Inclusion of lower percentage of egg yolk in

the milk based extender led to decreased motility, but this was confounded by the difference in

composition of extenders. The importance of increasing egg yolk concentration on resilience of

spermatozoa has been demonstrated. When cooled slowly, concentration of egg yolk (i.e., 10, 15,

and 20% of total volume) in TRIS citrate extender had no effect on motility. However, viability

of ram semen extended in 20% egg yolk was higher compared with 10% egg yolk. When cooled

rapidly or cold shocked, the higher percentage of egg yolk increased motility (Rajabi-Toustani et

al., 2014).

Low density lipoprotein is a fraction of egg yolk that binds bovine seminal plasma (BSP)

protein that prevents detrimental effects on sperm membrane (Manjunath et al., 2002). Inclusion

of 8% LDL extracted from egg yolk improved protection of sperm cells over inclusion of egg

yolk in cryo stored citrate extender (Bashawat et al., 2016). Semen stored in extender containing

8% LDL had the highest freeze-thaw sperm motility (62.66%) compared with 20% egg yolk

(54.18%), or 6 and 10% LDL supplementation (Bashawat et al, 2016). Similarly, 5 and 8% LDL

in TRIS based extender increased forward progressive motility of sperm cells that had been

thawed compared with 3% LDL or egg yolk alone (Gharibi et al., 2014). Progressive forward

motility of sperm cells extended with inclusion of 12% egg yolk or 5% LDL was similar up to 48

h, but was higher after 48 h for semen extended with 5% LDL compared with 12% egg yolk.

Thus, LDL used in an extender medium maintains sperm motility better than using egg yolk for

cooled or frozen storage of ram semen. However, LDL are not commercially available.

Low sodium choline-based medium (CJ2)

Under normal physiological conditions any excess sodium that enters the cell is expelled

by a sodium pump. When oocytes/spermatozoa are slowly frozen in cryoprotectants, water

14

comes out of the cell and sodium diffuses in. The sodium pump is gradually disabled with the

decrease in temperature leading to sodium accumulation inside the cell (Mazur, 1977).

Intracellular sodium accumulation damages cellular integrity and further development of oocytes

(Mazur, 1977; Stachecki and Willadsen, 1998a). It was discovered that choline could be used in

place of sodium as a major cation for cryopreservation of mouse oocytes. Oocytes frozen in

choline based extender had better survival, fertilization, and further development compared with

sodium based extender (Stachecki and Willadsen, 1998b). Similarly, replacing sodium with

choline in a medium for cryopreservation of metaphase II mouse oocytes caused lower pre-

freezing damage (Stachecki et al., 1998b).

Reasonably, choline is a component of the phospholipid bilayer and, therefore, could help

to stabilize cell plasma membrane for better protection of spermatozoa and oocytes. Very limited

studies are available in which choline based (CJ-2) semen extenders were used for extension and

cool storage of semen. Sperm motility parameters were evaluated after extension and cool

storage of bull semen using either caudal epididymal plasma or low sodium medium

supplemented with either a lipid-rich bovine serum albumin or egg yolk. Bull semen extended

and stored at 4° C using CJ-2 egg yolk had increased sperm motility parameters compared with

other semen extenders used (Delgado et al., 2009). Choline based extenders are not used for cool

storage of ram semen. It would be worthwhile to see if beneficial effects of choline based

extenders can be seen after cold storage of ram semen.

Ethylene glycol

Ethylene glycol is a cryoprotectant of choice for freezing embryos because of its low

molecular weight, which enables rapid influx into cells during equilibration and rapid efflux after

thawing (Bautista et al., 1997). Similar effects of ethylene glycol occurs across the plasma

membrane of sperm cells (Hammerstedt and Graham, 1992). Osmotic stress (difference in

15

relative permeability of cryoprotectants and water) is a major factor for spermatozoa damage

during semen cryopreservation (Gao et al., 1997). Sperm cells are sensitive to osmotic stress

during addition or removal of cryoprotectants and this sensitivity changes with the concentration

of cryoprotectants (Gao et al., 1997). Ball et al. (2001) used four different cryoprotectants (1 M;

glycerol, ethylene glycol, dimethylsulfoxide, or propylene glycol) on tyrode albumin lactate-

pyruvate medium (TALP) to evaluate the effect of addition and removal of these cryoprotectants

on osmotic stress after liquid storage of equine semen for 5 min at 22° C. Based on sperm

motility and viability, semen stored in ethylene glycol based TALP medium had the least

osmotic stress compared with other cryoprotectants (Ball et al., 2001).

The protective effect of cryoprotectants at various concentrations (glycerol 5%, ethylene

glycol 3% and 5%, and acetamide 3% and 5%; TRIS fructose media) on ram sperm cells was

measured after extension and freeze storage (Silva et al., 2012). No difference was found in

progressive motility, acrosomal integrity, and mitochondrial membrane integrity between semen

samples stored in ethylene glycol (3 or 5%) compared with 5% glycerol based medium.

However, those parameters were higher than samples extended and freeze-stored in 3 or 5%

acetamide based medium (Silva et al., 2012). In a similar study, cryoprotectants (glycerol 7%,

ethylene glycol 3, 5, or 7%, and DMSO 3, 5 or 7%) in TRIS based medium were examined

to extend and freeze-store ram semen. Semen stored in ethylene glycol (3 or 5%) had similar

progressive motility, viability, and membrane integrity compared with semen cryopreserved in

7% glycerol. The protective effect of these cryoprotectants was better than dimethyl sulfoxide

DMSO (Najafi et al., 2016). Both of the above studies were done in frozen extended semen, and

ethylene glycol or glycerol were found to be better cryoprotectants compared with DMSO or

16

acetamide for cryo storage of ram semen. Ethylene glycol at 3 or 5% can be used for

cryopreservation in a medium for freeze storage of ram semen.

Use of cryoprotectants for cool storage of extended semen were also tested at various

concentrations. Five cryoprotectants (dimethylacetamide, dimethyl sulfoxide, ethylene glycol,

glycerol or propylene glycol) at 2.5, 5 or 10% final concentration were added in TES-TRIS

fructose extender to extend and store ram semen at 4° C for 3 h. Semen stored in medium

containing 2.5% ethylene glycol had higher sperm progressive motility compared with other

cryoprotectants and concentrations used (Vásquez et al., 2013). Similarly, Carrascosa et al.

(2001) used 1.6% final concentration of either glycerol or ethylene glycol for storage of

Chinchila lanigera semen in TES-TRIS sodium citrate fructose medium at 4° C for up to 72 h.

Semen stored in ethylene glycol based medium had higher sperm motility, sperm viability, better

membrane and acrosomal integrity compared with semen stored in glycerol based medium.

Studies on use of ethylene glycol based medium for cold storage of ram semen are rare,

therefore, it would be interesting to see if a beneficial effect can be seen.

Penicillamine, Hypotaurine and Epinephrine (PHE)

Catecholamines (epinephrine), β-amino acid (hypotaurine), and serum albumin stimulate

sperm motility, viability, sperm capacitation, and acrosomal reaction (Bavister et al., 1979;

Meizel et al., 1980; Bavister, 1981). Penicillamine, hypotaurine, and epinephrine have different

modes of action but collectively enhance sperm motility, sperm capacitation, and acrosomal

reaction (Meizel, 1985). Penicillamine is a metal chelator and thus stimulates capacitation and

acrosomal reaction (Meizel, 1985). High K+ concentration could inhibit in vitro acrosomal

reaction, and use of hypotaurine partially reduces Na+, K+-ATPase activity, lowers intracellular

K+ and enhances in vitro fertilization (Meizel, 1985). Epinephrine activates α1-adrenergic

17

receptors and stimulates acrosomal reaction (Meizel, 1985). Leibfried and Bavister (1982)

conducted a series of experiments to determine the effects of epinephrine and hypotaurine on in

vitro fertilization of golden hamsters. Addition of 10 µM of hypotaurine and 1 µM of

epinephrine at the start of incubation in a culture media increased ova penetration by sperm

(71.7%) compared with using hypotaurine alone (6.6%). Addition of hypotaurine is necessary

prior to epinephrine to enable epinephrine to activate spermatozoa for ova penetration (Leibfried

and Bavister, 1982).

Supplementation of the combination penicillamine (10 µl), hypotaurine (1µl),

epinephrine (20 µl; PHE) compared with sodium metabisulfite (2 µM) or individual components

increased bovine sperm penetration when incubated with oocytes. However, the difference was

not seen after 14 h of incubation. It can be concluded that supplementation of PHE decreases

time interval for oocyte penetration by sperm, compared with individually using penicillamine,

hypotaurine or epinephrine (Susko-Parrish, et al., 1990). Miller et al. (1994) compared the effect

of addition of PHE, bovine oviduct epithelial cells (BOEC), or a combination of PHE + BOEC in

in vitro fertilization medium. The PHE (20 µM penicillamine, 10 µM hypotaurine, and 2 µM

epinephrine) was added to TALP fertilization medium. Addition of either PHE (62%) or BOEC

(57%) or the combination (PHE + BOEC) resulted in higher overall cleavage rate and more

embryos compared with no PHE and/or BOEC (Miller et al., 1994).

A study investigated the effect of PHE on sperm cell motility parameters after frozen-

thaw bovine semen. Pearson et al. (2007) compared computer assisted sperm analysis (CASA)

parameters of frozen thawed bovine semen cultured in sperm-TL medium with/without PHE.

Semen was incubated at 39° C and was evaluated every 2 h for 8 h. Semen stored in PHE

supplemented medium had higher progressive motility (PHE, 43.2 vs control, 35.7), path

18

velocity (PHE, 111.5 vs control, 99.4), progressive velocity (PHE, 100.0 vs control, 89.0), and

track speed (PHE, 166.3 vs control, 147.5) compared with no PHE (Pearson et al., 2007). All

these combined studies conclude that supplementation of PHE enhances sperm motility. To our

knowledge, no such studies have been conducted with PHE on cool stored ram semen.

b. Anti-Mullerian hormone as an endocrine marker to predict fertility.

Anti-Mullerian hormone

Anti-Mullerian hormone (AMH; also, known as mullerian inhibiting substance) is a

member of the transforming growth factor-β (TGF-β) family. It is a homodimeric disulfide-

linked glycoprotein, with a molecular weight of 140 kDa (Cate et al., 1986; A La Marca and

Volpe, 2006). Gene coding for AMH resides on short arm of human chromosome 19 (Cohen-

Haguenauer et al., 1987). In males, AMH is produced by sertoli cells as early as during testicular

differentiation (8-9 weeks of gestation in humans), whereas in females the hormone is secreted

by ovarian granulosa cells during late fetal life (36 – 38 weeks in human; Rajpert-De Meyts et

al., 1999).

Sexual differentiation in males and females

During early embryonic development, the genital ridge which arises from mesonephric

cells is bipotent (Wilson and Davies, 2007). This genital ridge develops into testes if a Y

chromosome is present. For this, transcription of Y-specific genes occurs resulting in production

of testes determining factor (Austin and Edwards, 1981). Testis-determining factor is responsible

for initiation of male sex differentiation in human. Absence of Y chromosome gives rise to an

ovary (Austin and Edwards, 1981). In humans, sex cords (Mullerian and Wolffian ducts) are

present within mesonephron, and remain similar in both males and females until seventh week of

gestation. In females, the wolffian duct regresses and the Mullerian duct forms the female

19

reproductive tract. Formation of the female reproductive tract does not depend on any ovarian

products. If genital ridges are removed at early embryonic development (before they develop into

testes or ovaries), a mammal develops into a female regardless of sex chromosomes carried, and

hence, development of female reproductive organs follows a default pathway (Alberts et al.,

2002). In males, two processes occur. First, Leydig cells produce testosterone which maintains

and promotes Wolffian ducts. The Wolffian duct differentiates into male tract and accessory

organs (vas deferens, seminal vesicles, and epididymis). Secondly, sertoli cells in testes secrete

AMH hormone which regresses mullerian duct (Munsterberg and Lovell-Badge, 1991).

In females, granulosa cells produce AMH (Vigier et al., 1984). Production of AMH in the

female fetus starts at third trimester (120 d gestation in sheep; Bezard et al., 1987). Ovarian

AMH hormone messenger RNA (mRNA) is expressed in granulosa cells from preantral and

small antral follicles. Corpora lutea, large antral follicles, oocytes, and primordial follicles lack

AMH hormone expression (Baarends et al., 1995). Weenen et al. (2004) studied the AMH

hormone expression in granulosa cells of human ovaries. Anti-Mullerian hormone protein was

observed in granulosa cells of primary follicles and continued until antral stage follicle (6 – 8

mm). The highest level of AMH was expressed in granulosa cells of preantral and small antral

follicles of less than 4 mm size. Scheetz (2010) analyzed bovine serum AMH concentration of

the ovulatory follicular wave (daily; 6 - 8 d prior to ovulation to the day of ovulation) and found

no difference in an individual during the last 6 - 8 d before ovulation. Therefore, it was

concluded that AMH hormone of an individual remains unchanged during an estrous cycle

(Scheetz, 2010). Anti-Mullerian hormone is highly variable between animals, but remains

unchanged for each animal when measured in 3 month intervals, indicating that this hormone

concentration remains constant in an individual animal over a long period of time (Rico et al.,

20

2009). Minimum variation of AMH occurred during the estrous cycle in 4 to 9 yr old Holstein

dairy cows, an indication of minimum variation with age during the reproductive life span

(Ireland et al., 2010).

About 1 million oocytes are present at birth, and that number decreases to less than half

once girls reach pubertal stage (Visser et al., 2006). With each follicular wave, follicles leave the

pool of primordial follicles and enter into the pool of growing follicles (Visser et al., 2006). After

puberty, a few of these growing follicles are rescued by follicle stimulating hormone (FSH),

while a majority will be lost as a result of atresia (Visser et al., 2006). In uniparous animals,

under the influence of luteinizing hormone (LH), only one among a few FSH-rescued follicles

become a dominant follicle and finally ovulate (Visser et al., 2006). Follicular reserve on the

ovary can be predicted by antral follicle count using an ultrasound (Visser et al., 2006). This

process involves transvaginal ultrasound examination. Transvaginal ultrasound examination for

antral follicle count requires technical expertise and is a time consuming process. Antral follicle

count is positively correlated with circulating AMH and number of morphologically healthy

oocytes in cattle (Ireland et al., 2008). High antral follicle count (> 25 follicles of more than 3

mm size) had 5 times greater number of morphologically healthy oocytes compared with low

antral follicle count (< 15 follicles; Ireland et al., 2008). Assessment of serum AMH

concentrations can be measured to predict follicular reserve in ovary. Positive correlations were

reported between AMH and first service pregnancy rates in dairy cows (Baruselli et al., 2015).

Additionally, dairy cows with lower AMH levels had a higher pregnancy loss between first and

second month of gestation (Baruselli et al., 2015). A positive correlation was also reported

between AMH and pregnancy rate in cows bred at estrus, leading to reinsemination and/or

natural service (Baruselli et al., 2015). In a recent study conducted in our lab, a positive

21

correlation between beef heifer AMH concentrations at first breeding and AI conception rate was

detected, and more cows were likely to conceive earlier in the breeding season when AMH

concentrations were higher (Rorie et al., 2017). In the same study, there was no correlation

between AMH concentrations measured at weaning and AI conception rate at first breeding,

overall pregnancy rate, and estrous response after synchronization (Rorie et al., 2017). Therefore,

based on this study we can conclude that the earliest age to measure AMH concentrations to

predict beef heifer fertility will be at breeding age.

Anti-Mullerian hormone and FSH

Anti-Mullerian hormone limits the number of primordial follicles entering the pool of

growing follicles (Durlinger et al., 2001). Growth of preantral and small antral follicles are FSH

dependent, and presence of AMH decreases FSH responsiveness of the follicle (Durlinger et al.,

2001). A study conducted with mice revealed that AMH deficient mice had normal reproductive

performance initially. However, primordial follicles were depleted at a faster rate compared with

normal mice (Durlinger et al., 2001). It is likely that the group of females with low AMH would

deplete primodial follicles early, resulting in early cessation of ovulation, and a shorter

reproductive life compared with animals with normal AMH level (Durlinger et al., 2001)

Anti-Mullerian hormone studies in ewes

In ewes anti-Mullerian hormone is high during the fetal stage compared with post-natal

concentrations (Bezard et al., 1987). Similarly, plasma AMH concentrations at 40 d of age in

ewe lambs is high compared with 3.6-month old ewes (Lahoz et al., 2012; Torres-Rovira et al.,

2014). Lahoz found vast differences in AMH concentrations between 1 and 5-month-old ewe

lambs, high at 1-month and lower at 5 mo of age (Lahoz, et al., 2016). Ovarian follicular

population, an indicator of fertility, is positively correlated with plasma AMH concentrations in

22

young lambs (Lahoz et al., 2016b). A positive correlation occurred between plasma AMH

concentration in 3.6-month ewe lambs and number of ovulations in response to equine chorionic

gonadotropin (eCG; Lahoz et al., 2012). In Rasa Aragonesa ewe lambs, a plasma AMH

concentration of 97 pg/ml measured at 3.6-months of age was determined as a cutoff value to

predict fertility at first mating (Lahoz et al., 2012). Plasma AMH concentrations higher than 97

pg/ml in prepubertal ewes had higher first mating conception rate compared with lower plasma

AMH concentration (50% vs 15%; Lahoz et al., 2012). High plasma AMH concentrations at 3.5-

months of age (97 pg/ml) were associated with a higher number of total lambs born per ewe until

3.5-years of age compared with ewes with lower AMH concentrations (97 ng/ml; Lahoz et al.,

2016a).

Summary and objective

Frozen storage and AI of ram semen results in low conception rates (Salamon and

Maxwell, 1995). Recommendations are mixed on storage temperature of cooled semen at 5º C or

15º C (O’ Hara et al., 2010; Mata-Campuzano et al., 2014). Differences may be dependent on the

supplements and storage duration. Addition of egg yolk and cryoprotectant prior to cooling, or

supplementation with penicillamine, hypotaurine, and epinephrine (PHE) before evaluation may

maintain or improve semen motility.

i) Therefore, the first objective of the current study was to compare four extenders

(milk, TRIS, TEST or CJ-2), and two temperatures (4 or 15° C) for extension and

storage of ram semen for up to 72 h using an equal amount of egg yolk, fructose, and

gentamicin.

23

ii) Second, to evaluate the effect of combinations of extenders (best two extenders from

objective i), ± ethylene glycol, egg yolk (5 or 20%), and ± PHE (penicillamine,

hypotaurine and epinephrine) on ram sperm motility parameters.

Ewes vary in their reproductive performance. Farmers can maximize their profit if fertile

ewes can be selected during early stages by using endocrine markers. Anti-Mullerian hormone is

produced by granulosa cells of preantral and small antral follicles of 1-3 mm in diameter (Bezard

et al., 1988; Monniaux et al. 2012). Ewe fertility at first mating was positively correlated with

plasma AMH concentrations measured at 3.6 months of age (Lahoz Crespo et al. 2012). High

plasma AMH concentrations at 3.5-month of age (> 97 pg/ml) was associated with a higher

number of lambs born per ewe as adults compared with ewes with lower AMH concentrations (<

97 pg/ml; Lahoz et al., 2016). The current experiment further explores how serum AMH

concentrations are correlated with estimated breeding values (EBV) such as number of lambs

born (NLB), number of lambs weaned (NLW), maternal weaning weight (MWWT), weaning

weight (WWT), and a USA hair index calculated by Sheep Genetics (Armidale, NSW, Australia)

through the National Sheep Improvement Program. Therefore, the objective of the study was to

determine correlations between serum concentrations of AMH and EBV for NLB, NLW,

MWWT, WWT, and the hair sheep index.

24

References

Alberts, B., A. Johnson, and J. Lewis. 2002. Primordial Germ Cells and Sex Determination in

Mammals. In Molecular Biology of the Cells. 4th edition. New York: Garland Science.

Almquist, J., R. J. Flipse, D. L. Thacker. 1954. Diluters for bovine semen. IV. Fertility of bovine

spermatozoa in heated homogenized milk and skim milk. J. Dairy Sci. 37:1303-1307.

Anel, L., M. Alvarez, F. Martinez-Pastor, V. Garcia-Macias, E. Anel, and P. de Paz. 2006.

Improvement strategies in ovine artificial insemination. Reprod. Dom. Anim. 41:30-42.

Anel, L., M. Kaabi, B. Abroug, M. Alvarez, E. Anel, J. C. Boixo, L. F. de la Fuente, P. de Paz.

2005. Factors influencing the success of vaginal and laparascopic artificial insemination

in Churra ewes: A field assay. Theriogenology. 63:1235-1247.

Austin, C. R., and R. G. Edwards (eds.). 1981. Mechanisms of sex differentiation in animals and

man. Academic Press, N. Y.

Baarends, W. M., J. Uilenbroek, P. Kramer, J. W. Hoogerbrugge, E. Van Leeuwen, A. P.

Themmen, J. A. Grootegoed. 1995. Anti-müllerian hormone and anti-müllerian hormone

type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal

development, the estrous cycle, and gonadotropin-induced follicle growth.

Endocrinology, 136:4951-4962.

Ball, B. A., A. T. Vo, and J. Baumber. 2001. Generation of reactive oxygen species by equine

spermatozoa. Am. J. Vet. Res. 62:508-515.

Baruselli, P. S., E. O. S. Batista, L. M. Vieira, A. H. Souza. 2015. Relationship between follicle

population, AMH concentration and fertility in cattle. Anim. Reprod. 12:487-497.

Bashawat, M., M. Moussa, M. R. Al-Merestani, M., S. Destrumelle, D. Tainturier. 2016. The use

of different low-density lipoproteins concentrations for developing a new extender for

Awassi ram’s semen. Sci. J. King Faisal Univ. (Basic Appl. Sci.). 17:21-30.

Bautista, J. A., Y. Takahashi, and H. Kanagawa. 1997. In vitro viability of mouse 8-cell embryos

vitrified in a simple solution of ethylene glycol. Jpn. J. Vet. Res. 45:67-73.

Bavister, B. D. 1981. Substitution of a synthetic polymer for protein in a mammalian gamete

culture system. J. Exp. Zool. 217:45-51.

Bavister, B. D., Chen, A. F. and P. C. Fu. 1979. Catecholamine requirement for hamster sperm

motility in vitro. J. Reprod. Fertil. 56:507-513.

Bezard, J., B. Vigier, D. Tran, P. Mauleon, and N. Josso. 1987. Immunocytochemical study of

anti-Müllerian hormone in sheep ovarian follicles during fetal and post-natal

development. J. Reprod. Fertil. 80:509-516.

25

Bolanos, J. R., J. W. Overstreet, and D. F. Katz. 1983. Human sperm penetration of zona-free

hamster eggs after storage of the semen for 48 hours at 2 degrees C to 5 degrees C. Fertil.

Steril. 39:536-541.

Broer, S. L., F. J. Broekmans, J. S. Laven, and B. C. Fauser, B. C. 2014. Anti-Müllerian

hormone: ovarian reserve testing and its potential clinical implications. Hum. Reprod.

Update. 20:688-701.

Burke, J. M. 2005. Lamb production of Dorper, Katahdin, and St. Croix bred in summer, winter,

or spring in the Southeastern US. Sheep Goat Res. J. 20:51-59.

Campbell, B. K., M. Clinton, and R.Webb. 2012. The role of anti-Müllerian hormone (AMH)

during follicle development in a monovulatory species (sheep). Endocrinology.

153:4533-4543.

Carrascosa, R. E., A. C Martini, M. F. Ponzio, J. M. Busso, A. A. Ponce, and J. L. Lacuara.

2001. Storage of Chinchilla lanigera semen at 4° C for 24 or 72 h with two different

cryoprotectants. Cryobiology. 42:301-306.

Cate, R. L., R. J. Mattaliano, C. Hession, R. Tizard, N. M. Farber, A. Cheung. 1986. Isolation of

the bovine and human genes for Müllerian inhibiting substance and expression of the

human gene in animal cells. Cell. 45:685-698.

Choong, C., and R. Wales. 1963. The use of various diluents for deep-freezing bull spermatozoa.

Aust. J. Biol. Sci. 16:896-904.

Cohen-Haguenauer, O., J. Y. Picard, J. Y. Mattei, S. Serero, N. Van Cong, M. F. N. Tand, D.

Guerrier, M. C. Hors-Cayla, N. Josso, J. Fresal. 1987. Mapping of the gene for anti-

Müllerian hormone to the short arm of human chromosome 19. Cytogenet. Genome Res.

44:2-6.

Dalgleish, D. G. 1998. Casein micelles as colloids: surface structures and stabilities. J. Dairy Sci.

81:3013-3018.

Davis, I. S., R. W. Bratton, and R. H. Foote. 1963. Livability of bovine spermatozoa at 5, -25,

and -85 C in Tris-buffered and citrate-buffered yolk glycerol extenders. J. Diary Sci.

46:333-336.

Delgado, P., T. Lester, and R. Rorie. 2009. Effect of a low-sodium, choline-based diluent on

viability of bovine sperm stored at refrigerator temperatures. Ark. Anim. Sci. Depart.

Rep. 77-79.

Desnoyers, L., and P. Manjunath. 1992. Major proteins of bovine seminal plasma exhibit novel

interactions with phospholipid. J. Biol. Chem. 267:10149-10155.

Donoghue, A. M., and G. J. Wishart. 2000. Storage of poultry semen. Anim. Reprod. Sci.

62:213-232.

26

Durlinger, A. L., M. J. Gruijters, P. Kramer, B. Karels, T. R. Kumar, M. M. M. M. Matzuk, U.

M. Rose, F. H. de Jong. J. T. Uilenbroek, J. A. Grootegoed. 2001. Anti-Mullerian

hormone attenuates the effects of FSH on follicle development in the mouse ovary.

Endocrinology. 142:4891-4899.

Evans, G., W. M. C. Maxwell. 1987. Salamon's Artificial Insemination of Sheep and Goats.

Butterworths, Sydney. 1-7.

Foot, R. H., and R. W Bratton. 1950. The fertility of bovine semen in extenders containing

sulfanilamide, penicillin, streptomycin, and polymyxin. J. Dairy Sci. 33:544-547.

Foote, R. H. 2002. The history of artificial insemination: selection notes and notables. J. Anim.

Sci. 80:1-10.

Foote, R. H., and R. W. Bratton. 1949. The fertility of bovine semen cooled with and without the

addition of citrate-sulfanilamide-yolk extender. J. Dairy Sci. 32:856-861.

Fukui, Y., and E. Roberts. 1978. Further studies on non-surgical intrauterine technique for

artificial insemination in the ewe. Theriogenology. 10:381-391.

Gao, D., P. Mazur, and J. K. Critser. 1997. Fundamental cryobiology of mammalian

spermatozoa. In: Karow A. M., and J. K. Critser (eds.), Reproductive Tissue Banking.

London: Academic Press.

Gharibi, S., A. Niasari-Naslaji, N. Poursasan, and A. A. Moosavi-Movahedi. 2014. Replacement

of salamon with shotor diluent and egg yolk with low density lipoprotein for chilled

storage of ram semen. Iran. J. Vet. Res. 15:279-284.

Gil, J., S. Fierro, O. Bentancur, and J. Olivera‐Muzante. 2011. Chilled storage of ram semen

improves with the addition of egg yolk and glycerol to milk‐based extenders. Reprod.

Dom. Anim. 46:503-507.

Gill, S. P., R. H. Hammerstedt, and R. P. Amann. 1999. Poultry artificial insemination:

Procedures, current status and future needs. In: Proc. Annu. Mtg. Soc. Theriogenology,

Nashville, TN. pp. 353-362.

Good, N. E., G. D. Winget, W. Winter, T. N. Connolly, S. Izawa, and R. M. Singh. 1966.

Hydrogen ion buffers for biological research. Biochemistry. 5:467-477.

Gourley D. D., and R. L. Riese. 1990. Laparoscopic artificial insemination in sheep. Vet. Clin.

North Am. Food Anim. Pract. 6:615-6133.

Graham, E. F., B. Crabo, and K. I. Brown. 1972. Effect of some zwitter ion buffers on the

freezing and storage of spermatozoa I. Bull1. J. Dairy Sci. 55:372-378.

27

Gündoğan, M. 2009. Short term preservation of ram semen with different extenders. Kafkas

Univ. Vet. Fak. Derg. 15:429-435.

Halbert G. W., H. Dobson, J. S. Walton, and B. C. Buckrell. 1990. The structure of the cervical

canal of the ewes. Theriogenology. 33:977-992.

Hammerstedt, R. H., and J. K. Graham. 1992. Cryopreservation of poultry sperm: the enigma of

glycerol. Cryobiology. 29:26-38.

Hollinshead, F.K., J. K. O’Brien, L. Gillan, M. Meyers, M., W. Maxwell, and G. Evans. 2004.

Liquid storage of flow cytometrically sorted ram spermatozoa. Theriogenology. 62:587-

605.

Holt, W. V., and R. North. 1984. Partially irreversible cold‐induced lipid phase transitions in

mammalian sperm plasma membrane domains: Freeze‐fracture study. J. Exp. Zool.

230:473-483.

Ireland, J. J., G. W. Smith, D. Scheetz, F. Jimenez-Krassel, J. K. Folger, J. L. Ireland, F. Mossa,

P. Lonergan, and A. C. Evans. 2010. Does size matter in females? An overview of the

impact of the high variation in the ovarian reserve on ovarian function and fertility, utility

of anti-Müllerian hormone as a diagnostic marker for fertility and causes of variation in

the ovarian reserve in cattle. Reprod. Fertil. Dev. 23:1-14.

Ireland, J., D. Scheetz, F. Jimenez-Krassel, A. Themmen, F. Ward, P. Lonergan, G. W. Smith, G.

I. Perez, A. C. O. Evans, and J. J. Ireland. 2008. Antral follicle count reliably predicts

number of morphologically healthy oocytes and follicles in ovaries of young adult cattle.

Biol. Reprod. 79:1219-1225.

Kershaw, C. M., M. Khalid, M. R. McGowan, K. Ingram, S. Leethongdee, G. Wax, and R. J.

Scaramuzzi. 2005. The anatomy of the sheep cervix and its influence on the transcervical

passage of an inseminating pipette into the uterine lumen. Theriogenology. 64:1225-

1235.

Kulaksiz, R., C. Cebi, and E. Akcay. 2012. The effect of different extenders on the motility and

morphology of ram sperm frozen or stored at 4 degrees C. Turk. J. Vet. Anim. Sci.

36:177-182.

La Marca, A., and A. Volpe. 2006. Anti‐Müllerian hormone (AMH) in female reproduction: is

measurement of circulating AMH a useful tool? Clin. Endocrinol. 64:603-610.

La Marca, A., V. Grisendi, and G. Griesinger. 2013. How much does AMH really vary in normal

women? Int. J. Endocrinol. 2013:1-8.

Lahoz, B., J. L. Alabart, and J. Folch. 2016a. Relationship between Anti-Müllerian hormone

(AMH) and the ovulatory response measured in three-month old ewe lambs with their

reproductive efficiency in adulthood. Asoc. Interprofes. Para el Des. Agr. 112:392-404.

28

Lahoz, B., J. L. Alabart, D. Monniaux, P. Mermillod, J. Folch. 2012. Anti-Mullerian hormone

plasma concentration in prepubertal ewe lambs as a predictor of their fertility at a young

age. BMC Vet. Res. 8:8-18.

Lahoz, B., J. L. Alabart, M. Cocero, D. Monniaux, E. Echegoyen, P. Sánchez, J. Folch. 2014.

Anti-Müllerian hormone concentration in sheep and its dependence of age and

independence of BMP15 genotype: an endocrine predictor to select the best donors for

embryo biotechnologies. Theriogenology. 81:347-357.

Lahoz, B., S. Fabre, J. H. Calvo, L. Drouilhet, P. Sanchez, P. Sarto, E. Echegoyen, J. Folch, D.

Monniaux, J. L. Alabart. 2016 b. Assessing the age-related relationship between plasma

anti-Mullerian hormone (AMH) and ovarian follicle population in prepubertal ewes

(abstract). 18th Int. Cong. Anim. Reprod. Nouzilly, France. 26-30, June 2016.

Leibfried, M. L., and B. D. Bavister. 1982. Effects of epinephrine and hypotaurine on in-vitro

fertilization in the golden hamster. J. Reprod. Fertil. 66:87-93.

López, A., L. Söderquist, and H. Rodriguez-Martinez. 1999. Sperm viability in ram semen

diluted and stored in three different extenders. Acta vet. Scand. 40:1-9.

MacDonald, B. J., and J. A. Foulkes. 1981. A spectrofluorometric investigation, using 1-anilino-

naphthalene-8-sulphonate, of the interaction between washed bovine spermatozoa and

seminal plasma or egg-yolk lipoprotein. J. Reprod. Fertil. 63:407-414.

Manjunath, P., L. Baillargeon, Y. Marcel, N. Seidah, M. Chrétien, and Chapdelaine. 1988.

Diversity of novel proteins in gonadal fluids. In: K. W. Mckerns, M. Chretien (eds.),

Molecular Biology of Brain and Endocrine Peptidergic Systems. New York: Plenum

Press:259-273.

Manjunath, P., V. Nauc, A. Bergeron, and M. Ménard. 2002. Major proteins of bovine seminal

plasma bind to the low-density lipoprotein fraction of hen's egg yolk. Biol. Reprod.

67:1250-1258.

Mata-Campuzano, M., M. Álvarez-Rodríguez, J. Tamayo-Canul, E. López-Urueña, P. de Paz, L.

Anel. 2014. Refrigerated storage of ram sperm in presence of Trolox and GSH

antioxidants: Effect of temperature, extender and storage time. Anim. Reprod. Sci.

151:137-147.

Maxwell, W. M. C. 1984. Current problems and future potential of artificial insemination

programs. In Reproduction in sheep. (ed. R. L. Lindsay and D. T. Pearce). Canberra, A.

C. T.: Australian Academy of Science and Australia Wool Corporation. pp. 291-298.

Maxwell, W. M. C., A. J. Landers, and G. Evans. 1995. Survival and fertility of ram

spermatozoa frozen in pellets, straws and minitubes. Theriogenology. 43:1201-1210.

29

Maxwell, W. M. C., and L. J. Hewitt. 1986. A comparison of vaginal, cervical and intrauterine

insemination of sheep. J. Agric. Sci. Camb. 106:191-193.

Maxwell, W. M. C., L. G. Butler, and H. R. Wilson. 1984. Intra-uterine insemination of ewes

with frozen semen. J. of Agric. Sci. 102:233-235.

Mazur, P. 1977. The role of intracellular freezing in the death of cells cooled at supraoptimal

rates. Cryobiology. 14:251-272.

Meizel, S. 1985. Molecules that initiate or help stimulate the acrosome reaction by their

interaction with the mammalian sperm surface. Dev. Dynam. 174:285-302.

Meizel, S., C. W. Lui, P. K. Working, and R. J. Mrsny. 1980. Taurine and hypotaurine: their

effects on motility, capacitation and the acrosome reaction of hamster sperm in vitro and

their presence in sperm and reproductive tract fluids of several mammals. Dev. Growth

Differ. 22:483-494.

Mickelsen, W. D., L. G. Paisley, J. F. Dahmen. 1981. The effect of season on the scrotal

circumference and sperm motility and morphology in rams. Theriogenology. 16:45-51.

Miller, G. F., D. W. Gliedt, J. M. Rakes, and R. W. Rorie. 1994. Addition of penicillamine,

hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or

in combination to bovine in vitro fertilization medium increases the subsequent embryo

cleavage rate. Theriogenology. 41:689-696.

Milovanov, V. K. 1964. Artificial insemination of livestock in the U.S.S.R Trans. by A. Birron

and Z. S. Cole. S. Monson, Jerusalem. Tech. Services, U.S. Dept. Commerce,

Washington, DC.

Monniaux, D., L. Drouilhet, C. Rico, A. Estienne, P. Jarrier, J. Touzé, J. Sapa, F. Phocas, J.

Dupont, R. Dalbiès-Tran, and S. Fabre. 2012. Regulation of anti-Müllerian hormone

production in domestic animals. Reprod. Fertil. Dev. 25:1-16.

Munsterberg, A., and R. Lovell-Badge. 1991. Expression of the mouse anti-Mullerian hormone

gene suggests a role in both male and female sexual differentiation. Development.

113:613-624.

Najafi, A., H. Daghigh-Kia, H. V. Dodaran, M. Mehdipour, M. Alvarez-Rodriguez. 2017.

Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-

based extenders in ram sperm cryopreservation. Anim. Reprod. Sci. 177:35-41.

O’Hara, L., J. Hanrahan, L. Richardson, A. Donovan, S. Fair, A. C. Evans, and P. Lonergan.

2010. Effect of storage duration, storage temperature, and diluent on the viability and

fertility of fresh ram sperm. Theriogenology. 73:541-549.

Pace, M., and E. Graham. 1974. Components in egg yolk which protect bovine spermatozoa

during freezing. J. Anim. Sci. 39:1144-1149.

30

Paulenz, H., L. Söderquist, R. Pérez-Pé, and K. Andersen Berg. 2002. Effect of different

extenders and storage temperatures on sperm viability of liquid ram semen.

Theriogenology. 57:823-836.

Paulenz, H., L. Söderquist, T. Ådnøy, K. Soltun, P. Saether, K. Fjellsøy, and K. A. Berg. 2005.

Effect of cervical and vaginal insemination with liquid semen stored at room temperature

on fertility of goats. Anim. Reprod. Sci. 86:109-117.

Paulenz, H., L. Söderquist, T. Ådnøy, O. H. Fossen, and K. A. Berg. 2003. Effect of milk-and

TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with

liquid semen. Theriogenology. 60:759-766.

Paulenz, H., T. Adnøy, O. Fossen, L. Söderquist, and K. A. Berg. 2002. Effect of deposition site

and sperm number on the fertility of sheep inseminated with liquid semen. Vet. Res.

150:299-302.

Paulenz, H., T. Ådnøy, O. H. Fossen, L. Söderquist. 2010. Effect on field fertility of addition of

gelatine, different dilution rates and storage times of cooled ram semen after vaginal

insemination. Reprod. Domest. Anim. 45:706-710.

Person, C. N., T. D. Lester, M. D. Person, and R. W. Rorie. 2007. Effects of penicillamine,

hypotaurine and epinephrine (PHE) on post-thaw bovine sperm parameters, as measured

by computer-assisted sperm analysis. Ark. Agri. Exp. Sta. Res. Ser. 553:47-50.

Phillips, P. H., and H. A. Lardy. 1940. A yolk-buffer pabulum for preservation of bull semen. J.

Dairy Sci. 23:399-404.

Plante, G., M. F. Lusignan, M. Lafleur, and P. Manjunath. 2015. Interaction of milk proteins and

Binder of Sperm (BSP) proteins from boar, stallion and ram semen. Reprod. Biol.

Endocrinol. 13:92-112.

Polge, C. 1956. The development of artificial insemination service for pigs. Anim. Breed.

24:209-217 (Abstr.).

Polge, C. 1968. Frozen semen and the A.I. Programme in Great Britain. In: Proc. 2nd Tech.

Conf. Artif. Insem. Reprod. NAAB, Columbia, MO. pp 46-51.

Polge, C., A. U. Smith, and A. S. Parkes. 1949. Revival of spermatozoa after vitrification and

dehydration at low temperature. Nature. 164:166.

Pope, C. E., Y. Z. Zhang, and B. L. Dresser. 1991. A simple staining method for evaluating

acrosomal status of cat spermatozoa. J. Zoo Wildl. Med. 22:87-95.

Purdy, P. H. 2006. A review on goat sperm cryopreservation. Small Rumin. Res. 63:215-225.

31

Quan, G. B., G. Q. Wu, Y. J. Wang, D. J. Li, Y. Ma, Q. H. Hong. 2016. Effects of the TRIS,

TES, or skim milk based extender on in vitro parameters of ram spermatozoa during

liquid storage. Small Rumin. Res. 134:14-21.

Rajabi-Toustani, R., M. Roostaei-Ali, and R. Motamedi-Mojdehi. 2014. Effect of different levels

of egg yolk on ram sperm coating and preserving at 5° C. Iran. J. of Vet. Res. 15: 168-

171.

Rajpert-De Meyts, E., N. Jørgensen, N. Græm, J. Muller, R. L. Cate, N. E. Skakkebæk. 1999.

Expression of anti-Mullerian hormone during normal and pathological gonadal

development: Association with differentiation of sertoli and granulosa cells 1. J. Clin.

Endocrinol. Metab. 84: 3836-3844.

Rehmana, F. U., C. Zhao, M. A. Shaha, M. S. Qureshib, and X. Wanga. 2013. Semen extenders

and artificial insemination in ruminants. Veterinaria. 1:1-8.

Ricker, J. V., J. J. Linfor, W. J. Delfino, P. Kysar, E. L. Scholtz, F. Tablin, J. H. Crowe, B. A.

Ball, and S. A. Meyers. 2006. Equine sperm membrane phase behavior: The effects of

lipid-based cryoprotectants. Biol. reprod. 74:359-365.

Rico, C., S. Fabre, C. Médigue, N. di Clemente, F. Clément, M. Bontoux, J. L. Touze, M.

Dupont, E. Briant, B. Remy, J. F. Beckers, and D. Monniaux. 2009. Anti-müllerian

hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can

help to predict superovulatory responses in the cow. Biol. Reprod. 80:50-59.

Rorie, R., H. Newberry, T. D. Lester, M. Acharya, and C. Hansen. 2016. Anti-müllerian

hormone at weaning and breeding as a predictor of beef heifer fertility. Reprod. Fertl.

Dev. 29:111-112 (Abstr.).

Salamon, S. 1976. Artificial insemination of sheep. Publicity Press, Chippendale. pp 61-64.

Salamon, S., and D. Visser. 1972. Effect of composition of Tris-based diluent and of thawing

solution on survival of ram spermatozoa frozen by the pellet method. Aust. J. Biol. Sci.

25:605-618.

Salamon, S., and W. M. C. Maxwell. 1995. Frozen storage of ram semen I. Processing, freezing,

thawing and fertility after cervical insemination. Anim. Reprod. Sci. 34:185-249.

Salamon, S., and W. M. C. Maxwell. 2000. Storage of ram semen. Anim. Reprod. Sci. 62:77-

111.

Salisbury, G. W., H. K. Fuller, and E. L. Willett. 1941. Preservation of bovine spermatozoa in

yolk-citrate diluent and field results from its use. J. Dairy Sci. 24:905-910.

Salvador, I., M. A. Silvestre, J. Yániz, M. P. Viudes-de-Castro, J. Bernacer, and E. A. Gómez.

2005. Effect of solid storage at 5° C on sperm motility of goat semen by addition of

gelatin. Reprod. Domest. Anim. 40: 381 (abstr.).

32

Scheetz, D. M. 2010. Regulation and role of anti-Mullerian hormone in bovine reproduction

(Doctoral dissertation). Retrieved from UMI ProQuest Database. (UMI No. 1487140).

Seidel, G. E., J. L. Schenk, L. A. Herickhoff, S. P. Doyle, Z. Brink, R. D. Green, D. G. Cran.

1999. Insemination of heifers with sexed sperm. Theriogenology. 52:1407-1420.

Senger P. L. 2003. Pathway to pregnancy and parturation. Pullman, WA. Current Concepts, Inc.

pp 36-56.

Shannon, P., B. Curson, and A. P. Rhodes. 1984. Relationship between total spermatozoa per

insemination and fertility of bovine semen stored in Caprogen at ambient temperature. N.

Z. J. Agric. Res. 27:35-41.

Silva, E., J. Cajueiro, S. Silva, A. Vidal, P. Soares, and M. Guerra. 2012. In vitro evaluation of

ram sperm frozen with glycerol, ethylene glycol or acetamide. Anim. Reprod. Sci.

132:155-158.

Stachecki, J. J., J. Cohen, and S. M. Willadsen. 1998 b. Cryopreservation of unfertilized mouse

oocytes: the effect of replacing sodium with choline in the freezing medium.

Cryobiology. 37:346-354.

Stachecki, J. J., J. Cohen, and S. Willadsen. 1998 a. Detrimental effects of sodium during mouse

oocyte cryopreservation. Biol. of Reprod. 59:395-400.

Susko-Parrish, J. L., M. B. Wheeler, R. L. Ax, N. L. First, and J. J. Parrish. 1990. The effect of

penicillamine, hypotaurine, epinephrine and sodium metabisulfite, on bovine in vitro

fertilization. Theriogenology. 33:333 (Abstr.).

The Baalands. 2017. Katahdin frequently asked question. Available online.

http://www.baalands.com/#!katahdinfaqs/cwoc. Accessed on 24th April 2017.

Torres-Rovira, L., A. Gonzalez-Bulnes, S. Succu, A. Spezzigu, M. E. Manca, G. G. Leoni, G. G.,

M. Sanna, S. Pirino, M. Gallus, S. Naitana, F. Berlinguer. 2014. Predictive value of antral

follicle count and anti-Müllerian hormone for follicle and oocyte developmental

competence during the early prepubertal period in a sheep model. Reprod. Fertil. Dev.

26:1094-1106.

Vanimisetti, H. B., D. R. Notter, and L. A. Kuehn. 2007. Genetic (co) variance components for

ewe productivity traits in Katahdin sheep. J. Anim. Sci. 85:60-80.

Vásquez, J. H., V. H. Núñez, E. A. Florentini, J. M. Gonzales, L. A. Camargo, and M. E.

Valdivia. 2013. Effects of five cryoprotective agents on quality of sheep epididymal

spermatozoa during pre-freezing. Livest. Sci. 152: 94-99.

http://www.baalands.com/#!katahdinfaqs/cwoc

33

Vigier, B., J. Y. Picard, D. Tran, L. Legeai, and N. Josso. 1984. Production of anti-Mullerian

hormone: another homology between sertoli and granulosa cells. Endocrinology.

114:1315-1320.

Visser, D., and S. Salamon. 1973. Fertility of ram spermatozoa frozen in a TRIS-based diluent.

Aust. J. Biol. Sci. 26:513-515.

Visser, J. A., F. H. de Jong, J. S. Laven, and A. P. Themmen. 2006. Anti-Müllerian hormone: a

new marker for ovarian function. Reproduction. 131:1-9.

Watson, P. F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa

and the assessment of their post-thawing function. Reprod. Fertl. Dev. 7: 871-891.

Weenen, C., J. S. Laven, A. R. Von Bergh, M. Cranfield, N. P. Groome, J. A. Visser, P. Kramer,

B. C. Fauser, and A. P. N. Themmen. 2004. Anti-Mullerian hormone expression pattern

in the human ovary: potential implications for initial and cyclic follicle recruitment. Mol.

Hum. Reprod. 10:77-83.

White, I. G. 1993. Lipids and calcium uptake of sperm in relation to cold shock and preservation:

a review. Reprod. Fertil. Dev. 5:639-658.

Wildeus, S. 2014. Sire breed differences in pregnancy rate of hair sheep ewes following liquid

semen vaginal artificial insemination. J. Anim. Sci. 39-39 (Abstr.).

Wildeus, S., and E. J. Chozu. 2010. Liquid semen vaginal AI in three hair sheep breed during

transition to seasonal breeding. J. Anim. Sci. 88(suppl. 1):34-35 (Abstr.).

Wildeus, S., D. Wright, and D. O’Brien. 2016. Use of liquid semen artificial insemination in

Katahdin sheep in a commercial farm setting. J. Anim. Sci. 95(suppl. 1): 51.

Wilson, C., and D. Davies. 2007. The control of sexual differentiation of the reproductive system

and brain. Reproduction. 133:331-359.

Wusiman, A., Y-P. Wang, K. Ren, G-B. Zhou, X-W. Fu, L. Suo, Z-Q. Fan, L. Wang, and S-E.

Zhu. 2012. Semen storage at 23, 4 or-196° C and its application to artificial insemination

in small-tail Han sheep. Asian J. Anim. Vet. Adv. 7:299-308.

Yániz, J. L., J. A. Mateos, and P. Santolaria. 2011. Zwitterionic buffers preserve ram semen

quality more efficiently than TRIS during storage at 15° C. Small Rumin. Res. 95:54-60.

Yániz, J. L., P. Santolaria, M. A. Marco-Aguado, and F. López-Gatius. 2008. Use of image

analysis to assess the plasma membrane integrity of ram spermatozoa in different

diluents. Theriogenology. 70:192-198.

34

Chapter III

Effect of semen extender and storage temperature on ram sperm motility over time

Abstract

The objective was to determine the effect of semen extender and storage temperature on

motility of ram semen stored up to 72 h. Semen was collected by electroejaculator from 5

Katahdin rams. Each semen collection was divided and extended using four extenders: milk,

TRIS, TEST, and CJ-2, diluted to 600 million sperm/ml, and loaded to 0.25 ml straws for storage

at 4 and 15° C. All extenders were supplemented with 5% (v/v) egg yolk. Sperm motility

parameters were measured at 0, 24, 48, and 72 h of storage using computer an assisted sperm

analysis (CASA) system. Data were analyzed using the PROC MIXED procedure of SAS, with

each semen collection as the experimental unit. Variables in the model included extenders,

temperature, hours (repeated measures) and interactions. Most of the sperm motility parameters

were higher after storage at 4° C compared with 15° C. Ram semen extended using milk or TRIS

and stored at 4° C for 3 d maintained similar sperm motility parameters (P > 0.05). Semen

extended using CJ-2 or TEST extenders and stored at 4° C for 3 d had lower total motility and

path velocity (VAP; P < 0.05) compared with milk or TRIS, while most of the remaining sperm

motility parameters were similar (P > 0.05). For ram semen stored at 15° C for 3 d, TRIS had

higher sperm sperm motility parameters (P < 0.05) compared with other extenders. Milk or

TEST extender maintained intermediate motility, while CJ-2 extender had lowest sperm motility

parameters (P < 0.05) at 15° C. There was an extender × hour interaction (P < 0.05) on track

speed (VCL), path velocity (VAP), progressive velocity (VSL), beat frequency (BCF), lateral

amplitude (ALH), and straightness (STR). Such interactions are due to greater reduction of

sperm motility parameters at 48 and 72 h of storage for CJ-2 or milk extenders compared with

TEST or TRIS extenders. Within 24 h, percentage of progressive motile sperm decreased sharply

35

regardless of extenders or storage temperatures. Progressive motility of spermatozoa is positively

correlated to fertilizing capacity. Therefore, further research is needed to increase progressive

motility.

Keywords: Extenders, ram semen, storage temperature

Introduction

Artificial insemination (AI) of ewes with frozen ram semen often results in low

conception rates (20-30%; Salamon and Maxwell, 1995). Fresh diluted and chilled ram semen is

an alternative to frozen storage (Salamon and Maxwell, 1995). Better motility of ram semen was

found after storage at 4° C for 2 to 3 d compared with frozen storage for several weeks

(Wusiman et al., 2012; Kulaksiz et al., 2012). Cervical AI conception rate was higher when ram

semen extended with sodium citrate was stored at 4 or 23° C for 1 to 3 d compared with several

weeks storage at -196° C (Wusiman et al., 2012). Motility of ram semen was improved when

stored 5° C (O’Hara et al., 2010), or at 15° C (Mata-Campuzano et al., 2014). Differences in

motility among experiments may be due to the type of extender and storage duration.

Milk or tris[Hydroxymethyl]aminomethane (TRIS), based extenders are the most

common extenders used for liquid storage of ram semen (Salamon and Maxwell, 2000). The

TEST (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid i.e., TES titrated with TRIS)

has been evaluated in other species but not rams (Bolanos et al., 1983; Pope et al., 1991). The

CJ-2 extender was initially formulated to overcome harmful effects of sodium for

cryopreservation of oocytes (Mazur, 1977; Stachecki and Willadsen, 1998). Bull semen extended

in CJ-2 egg yolk extender maintained acceptable motility up to 7 d of storage at 4° C (Delgado et

al., 2009. There are no reports for using CJ-2 extender for extension and chilled storage of ram

36

semen. Therefore, the objective of the current study was to compare four different extenders

(milk, TRIS, TEST or CJ-2) stored at two different temperatures (4 or 15° C) for 72 h.

Materials and methods

This trial was conducted at the University of Arkansas, Fayetteville, AR. All animal

procedures were approved by Institutional Animal Care and Use Committee (Protocol # 15019).

Animal management

Five mature Katahdin rams were clinically healthy and raised on mixed tall fescue

(Schedonorus arundinacea) and bermudagrass (Cynodon dactylon) pasture. All rams had access

to water, and mineral salt, and 0.5 kg/d of corn/soybean meal (17% CP, 82% TDN on DM basis)

per ram. Semen was collected by electroejaculation into 15 ml polystyrene tubes, screw capped

and placed immediately into a thermo-flask (Lab-line Instruments Inc., Melrose Park, IL)

containing water maintained at 35° C. Semen samples were transported to the laboratory for

analysis.

Evaluation of fresh semen by computer assisted sperm analysis (CASA)

All semen samples were evaluated within 2 h post collection, using a computer assisted

sperm analysis (CASA; Hamilton-Thorne, IVOS, Beverly MA) with 12 TOX IVOS software.

Sperm motility parameters were analyzed using the default analysis settings recommended by

manufacturer for ram semen. Semen samples were diluted in CJ-2 extender without egg yolk at

1:100 (semen: extender ratio). About 3.5 µl of diluted sample was placed in disposable 20 µm

deep, 2-chambered IVOS slide (Cytonix, Beltsville, MD). At least four fields were scanned, with

30 video frames captured per field at a frame rate of 60 Hz. A minimum of 400 sperm cells were

counted in each sample. Variables measured with CASA were motility (%), progressive motility

(%), track speed (VCL, µm/sec), path velocity (VAP, µm/sec), progressive velocity (VSL,

37

µm/sec), beat frequency (BCF, MHz), lateral amplitude (ALH, µm), linearity (LIN, %), and

straightness (STR, %; Table 1).

Preparation of semen extenders

Milk extender

All the ingredients were from Sigma Company unless otherwise mentioned. Locally

purchased 10 g of organic skim milk powder (NOW Real Food®, organic, non-fat dry milk

powder) was dissolved in a total volume of 95 ml of lukewarm Milli-Q water in a 250-ml beaker.

The solution was constantly stirred for 10 min using a magnetic stirrer while heated at 95° C on a

hot plate (VWR, West Chester, PA model 371). The solution was cooled to room temperature

and then 0.5 g of fructose and 0.5 ml of gentamicin (10 mg/ml; Life Technologies, Gibco®) were

added. The stock solution was stored at 4° C. On the day of semen collection, 5 ml of egg yolk

from USDA certified organic egg was added to the stock solution, mixed thoroughly and

warmed to 35° C.

TEST extender

The TES buffer of 7.5 g was dissolved in 100 ml Milli-Q water in a 250-ml beaker. In

another beaker, 3.93 g of TRIS (tris[Hydroxymethyl] aminomethane) was dissolved in 100 ml

Milli-Q water. The TES solution was titrated with TRIS to a pH of 7.2, using a pH meter (Model

420A, Orion Laboratory Products Division Inc., Boston, MA). Osmolarity of the titrated solution

was 318 mOsmkg-1 determined with a computerized micro-osmometer (Multi-Osmette 2430,

Precision Systems, Natick, MA, USA). One half gram fructose and 0.5 ml of gentamicin (10

mg/ml; Life Technologies, Gibco®) was added to 100 ml of titrated solution. The stock solution

was filtered through a 0.45 µm sterile Gelman Acrodisk filter. The filtered stock solution was

38

stored at 4º C. On the day of semen collection, egg yolk was added to 95 ml of the stock solution

as described for milk extender.

TRIS extender

The extender was prepared by adding 3.634 g of TRIS (tris[Hydroxymethyl]

aminomethane) and 1.99 g of citric acid to 100 ml Milli-Q water in a 250-ml beaker. The pH of

the resulting solution was 7.0 measured using a pH meter (Model 420A, Orion Laboratory

Products Division Inc., Boston, MA). Fructose, gentamicin and egg yolk was added and the

extender was made ready for extension as described for TEST.

CJ-2 extender

Choline extender (CJ-2) was prepared according to the method described by Stachecki et

al. (1998) with modification. Gentamicin was used instead of kanamycin sulfate. Lactic acid,

hemi-calcium salt was substituted for CaCL2*2H2O. Glucose was substituted with fructose, and

phenol red was not added. The pH and osmolarity were 7.55 and 280 mOsm, respectively. To

prepare CJ-2 extender, KCL (0.2 g), KH2PO4 (0.2 g), Na2HPO4 (1.1 g) and MgCl2*6H20 (0.1 g),

was added to a total volume of 1 liter of Milli-Q water and stirred until completely dissolved.

Lactic acid (calcium lactate; 19.259 g) was added very slowly and stirred continuously to prevent

calcium precipitation by K2HPO4. Choline chloride (C5H14NOCl) was added last. The stock

solution was then filtered through a 0.20 µm sterile Gelman Acrodisk filter and stored at 4° C.

Fructose, gentamicin and egg yolk was added and the extender was prepared as described for

TEST.

Preparation of semen for storage

Semen was initially extended to 150 million cells/0.25 ml in each extender. Extended

semen (50 µl aliquots) was then stored in 0.25 ml semen straws (IMV, Paillette Crista, France).

39

However, by initially filling straws with 50 µl respective extender, separating with 50 µl volume

of air, injecting the 50 µl of experimental extended semen, separated by another 50 µl volume of

air, then 50 µl of extender to insulate the extended semen. Total 24 straws were prepared from

single semen collection per ram. The straw was sealed with an electric sealer (H. W. Andersen

Products, Inc., Oyster Bay, NY). Half of the straws were placed in water in a laboratory flask

(Sigma Chemical Co, St. Louis, MO) at 35° C. This flask was then placed in a refrigerator at 4°

C. Remaining straws were similarly placed in anther flask (Sigma Chemical Co, St. Louis, MO)

which was then placed in a cooler (Koolatron PC3, Koolatron, Brantford, Ontario, Canada)

maintained at 15° C. Extended semen was cooled to desired temperature over 2 to 3 h period

(Table 2).

Evaluation of extended semen using CASA

Extended semen was evaluated by IDENT fluorescence method. The IDENT STAIN

(Hoechst 33342) illuminates only DNA containing objects. Minimum and maximum static size

and intensity gates were used to eliminate the chance of counting any other cells. The

illumination intensity for fluorescent illumination was set up between 2050 and 4050. The

chilled extended semen was pre-warmed in a thaw jug maintained at 35° C for 15 sec. The straw

was wiped dry and cut with scissor adjacent to extended semen. Extended semen was removed

with pipet and kept in a well plate. About 3.5 µl of extended semen was pipetted and mixed with

pre-warmed 95 µl of corresponding extender (without egg yolk) in a 0.5 ml Eppendorf tube, then

5 µl of Hoechst 33342 stain (10 µg/ml) was added to the diluted semen and stored in heating

block maintained at 35° C for 15 min before analysis (IVOS IDENT illumination option). For

semen samples diluted using milk extender, 10 µl of Hoechst 33342 stain was added. After 15

min of equilibration with the stain, the sample was analyzed using IVOS system. About 3.5 µl of

40

diluted semen was placed in disposable 20 µm deep, 2-chambered IVOS slide (Cytonix,

Beltsville, MD). The semen analysis procedure was followed as described for fresh semen.

Extended semen was analyzed every 24 h for 7 d. The IVOS machine setting for ram semen

motility analysis is given in a Table 3.

Statistical analysis

Semen motility parameters were analyzed at 24, 48 and 72 h after storage. Data were

analyzed as repeated measures using MIXED procedure of SAS (Littell et al., 1996), with semen

collection of each ram as the experimental unit. Variables in the model included extender,

temperature, hour (as a repeated measures of 24, 48, and 72 h), and all 2 and 3 way interactions.

Non-significant (P > 0.1) interactions were removed from the model. The final model included

extender, temperature × extender, and extender × hour interactions. Data are presented as least

squares means ± standard error. Means separation occurred using PDIFF option of SAS.

Dependent variables measured with IVOS were total motility, progressive motility, VCL, VAP,

VSL, BCF, ALH, LIN, and STR.

Results

Total motility of fresh semen was 48.4%. Total motility decreased to 43.6 ± 2.4, 31.1 ±

2.4, and 27.2 ± 2.4% at 24, 48, and 72 h respectively (P < 0.01). There was a temperature ×

extender interaction (P < 0.01), mainly due to decreased motility of sperm cells stored in CJ-2

and milk based extenders at 15° C. Total motility of sperm cells stored at 4° C in TRIS or milk

was higher compared with TEST or CJ-2 (P < 0.05; Table 4). Extended semen stored at 4° C

tended (P = 0.08) to have a higher sperm motility compared with that stored at 15° C. Motility of

sperm cells stored at 15° C was highest in TRIS compared with other extenders (P < 0.05).

41

Progressive motility of non-extended semen was 35.2%. Progressive motility declined to

8.6 ± 0.7, 5.3 ± 0.7, and 3.2 ± 0.7% at 24, 48, and 72 h, respectively (P < 0.01). There was a

temperature × extender interaction (P < 0.01) mainly due to a decrease in progressive motility of

sperm cells stored in CJ-2 extender at 15° C. Progressive motility of sperm cells stored at 4° C

using TRIS, CJ-2, and milk were similar (P > 0.05) but higher compared with TEST (P < 0.05).

Track speed of fresh semen was 226.3 µm/sec. Mean VCL at 24, 48, and 72 h declined to

116.6 ± 4.2, 95.9 ± 4.2, and 82.1 ± 4.2 µm/sec, respectively (P < 0.01). There was a trend toward

extender × hour interaction on VCL (P = 0.060; Table 5), mainly due to a greater reduction of

VCL on semen stored in CJ-2 and milk extender at 48 and 72 h of storage. A temperature ×

extender interaction on VCL was observed (P < 0.01), mainly due to decreased VCL of sperm

cells extended using CJ-2 or milk and stored at 15° C. Track speed of sperm cells stored at 4° C

was similar across extenders (P > 0.05).

Path velocity of fresh semen was 155.7 µm/sec. Overall VAP at 24, 48 and 72 h was 61.8

± 2.4, 50.4 ± 2.4, and 42.5 ± 2.4 µm/sec, respectively (P < 0.01). An extender × hour interaction

was observed on VAP of sperm cells (P = 0.023), mainly due to a reduction of VAP when stored

in CJ-2 and milk based extenders at 48 and 72 h, respectively. A temperature × extender

interaction on VAP was observed (P < 0.01), mainly due to decreased VAP of sperm cells stored

in CJ-2 or milk and stored at 15° C. Path velocity of sperm cells stored at 4° C using CJ-2, milk,

and TRIS was similar but higher than sperm cells stored in TEST (P < 0.05).

Progressive velocity of fresh sperm cells was 139.3 µm/sec. Overall VSL of sperm cells

at 24, 48, and 72 h declined to 44.5 ± 1.7, 36.1 ± 1.7, 29.1 ± 1.7 µm/sec, respectively (P < 0.01).

An extender × hour interaction was observed for VSL (P = 0.023), mainly due to a reduction of

VSL of sperm cells stored in CJ-2 or milk extenders at 48 and 72 h, respectively. An extender ×

42

temperature interaction was also observed (P < 0.01), mainly due to a decrease of VSL of sperm

cells stored in CJ-2, milk or TEST extenders at 15° C. Progressive velocity of sperm cells stored

at 4° C using CJ-2, milk, and TRIS was similar but higher than sperm cells stored in TEST (P <

0.05).

Beat frequency of fresh sperm was 33.4 MHz. Overall, BCF at 24, 48, and 72 h declined

to 34.4 ± 1.5, 29.3 ± 1.5, and 26.4 ± 1.5 MHZ, respectively (P < 0.01). An extender × hour

interaction was observed on BCF (P = 0.005), mainly due to a reduction of BCF of sperm stored

in CJ-2 or milk extenders at 48 and 72 h, respectively. There was an extender × temperature

interaction (P = 0.01) due to a decrease of BCF of sperm cells stored in CJ-2 or milk based

extenders at 15° C. Beat frequency of sperm cells stored at 4° C was similar across extenders (P

> 0.05).

Lateral amplitude of fresh semen was 6.88 µm. Overall ALH at 24, 48, and 72 h of

storage declined to 6.1 ± 0.2, 5.3 ± 0.2, and 4.8 ± 0.2 µm, respectively (P = 0.011). There was an

extender × temperature interaction (P = 0.03), mainly due to a decrease in ALH in CJ-2 or milk

based extenders at 15° C. Lateral amplitude of sperm cells stored at 4° C was lower in CJ-2 and

milk compared with TEST or TRIS extenders (P < 0.05).

Linearity of fresh semen was 65%. Linearity at 24, 48, and 72 h was 38.3 ± 1.7, 32.7 ±

1.7, and 29.5 ± 1.7 %, respectively (P > 0.05). An extender × hour interaction was observed on

LIN of sperm cells (P = 0.023), mainly due to a reduction of LIN of sperm cells stored in CJ-2 or

milk based extenders at 48 and 72 h, respectively. Temperature × extender interaction on LIN

was observed (P < 0.01), mainly due to a decrease of LIN of sperm cells stored in CJ-2 at 15° C.

Linearity of sperm cells stored at 4° C was similar across extenders (P > 0.05).

43

Straightness of fresh sperm cells was 87%. Overall STR of sperm cells at 24, 48, and 72 h

declined to 66.6 ± 2.8, 50.4 ± 2.4, and 42.5 ± 2.4, MHz, respectively (P = 0.01). An extender ×

hour interaction was observed on STR of sperm cells (P = 0.003), mainly due to a reduction of

STR of sperm cells stored in CJ-2 or milk extenders at 48 and 72 h of storage. There was an

extender × hour interaction, mainly due to a decrease of STR of sperm stored in CJ-2 or milk at

15° C (P < 0.05). Straightness of sperm cells stored at 4° C was similar across extenders (P >

0.05).

Discussion

Effective technologies are needed to move sheep germplasm readily between farms to

facilitate genetic improvement and to allow the use of superior genetic resources. Artificial

insemination enables fast spread of quality genetic traits between farms. Artificial insemination

with frozen ram semen results in low conception rates (20-30%; Salamon and Maxwell, 1995).

Fresh diluted and cold stored semen is an alternative to frozen storage (Salamon and Maxwell,

1995). Ram semen stored at 4° C for a short period (2 to 3 d) of time was more beneficial than

frozen storage for a long-period of time (-196° C for several weeks; Wusiman et al., 2012;

Kulaksiz et al., 2012). Studies on liquid storage of ram semen concluded that semen can

successfully be cold stored at 5 or 15° C for 2 to 3 d (Hollinshead et al., 2004; Mata-Campuzano

et al., 2014). Storage at 5° C maintained viability over storage at 15° C when stored for 72 h (O’

Hara et al., 2010). However, a different result was reported when stored for up to 48 h (Mata-

Campuzano et al., 2014). Use of different extenders, supplements and storage durations in those

studies could have impacted results. In the current study, we compared four different extenders

(milk, TEST, TRIS, and CJ-2) at two storage temperature (5 or 15° C) for extension and storage

of ram semen, using similar concentration of egg yolk, fructose, and gentamycin in each

44

extender. Semen was evaluated before extension and every 24 h up to 72 h. Most of the CASA

motility parameters were higher after storage at 4 compared with 15° C in all the extenders.

However, if ram semen is to be stored at 15° C, then our data from the current study indicates

that semen stored at 15° C for 3 d using TRIS extender had better sperm motility parameters

compared with milk, TEST, or CJ-2 based extenders. Based on results obtained from the current

study, it is suggested that ram semen stored at 4 is superior over storage at 15º C for 3 d.

Milk or TRIS based extenders are the most common extenders used for extension of ram

semen (Lopaz et al., 1999; Paulenz et al., 2002; Hollinshead et al., 2004; Gundogan et al., 2009;

Quan et al., 2016). The TEST based extender was used for extension of semen in other species,

but not commonly used for the extension of ram semen (Bolanos et al., 1983; Pope et al., 1991).

The CJ-2 extender was developed to avoid intercellular damage caused by sodium in sodium

based extenders (Mazur, 1977; Stachecki and Willadsen, 1998a; Stachecki and Willadsen,

1998b). The use of CJ-2 based extender for cool storage of bull semen was tested previously

(Delgado et al., 2009), but to our knowledge it has not been tested for cool storage of ram semen.

In general, the current study shows that ram semen stored at 4° C using milk or TRIS based

extenders maintained similar but better motility parameters compared with storage using TEST

or CJ-2 extenders. There was no difference in most motility parameters using TRIS or milk

based extenders for extension and storage of ram semen at 4° C for 3 d in the current study, these

results are similar to that reported by Gundogan et al. (2009), but contrary to results reported by

others (Paulenz et al., 2002; Hollinshead et al., 2004; Kulaksiz et al., 2012; Quan et al., 2016;

Yaniz et al., 2011). Some studies reported ram semen extended in milk extender maintained

better motility over TRIS (Hollinshead et al., 2004; Wusiman et al., 2012) after storage at 4° C

while others reported better motility using TRIS extender (Paulenz et al., 2002; Lopez et al.,

45

1999; Quan et al., 2016). Differences observed between treatments could be due to use of

different percentage of egg yolk and other supplements in the extender medium. Additionally,

duration of storage differed. Hollinshead et al. (2004) used 6 million spermatozoa in 0.5 ml

extender to store in 5 ml sterile sealed tubes to evaluate after 6 h of storage at 4° C, whereas,

Mata-campuzano et al. (2014) stored at 15° C for 48 h.

In the current study, we used a similar concentration of egg yolk, antibiotics, and fructose

in each of the extenders. Semen was evaluated at a constant interval of 24 h for 3 d. Ram semen

stored using milk or TRIS based extenders maintained similar sperm motility parameters but

better compared with TEST or CJ-2 extenders when stored at 4° C for 3 d.

Milk extender with 5% egg yolk resulted in better conception rate compared with TRIS

extended with 20% egg yolk when ewes were vaginally inseminated with fresh extended semen

(Paulenz et al., 2003). In a preliminary trial, we artificially inseminated 19 Gulf Coast Native

ewes with fresh semen extended in milk with 5% egg yolk or TRIS with 5% egg yolk. The same

percentage of ewes conceived in both groups (milk: 5/9; TRIS: 5/9).

In the current study, there was an extender by storage time interaction in most sperm

motility parameters measured. Motility parameters were similar up to 24 h of storage, but after

that sperm motility declined more rapidly in milk or CJ-2 extenders compared with TEST or

TRIS extender. Such a decrease may be due to a greater reduction at the 15° C temperature using

milk or CJ-2 extenders. Consistent with our results, TEST or TRIS extenders had similar motility

parameters after extension and storage of rabbit semen at 15° C (Roca et al., 2000) for 96 h. A

greater reduction of motility in milk compared with TRIS based extenders in the current study is

similar to that reported by Gundogan et al. (2009), who reported 50% of ram sperm extended in

TRIS up to 4 d compared with similar motility up to only 3 d in milk extender.

46

In the current study, most of sperm motility parameters extended using CJ-2 and milk

decreased sharply after storage at 15° C compared with TRIS or TEST extender. However, such

decreases are not very dramatic after storage at 4° C. It is not known why a greater reduction of

motility of sperm cells was seen after storage using CJ-2 extenders at 15° C. Choline based

extender provided better medium for storage of oocytes compared with traditional sodium

containing medium (Mazur, 1977; Stachecki and Willadsen, 1998a; Stachecki and Willadsen,

1998b). Choline is a component of the phospholipid bilayer and helps in stabilizing cell plasma

membrane for protection of spermatozoa and oocytes. Bull semen extended and cold-stored at 4°

C for 7 d using CJ-2 extender had equal or higher sperm motility parameters compared with

semen stored in synthetic caudal epididymal plasma (Delgado et al., 2009).

In conclusion, most motility parameters were significantly higher after extension and

storage at 4 compared with 15° C. Ram semen stored using milk or TRIS based extenders at 4° C

can maintain similar motility parameters but better compared with TEST or CJ-2 extenders after

storage for 3 d. In the current study, ram semen stored at 15° C for 3 d using TRIS extender had

better sperm motility parameters compared with milk, TEST or CJ-2 extender. Milk and TEST

based extenders had intermediate efficacy for maintaining motility at 15° C. In contrast, CJ-2

based extenders had drastic reduction of most motility parameters after storage at 15° C.

Combined results from both temperatures shows most of the motility parameters were similar up

to 24 h of storage but after that sperm motility declined more rapidly in milk or CJ-2 extenders

compared with TEST or TRIS extender. Such a decrease is due to a greater reduction after 24 h

using milk and CJ-2 extenders and storage at 15° C. Considering sperm progressive motility,

which is also a predictor of in vivo fertility, milk and TRIS extenders stored at 4° C had similar

but higher progressive motility compared with ram sperm extended in CJ-2 and TEST extenders.

47

However, progressive motility of ram sperm declined by 75% when stored at 4° C for 24 h, and

continued to decline over time regardless of extenders used. Further studies can be done to

supplement milk or TRIS based extenders for extension and storage of ram semen at 4° C.

48

References

Bolanos, J. R., J. W. Overstreet, and D. F. Katz. 1983. Human sperm penetration of zona-free

hamster eggs after storage of the semen for 48 hours at 2 degrees C to 5 degrees C. Fertil.

Steril. 39:536-541.

Delgado, P., T. Lester, and R. Rorie. 2009. Effect of a low-sodium, choline-based diluent on

viability of bovine sperm stored at refrigerator temperatures. Ark. Anim. Sci. Depart.

Rep. 77-79.

Gündoğan, M. 2009. Short term preservation of ram semen with different extenders. Kafkas

Univ. Vet. Fak. Derg. 15:429-435.

Hollinshead, F., J. K O’Brien, L. Gillan, M. Meyers, W. M. C. Maxwell, and G. Evans. 2004.

Liquid storage of flow cytometrically sorted ram spermatozoa. Theriogenology, 62:587-

605.

Kulaksiz, R., C. Cebi, and E. Akcay. 2012. The effect of different extenders on the motility and

morphology of ram sperm frozen or stored at 4 degrees C. Turk. J. Vet. Anim. Sci. 36:

177-182.


diluted and stored in three different extenders. Acta Vet. Scand. 40:1-9.

Mata-Campuzano, M., M. Álvarez-Rodríguez, J. Tamayo-Canul, E. López-Urueña, P. de Paz, L.

Anel, and F. Martinez-Pastor. 2014. Refrigerated storage of ram sperm in presence of

Trolox and GSH antioxidants: Effect of temperature, extender and storage time. Anim.

Reprod. Sci. 151:137-147.

Mazur, P. 1977. The role of intracellular freezing in the death of cells cooled at supraoptimal

rates. Cryobiology. 14:251-272.

O’Hara, L., J. Hanrahan, L. Richardson, A. Donovan, S. Fair, A. C. Evans, and P. Lonergan.

2010. Effect of storage duration, storage temperature, and diluent on the viability and

fertility of fresh ram sperm. Theriogenology. 73:541-549.

Paulenz, H., L. Söderquist, R. Pérez-Pé, and K. Andersen Berg. 2002. Effect of different

extenders and storage temperatures on sperm viability of liquid ram semen.

Theriogenology. 57:823-836.

Paulenz, H., L. Söderquist, T. Ådnøy, O. H. Fossen, and K. A. Berg. 2003. Effect of milk-and

TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with

liquid semen. Theriogenology. 60:759-766.

Pope, C. E., Y. Z. Zhang, and B. L. Dresser. 1991. A simple staining method for evaluating

acrosomal status of cat spermatozoa. J. Zoo Wildl. Med. 22:87-95.

49

Quan, G. B., G. Q. Wu, Y. J. Wang, D. J. Li, Y. Ma, and Q. H. Hong. 2016. Effects of the Tris,

Tes, or skim milk based extender on in vitro parameters of ram spermatozoa during liquid

storage. Small Rum. Res. 134: 14-21.

Roca, J., S. Martinez, J. M. Vasquez, X. Lucas, I. Parrills, and E. A. Martinez. 2001. Viability

and fertility of rabbit spermatozoa diluted in Tris-buffer extender and stored at 15° C.

Anim. Reprod. Sci. 64:103-112.

Salamon, S., and W. M. C. Maxwell. 1995. Frozen storage of ram semen I. Processing, freezing,



111.

Stachecki, J. J., J. Cohen, and S. M. Willadsen. 1998 b. Cryopreservation of unfertilized mouse

oocytes: the effect of replacing sodium with choline in the freezing medium.

Cryobiology. 37:346-354.

Stachecki, J. J., J. Cohen, and S. Willadsen. 1998 a. Detrimental effects of sodium during mouse

oocyte cryopreservation. Biol. Reprod. 59:395-400.

Wusiman, A., Y. P. Wang, K. Ren, G. B. Zhou, X. W. Fu, L. Suo, Z. Q. Fan, L. Wang, and S. E.

Zhu. 2012. Semen Storage at 23, 4 or -196° C and its application to artificial

insemination in small-tail Han sheep. Asian J. Anim. Vet. Adv. 7:299-308.

Yániz, J. L., J. A. Mateos, and P. Santolaria. 2011. Zwitterionic buffers preserve ram semen

quality more efficiently than TRIS during storage at 15° C. Small Ruminant Res. 95:54-

60.

50

Table 1. CASA parameters

Parameters Description

Total motility (%) Path velocity ≥ 30 µm/sec and progressive velocity ≥ 15

µm/sec

Progressive motility (%) Path velocity ≥ 50 µm/sec and straightness ≥ 70%

Track speed (VCL, µm/sec) Average velocity measured over the actual point-to-point

track.

Path velocity (VAP, µm/sec) Average velocity of the smoothed (best-fit) sperm path.

Progressive velocity (VSL,

µm/sec)

Average velocity over straight line from the beginning to

the end of the sperm track.

Beat frequency (BCF, MHz) Frequency of sperm head crossing the sperm average path

in either direction.

Lateral amplitude (ALH,

µm)

Mean width of the head oscillation as the sperm swims.

Linearity (LIN, %) Departure of actual sperm path from straight line

(VSL/VCL).

Straightness (STR, %) Departure of average sperm path from a straight line

(VSL/VAP).

51

Table 2. Hourly change in temperature of semen filled straws kept inside Koolatron cooler

maintained at 15° C or laboratory refrigerator at 4° C. Temperature was measured till 3 h after

storage.

Hour after storage Koolatron cooler at 15° C Laboratory refrigerator at 4° C

0 35.0° C 35.0° C

1 24.2° C 14.8° C

2 19.7° C 7.7° C

3 17.6° C 4.8° C

52

Table 3. IVOS machine settings for ram semen motility analysis

Parameter Setting

Frames per second 60

Number of frames 30

Minimum contrast 60

Minimum size 5

Default cell size 5

Default cell intensity 55

Path velocity (VAP) 75.0

Straightness (STR) 80

Slow cells Static

VAP cut off 21.9

VSL cut off 6.0

Minimum intensity gate 0.25

Maximum intensity gate 1.50

Minimum size gate 0.60

Maximum size gate 8.00

Minimum elongation gate 0

Maximum elongation gate 95

UV fluorescent objective 10×

Target photometer 45-55

Field type Dark

Chamber depth 20

53

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.

Sperm motility Storage Extender treatment

characteristics temperature CJ-2 Milk TEST TRIS SE

Total motility 4° C 30.1b,x 40.6ab,x 30.4b,x 44.5a,x 4.1

15° C 9.1c,y 20.1c,y 41.3b,x 55.2a,x 4.1

Progressive motility 4° C 6.8a,x 7.8a,x 2.8b,x 7.4a,y 1.1

15° C 1.0c,y 4.9b,x 3.8bc,x 11.1a,x 1.1

Track speed 4° C 101.1a,x 116.1a,x 103.4a,x 118.1a,x 6.9

15° C 43.6c,y 66.1b,y 105.5a,x 131.7a,x 6.9

Path velocity 4° C 56.3ab,x 65.5a,x 50.7b,x 60.6ab,x 3.9

15° C 22.9d,y 38.3c,y 51.9b,x 66.2a,x 3.9

Progressive velocity 4° C 42.5a,x 45.2a,x 34.1b,x 42.9a,x 2.9

15° C 17.1c,y 29.6b,y 34.4b,x 46.5a,x 2.9

Beat frequency 4° C 33.3a,x 34.1a,x 32.5a,x 34.7a,x 2.5

15° C 18.2b,y 22.8b,y 31.1a,x 33.5a,x 2.5

Lateral amplitude 4° C 5.1b,x 5.4ab,x 6.6a,x 6.6a,x 0.4

15° C 2.8b,y 3.6b,y 6.4a,x 6.7a,x 0.4

Linearity 4° C 39.2a,x 40.3a,x 35.1a,x 36.8a,x 2.1

15° C 18.4b,y 29.7a,y 33.8a,x 34.7a,x 2.1

Straightness 4° C 65.6a,x 67.2a,x 65.1a,x 67.1a,x 4.5

15° C 32.5c,y 48.3b,y 63.7a,x 63.6a,x 4.5

Data expressed are the least square means and standard error (SE) for sperm motility parameters

taken at 24, 48, and 72 h after storage. abcdValues within the same row with unlike superscripts are different (P < 0.05). xyValues within the same column with unlike superscripts differ (P < 0.05).

54

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.

Extenders Time of evaluation

24 h 48 h 72 h SE

Track speed

CJ-2 109.9a,x 59.8b,z 47.2b,z 8.5

Milk 111.2a,x 89.8a,y 72.1b,y 8.5

TEST 110.5a,x 106.2a,xy 96.8a,x 8.5

TRIS 134.6a,x 127.8a,x 112.2a,x 8.5

Path velocity

CJ-2 60.9a,xy 32.9b,z 25.1b,z 4.8

Milk 65.1a,xy 51.1b,y 39.7b,y 4.8

TEST 53.7a,xy 52.1a,y 48.1a,x 4.8

TRIS 67.5a,x 65.6a,x 57.1a,x 4.8

Progressive velocity

CJ-2 46.1a,x 25.3b,z 17.8b,yz 3.5

Milk 49.4a,x 37.1b,y 25.6c,y 3.5

TEST 35.4a,y 34.7a,y 32.8a,xy 3.5

TRIS 47.1a,x 47.3a,x 39.7a,x 3.5

Beat frequency

CJ-2 38.1a,x 22.8b,y 16.4b,y 3.6

Milk 34.4a,x 27.9ab,xy 23.1b,y 3.6

TEST 32.1a,x 31.5a,x 31.8a,x 3.6

TRIS 33.2a,x 35.1a,x 34.4a,x 3.6

Linearity

CJ-2 40.7a,xy 25.9b,y 19.9b,y 3.4

Milk 44.7a,x 33.5ab,xy 26.9b,xy 3.4

TEST 33.2a,y 34.2a,xy 36.6a,x 3.4

TRIS 37.7a,y 37.2a,x 35.5a,x 3.4

Straightness

CJ-2 69.7a,x 43.3b,z 34.2b,z 5.6

Milk 72.1a,x 56.1b,xy 45.5b,yz 5.6

TEST 62.8a,x 64.3a,x 66.1a,x 5.6

TRIS 62.2a,x 68.5a,x 65.4a,x 5.6

Data expressed are the least square means and standard error (SE) for parameters taken at 24, 48,

and 72 h for extender by hour interactions. abcdValues within the same row with unlike superscripts are different (P < 0.05). xyzValues within the same column with unlike superscripts differ (P < 0.05).

55

Chapter IV: Evaluation of semen extenders for short-term storage of ram semen at 4° C

Abstract

The objective of the study is to evaluate the effects of extender, ethylene glycol (EG), egg

yolk and penicillamine, hypotaurine and epinephrine (PHE) on ram sperm progressive motility

during storage. Semen collected from 9 mature rams by electroejaculation was distributed across

treatments consisting of either TRIS or milk extenders supplemented with 5 or 20% (v/v) egg

yolk, ± EG, and ± PHE. For each semen collection, TRIS extender was prepared from 4×

solution so that TRIS, citric acid and fructose concentration were constant at 300, 94.7, 27.8

mM, respectively, regardless of semen dilution factor. A 4× milk extender was also used so that

the extender contained 10% (w/v) milk powder, regardless of semen dilution factor. Semen was

diluted in extender to a final concentration of 300 million sperm/ml in 1.5 ml tubes, and cooled

to 4° C over a 2 to 3 h period. Semen was evaluated initially and daily for 3 d, using computer

assisted sperm analysis. Data were analyzed using the PROC MIXED procedure of SAS, with

ram as the experimental unit. Variables in the model included extenders, supplements, hour

(repeated measures) and interactions. Milk extender maintained better motility parameters over

time compared with TRIS (P < 0.05). Addition of either 5 or 20% egg yolk in the extender had

no effect on motility parameters (P > 0.05). However, milk with 20% egg yolk had the highest

track speed and path velocity followed by TRIS with 20% egg yolk, TRIS with 5% egg yolk, and

milk with 5% egg yolk, respectively (P < 0.05). There was an EG × hour interaction. At 72 h of

storage there was a rapid decline in sperm motility parameters in extenders without EG (P <

0.05). Addition of PHE before semen analysis increased progressive motility (P < 0.05), beat

frequency (P < 0.05), and tended to increase total motility (P = 0.058) and straightness (P =

56

0.078) of sperm cells. Results indicated that milk extender supplemented with 1% EG, PHE and

either 5 or 20% egg yolk can be used to protect ram semen at 4° C for 72 h.

Keywords: cold-storage, ethylene glycol, PHE, ram semen

Introduction

Typically, cryopreservation of ram semen results in poor viability post-thaw (Salamon

and Maxwell, 1995; Salamon and Maxwell, 2000). An alternative to cryopreservation is

temporary storage of ram semen at 4° C (Quan et al., 2016). Milk and TRIS extenders with 5%

egg yolk are commonly used for liquid storage of ram semen (Salamon and Maxwell, 2000;

Camara et al., 2011). However, preliminary studies found that progressive motility of ram sperm

declined up to 75% when stored at 4° C for 24 h, and continued to decline over time when using

extenders supplemented with 5% egg yolk (Acharya et al., 2016). Inclusion of egg yolk in the

extender protects ram sperm cells during cold storage of ram semen (Gil et al., 2011). The

literature is void of studies on optimal percentage of egg yolk to maintain ram sperm motility for

cool storage. If a majority of reduction in progressive motility occurs during the initial cooling

and storage of ram semen, then additional egg yolk and cryoprotectant prior to cooling, and

addition of supplements before evaluation might maintain or improve progressive motility.

Penicillamine, hypotaurine, and epinephrine (PHE) have different mode of action, but

collectively enhance sperm motility, sperm capacitation, and acrosomal reaction (Meizel, 1985).

Addition of PHE in the extender decreased time interval for oocyte penetration by sperm (Susko-

Parrish, et al., 1990). Frozen thawed and cultured bull semen in semen extender with PHE

increased most sperm motility parameters compared with absence of PHE (Pearson et al., 2007).

To our understanding there are no studies which compared the effect of addition of PHE on

sperm motility parameters after cool storage of ram semen.

57

Ethylene glycol (EG) was initially used as a cryoprotectant of choice for freezing

embryos and freeze-storage of ram semen (Bautista et al., 1997). Ethylene glycol causes the least

osmotic stress to sperm cells during cryopreservation compared with other cryoprotectants (Gao

et al., 1997; Ball et al., 2001). Addition of 1.6% EG in the extender medium to extend Chincila

lanigera semen increased sperm motility after storage for 72 h at 4° C compared with no EG

added. Addition of EG in the extender medium for cold storage of ram semen has yet to be

tested. The objective of this study was to evaluate the effects of different combinations of EG,

egg yolk, and PHE in milk or TRIS extenders on ram sperm progressive motility during storage

at 4° C for 72 h.


The trial was conducted at the University of Arkansas, Fayetteville. All animal


Animal management

Seven Katahdin and 2 Suffolk rams greater than 1 year of age were used for the study.

All rams were clinically healthy and free of internal and external parasites. Katahdin rams were

raised on tall fescue (Schedonorus arundinacea) pasture, whereas Suffolks rams were maintained

on mixed pasture containing white ladino clover (Trifolium repens) and bermudagrass

(Cyanodon dactylon). Animals had free access to drinking water, and mineral mixture and

additionally supplemented with 0.5 kg of sheep ration (17% CP, 82% TDN on DM basis)/ram/d.

Semen was collected by electroejaculation in 15 ml conical polystyrene tubes (Scientific USA,

Orlando, FL), screw capped and placed immediately into a thermo-flask (Lab-line Instruments

Inc., Melrose Park, IL) with water maintained at 30° C. Semen samples were transported to the

58

laboratory for analysis. All semen samples were evaluated within 2 h post collection using

computer assisted sperm analysis (CASA).

Evaluation of fresh semen by computer assisted sperm analysis (CASA)

All semen analysis and extension was conducted in a culture room maintained at 30° C.

A CASA system (Hamilton-Thorne, IVOS, Beverly MA) with 12 TOX IVOS software was used

for semen analysis. Sperm motility parameters were analyzed using default analysis setting

recommended by the manufacturer for ram semen. For analysis, semen samples were diluted in

TRIS extender (without egg yolk) at 1:100 (semen: extender) and 3.5 µl of diluted sample was

placed in a disposable 20 µm deep 2-chambered IVOS slide (Cytonix, Beltsville, MD). At least

four fields were scanned, with 30 video frames captured per field at a frame rate of 60 Hz. A

minimum of 400 sperm cells were counted for each sample analysis. Variables measured

included motility (%), progressive motility (%), track speed (VCL, µm/sec), path velocity (VAP,

µm/sec), progressive velocity (VSL, µm/sec), beat frequency (BCF, MHz), lateral amplitude

(ALH, µm), linearity (LIN, %), and straightness (STR, %).

Experimental treatments

Semen samples with less than 40% total motility were excluded. Semen was distributed

over 8 different treatments, 1) milk + 5% (v/v) egg yolk; 2) milk + 20% (v/v) egg yolk; 3) milk +

5% (v/v) egg yolk + 1% EG; 4) milk + 20% (v/v) egg yolk + 1% EG; 5) TRIS + 5% (v/v) egg

yolk; 6) TRIS + 20% (v/v) egg yolk; 7) TRIS + 5% (v/v) egg yolk + 1% EG; 8) TRIS + 20%

(v/v) egg yolk + 1% EG. Semen was extended at a rate of 300 million sperm cells/ml and stored

in 1.5 ml Eppendorf tubes. Fructose and antibiotics were added at the time of preparation of 4×

milk or 4× TRIS citrate extenders. The PHE 40 µl/1000 µl of extended semen was added 5 min

before evaluation.

59

Extenders

Milk and TRIS extender

For the 4× milk extender, skim milk powder (40g; NOW Real Food®, organic, non-fat

dry milk powder) was dissolved in 100 ml of purified water (Milli-Q) in a 250-ml beaker by

constantly stirring heating to 95° C for 10 min (VWR, West Chester, PA, model 371 hot plate

stirrers). Once cooled, 2 g of fructose (Sigma Chemical Co, St. Louis, MO) and 2 ml of

gentamicin reagent (10 mg/ml; Life Technologies, Gibco®, Waltham, MA) were added.

To prepare the 4× TRIS extender, tris[Hydroxymethyl]aminomethane (TRIS; 14.5 g;

Sigma Chemical Co, St. Louis, MO) and citric acid (8.0g; Sigma Chemical Co, St. Louis, MO)

were dissolved in 100 ml water in a 250-ml beaker. Fructose (2 g; Sigma Chemical Co, St.

Louis, MO) and 2 ml of gentamicin reagent (10 mg/ml; Life Technologies, Gibco®, Waltham,

MA) were added. The stock solution was filtered (Gelman 0.20 µm sterile Acrodisk filter; Sigma

Chemical Co, St. Louis, MO). Once the dilution factor of fresh semen was determined, egg yolk

(5 or 20% v/v), ethylene glycol, and purified water was added to 4× milk or TRIS and mixed

homogenously at 30° C for extension of fresh semen (maintained at 30° C).

Determination of semen extension rate

The concentration of sperm cells in a fresh semen sample determined by CASA analysis

was multiplied by the dilution factor and volume of ejaculate. Semen was extended to 150

million cells/0.25 ml. Each semen sample was diluted with each extender combination.

Semen extension using extenders

A 4× concentration of milk (constituted of milk, fructose, and gentamycin) or TRIS

extender (constituted of TRIS, citric acid, fructose, and gentamycin) was prepared at first. After

determination of semen concentration, the appropriate amount of egg yolk, EG, and purified

60

(Milli-Q) water, and 4× milk or TRIS was added to known concentration of semen. Prepared

extender was added to semen. Each time semen was extended to the constant concentration (300

million sperm cells/ml). For each semen extension, TRIS extender was prepared from 4×

solution so that the TRIS, citric acid and fructose concentration were constant 300, 94.7, 27.8

mM, respectively, regardless of semen dilution factor. A 4× milk extender was also used so that

the extender contained 10% (w/v) milk powder, regardless of semen dilution factor. Both

extenders were supplemented with 50 µg/ml of gentamicin. Egg yolk (5 or 20% v/v) and ± 1%

EG was added at the time of extension. Extended semen samples were stored in 1.5 ml

Eppendorf tubes and gradually cooled to 4° C in a laboratory refrigerator. Semen was cooled to

4° C over a 3 h period. Cooling rate was recorded using Physitemp Model BAT-12 temperature

recorder. Slow cooling rate was achieved by putting Eppendorf tubes in 300 ml laboratory

beaker containing water at 30° C and placing it into a refrigerator.

Preparation of PHE

A penicillamine, hypotaurine and epinephrine (PHE) stock solution was prepared by

adding 5 ml penicillamine stock (2.98 g in 10 ml of 0.8% saline), 5 ml hypotaurine stock (1.09

mg in 0.8% saline) and 4 ml epinephrine stock (165 mg 60% sodium lactate and 50 mg sodium

meta-bisulfite in 50 ml of water; pH was adjusted to 4.0 with 1 N HCL; 1.83 mg epinephrine was

added to 40 ml of this solution in 6 ml 0.8% saline). The final solution was comprised of 20 µl

penicillamine, 10 µl hypotaurine, 2 µl epinephrine, 21 µl sodium meta-bisulfite and 118 µl

sodium lactate. The PHE stock solution was stored in 1.0 ml cryogenic vials (Vangard Cryos,

Sumitomo Bakelite Co., Ltd., Japan) at -20 °C. On the day of semen analysis, 12.5 µl of thawed

PHE stock solution was mixed with 315 µl of diluted semen samples (300 µl of 1× TRIS citrate

61

extender + 15 µl of extended semen) immediately before CASA analysis. Note: the procedure for

PHE preparation was adopted from Rick Rorie (personal communication).

Evaluation of extended semen

Extended semen was evaluated by IDENT fluorescence method every 24 h up to 72 h

(CASA setting; Table 1). Ident stain (Hoechst 33342) was used to illuminate DNA containing

objects. An aliquot of 15 µl of chilled semen was diluted with pre-warmed 300 µl of 1× TRIS

extender (at 35° C) in a 1.5 ml Eppendorf tube. About 12.5 µl of PHE was added to the PHE

treatment samples. The amount of PHE required to stain a given volume of extended semen was

described previously (rate of 40 µl of PHE/ml of diluted semen; Miller et al., 1994; Pearson et

al., 2007). Diluted semen sample was stained (Hoechst 33342) before analysis (IVOS IDENT

illumination option). About 5 µl of stain (10 µg/ml) was added to the diluted semen and

maintained in a heating block at 35° C for 15 min before analysis. For milk egg yolk extender,

10 µl of stain was added. Samples were warmed in a digital heatblock (VWR International,

USA) for 15 min at 35° C. After 15 min of equilibration with stain at 35° C, samples were

analyzed using CASA. About 3.5 µL of extended semen was placed in a disposable 2-chamber,

20 µm deep IVOS slide (Cytonix, Beltsville, MD). At least four fields were scanned, with 30

video frames captured per field at a frame rate of 60 Hz. A minimum of 400 sperm cells were

counted in each sample analysis. Variables measured were total motility, progressive motility,

VCL, VAP, VSL, BCF, ALH, LIN, and STR (Table 2).


Data were analyzed as repeated measures using MIXED procedure of SAS (Littell et al.,

1996; SAS Institute, Carey, NC) with ram as the experimental unit. Variables in the model

included extenders (milk or TRIS), 5 or 20% egg yolk, 0 or 1% EG, and 0 or 40 µl PHE/1000 µl

62

of diluted semen and all 2, and 3 way interactions. Non-significant (P > 0.1) interactions were

removed from the model (P > 0.1). The final model included extender, egg yolk, EG, PHE,

ethylene glycol × hours, ethylene glycol × extender, and egg yolk × extender interactions.

An autoregressive covariance structure was used. Data are presented as least square means and

standard error. Means were separated using the PDIFF option of SAS. If complex interactions

were not significant they were removed. Variables analyzed using CASA were motility,

progressive motility, VCL, VAP, VSL, BCF, ALH, LIN, and STR.

Results

Total Motility of fresh sperm cell was 67%. Motility declined to 53.1 ± 1.6, 46.2 ± 1.6,

and 40.7 ± 1.6% after 24, 48, and 72 h after extension and storage (P < 0.01). Motility was

higher with milk compared with TRIS extender (P < 0.01; Table 3). Supplementation of either 5

or 20% egg yolk resulted in similar total motility (P = 0.463; Table 4). Supplementation of EG

increased total motility compared to no addition (P < 0.01; Table 5). Addition of PHE before

semen analysis tended to increase (P < 0.0589; Table 6) total motility compared with no PHE

addition. There was an EG × hour interaction (P < 0.01; Table 7) due to decreased sperm

motility without EG at 72 h.

Progressive motility of fresh sperm cells was 38.6% and declined to 24.4 ± 0.9, 20.1 ±

0.9, and 17.1 ± 0.9 after 24, 48, and 72 h of storage (P < 0.01). There was a trend toward an

extender × EG interaction (P = 0.0793) in that semen extended in milk supplemented with EG

had higher progressive motility compared with TRIS with EG or without EG (P < 0.05; Table 8).

Semen extended using milk + EG tended to have higher sperm progressive motility compared

with milk without EG (P = 0.055). There was an EG × hour interaction (P < 0.05) mainly due to

decreased progressive motility in extenders without EG at 72 h of storage. Sperm progressive

63

motility was similar between extenders with 5 or 20% egg yolk (P = 0.721). Addition of PHE

before semen analysis increased (P < 0.01) sperm progressive motility compared with no PHE

addition.

Track speed of fresh sperm cells was 202.8 µm/sec. Track speed declined to 192.5 ± 5.2,

186.8 ± 5.2, and 174.0 ± 5.2 µm/sec after 24, 48, and 72 h of storage (P < 0.05). There was a

trend toward extender × egg yolk-percentage interaction (P = 0.069; Table 9) on VCL. Storage

of semen in milk + 20% egg yolk had higher VCL compared with milk + 5% egg yolk (P <

0.05), but, was similar between TRIS + 5% egg yolk and TRIS + 20 % egg yolk (P > 0.05).

There was an EG × hour interaction (P = 0.036) mainly due to decreased VCL in extenders

without EG at 72 h of storage. Addition of PHE before semen analysis had no effect on VCL

compared with no PHE addition (P = 0.161).

Path velocity of fresh sperm cells was 130.4 µm/sec. Path velocity declined to 106.3 ±

2.4, 101.5 ± 2.3, and 92.1 ± 2.3 µm/sec after 24, 48, and 72 h of storage (P < 0.05). There was a

trend toward a significance interaction for VAP observed in an extender × egg yolk-percentage

(P = 0.056). Milk + 20% egg yolk had higher VAP compared with milk + 5% egg yolk, but VAP

was similar between TRIS + 5 or 20% egg yolk (P < 0.05). There was an EG × hour interaction

(P < 0.01) mainly due to a decrease in VAP in extenders without EG at 72 h of storage. VAP

was similar whether PHE was added or not before semen analysis (P = 0.276).

Progressive velocity of fresh sperm cells was 109.6µm/sec. Progressive velocity declined

to 82.1 ± 1.7, 78.0 ± 1.7, and 70.7 ± 1.7 µm/sec after 24, 48, and 72 h of storage, respectively (P

< 0.01). An extender × EG interaction was seen on sperm VSL (P < 0.01). Semen stored in milk

with or without EG had a higher VSL compared with TRIS with or without EG (P < 0.05), but

TRIS supplemented with EG had higher VSL compared with TRIS without EG (P < 0.05).

64

There was an EG × hour interaction on sperm VSL mainly due to decreased VSL in extenders

without EG after 72 h of storage (P < 0.01). The concentration of egg yolk (P = 0.163) or PHE

had no effect on VSL (P = 0.541).

Beat frequency of fresh sperm cells was 38.7 MHz. Beat frequency at 24, 48, and 72 h

was 31.9 ± 0.7, 31.0 ± 0.7, and 32 ± 0.7 MHz, respectively (P = 0.614). An extender × EG

interaction was observed on sperm BCF (P < 0.05). Semen extended using milk with or without

EG, and TRIS + EG had similar BCF (P > 0.05), which were higher than TRIS without EG (P <

0.05). BCF was similar between extenders with 5 and 20% egg yolk (P = 0.163). Addition of

PHE compared with no PHE before semen analysis increased BCF (P < 0.01).

Linearity of fresh sperm cells was 55%. Linearity of sperm cells declined to 47 ± 0.9, 45

± 0.9, and 40 ± 0.9% at 24, 48, and 72 h, respectively (P < 0.01). An extender × EG interaction

was observed on LIN (P = 0.006). Linearity of sperm cells was similar after semen storage in

milk with or without EG and TRIS EG extenders (P > 0.05), which were higher compared with

TRIS without EG (P < 0.05). An EG × hour interaction was observed on LIN, mainly due to

decreased LIN of sperm stored in EG non-supplemented extenders at 72 h of storage (P =

0.043). There was no difference in LIN between extenders with 5 or 20% egg yolk (P = 0.185).

Addition of PHE before semen analysis did not influence LIN compared with no PHE addition

(P = 0.267).

Lateral amplitude (ALH) of fresh sperm cells was 6.37 µm. Lateral amplitude of sperm

cells at 24, 48, and 72 h was 7.26 ± 0.1, 7.39 ± 0.1, and 6.92 ± 0.1µm respectively (P = 0.2374).

There was a trend toward an interaction between EG × hour (P < 0.092), mainly due to reduced

ALH of sperm cells stored without EG at 72 h (P < 0.059). Lateral amplitude was higher in

TRIS extender compared with milk (P < 0.01). Sperm ALH was similar between extenders with

65

5 and 20% egg yolk (P = 0.231). Similarly, addition of PHE before semen analysis had no effect

on ALH (P = 0.267).

Straightness of fresh sperm cells was 81%. Straightness of sperm cells declined to 77 ±

1.4, 75 ± 1.4, and 70 ± 1.4% at 24, 48, and 72 h, respectively (P < 0.01). An extender × EG

interaction was observed on STR (P < 0.01) in that TRIS without EG was lower than that of

milk with or without EG and TRIS + EG (P > 0.05). An EG × hour interaction was observed on

sperm STR mainly due to decreased STR in EG non-supplemented extenders at 72 h of storage

(P = 0.027). Straightness was similar in extenders with 5 or 20% egg yolk (P = 0.231). Addition

of PHE before semen analysis tended to increase sperm STR compared with no PHE addition (P

= 0.078).

Discussion

Ram semen extended and stored at 4° C using milk or TRIS maintained better sperm

motility compared with TEST and CJ-2 extender (Acharya et al., 2017; unpublished data).

Storage at 4 maintained better sperm motility parameters compared with storage at 15° C.

(Acharya et al., 2017; unpublished data). Although milk or TRIS extenders maintained better

motility compared with TEST and CJ-2 extenders at 4° C, the overall progressive motility

declined ~ 75% within 24 h of storage and continued to decline over time using extenders

supplemented with 5% egg yolk. If the majority of reduction in progressive motility occurs

during the initial cooling and storage of ram semen, then additional egg yolk and cryoprotectant

at the time of extension or supplementation of PHE before evaluation might maintain or improve

motility of ram semen. In the current study, milk extender maintained better sperm motility

parameters over time compared with TRIS extender. Addition of either 5 or 20% egg yolk in

extenders had no effect on sperm motility parameters. Addition of EG in the extender before

66

storage increased total motility, progressive motility, and BCF of sperm cells compared with no

addition. Addition of PHE before semen analysis increased VSL and BCF, and tended to

increase total motility and STR of sperm cells compared with no PHE addition.

Several studies have been reported on sperm quality between were done in the past to

compare milk and TRIS extenders for extension and chilled storage of ram semen, but with

inconsistent results. Some authors reported superiority of milk over TRIS (Hollinshead et al.,

2004), while others reported TRIS maintained better motility over milk (Quan et al., 2016). Such

inconsistent results could be due to differences in supplements in the extender, storage volume,

storage durations, concentration of sperm cell in the extender, and use of sorted or non-sorted

semen. For example, Quan et al. (2016) used 15 ml extended semen to store in 15 ml plastic vials

at a dilution rate 100 million spermatozoa/ml, while Hollinshead et al. (2004) used sorted semen

for extension and cold storage. Sorted semen was extended at 6 million spermatozoa in 0.5 ml

extender to store in 5 ml sterile sealed tubes to evaluate after 6 h of storage at 4° C (Hollinshead

et al., 2004). Therefore, none of the studies can be used for generalization of superiority of one

extender over other (milk or TRIS). In the current study, ram semen was extended to a

concentration of 300 million sperm cells/ml to match with a standard of 150 million sperm

cells/0.5 ml insemination dose. TRIS, citric acid, and fructose concentrations in the medium was

constant, regardless of semen dilution factor. Similarly, milk extender contained constant amount

of 10% (w/v) milk powder regardless of semen dilution factor. Extended semen was kept in 1.5

ml Eppendorf tubes to store in 300 ml beaker filled with water at 30° C before storage to achieve

slow cooling rate. Semen was evaluated every 24 h up to 72 h of storage at 4° C. Milk

maintained higher sperm motility parameters compared with TRIS extender and with the

67

addition of cryoprotectant and supplements, progressive motility of sperm cell was ~ 60% of the

initial value after storage at 4° C for 72 h.

During the process of cooling extended semen for cold storage, massive rearrangements

and leakage of sperm plasma membrane occurs which could lead to sperm cell death (Holt and

North, 1984). Inclusion of egg yolk in the culture medium protects ram spermatozoa from cell

death (Gil et al., 2011). It is still unclear about the components of egg yolk and mechanism for

protection of ram sperm cells. Inclusion of TRIS + 20% egg yolk better protected sperm cells

against cold shock compared with inclusion of TRIS + 15% egg yolk, and TRIS + 10% egg yolk

(Rajabi-Toustani et al., 2014). Similarly, Lopez et al. (1999) reported better sperm motility after

storage in TRIS or sodium citrate extender with 20% compared with milk and 5.5% egg yolk.

However, the study was confounded as different extenders and different egg yolk percentages

were used (Lopez et al., 1999). In the current study, milk or TRIS extenders were compared

using either 5 or 20% egg yolk in extender medium. There was no difference in motility

parameters using 5 or 20% (v/v) of egg yolk in either of the extender. However, inclusion of

20% egg yolk in milk extender led to a higher VCL and VAP, followed by TRIS + 20% egg

yolk, TRIS + 5% egg yolk and milk 5% egg yolk.

Ethylene glycol is a cryoprotectant of choice for freezing embryos because of its low

molecular weight, which enables rapid influx into cells during equilibration and rapid efflux after

thawing (Bautista et al., 1997). Similar effects of ethylene glycol occurs across plasma

membrane of sperm cells (Hammerstedt and Graham, 1992). Ethylene glycol at 2.5%

concentration in TEST extender effectively protected ram semen stored for 3 h at 4° C compared

with DMSO, propylene glycol, dimethylacetamide, and glycerol at 2.5, 5, and 10 %

concentrations (Vasquez et al., 2013). Inclusion of 1.6% EG in TEST extender was beneficial for

68

extension and storage of Chincila lanigera semen at 4° C for 72 h. Use of EG for extension and

chill storage of ram semen is not available. In the current study, addition of 1% EG in the

extender for extension and storage of ram semen at 4° C for 72 h increased most sperm motility

parameters compared with no EG addition. An EG × hour interaction also occurred in most

sperm motility parameters. Such interactions are due to greater reduction of sperm motility

parameters at 72 h of storage in extenders without EG.

Conception rate was compared using fresh or cool stored ram semen during August in

Midwestern US. Semen was extended using milk extender (with 20% egg yolk and 1% EG) and

inseminated immediately after extension (n = 27) or stored at 4° C for up to 36 h before

insemination (n = 28). The conception rate in ewes inseminated using stored semen was 31%

while fresh extension and insemination resulted 37% conception rate.

Penicillamine, hypotaurine, and epinephrine have different modes of action but

collectively enhance sperm motility, sperm capacitation, and acrosomal reaction (Meizel, 1985).

Addition of PHE in the medium decreased time interval for oocyte penetration by sperm (Susko-

Parrish, et al., 1990). Frozen thawed and cultured bull semen in PHE-added medium increased

most sperm motility parameters compared with no PHE (Pearson et al., 2007). Studies reporting

the effects on PHE on sperm motility parameters of extended ram semen are rare. In the current

study, addition of PHE before semen analysis increased progressive motility and BCF of sperm

cells and tended to increase total motility and STR of sperm cells. In a separate study, we

compared effect of addition or no addition of PHE (40 µl PHE/ 1000 µl extender) in milk

extender before AI. A total of 27 ewes were artificially inseminated with extended, and cold

stored ram semen for 12 to 24 h at 4° C. The PHE was added in extended semen 5 min before AI

69

in PHE group (n = 14) or no PHE addition in control group (n = 13). The conception rate in the

PHE added group was 50% while only 23% conceived in the no PHE group.

In conclusion, milk extender maintained better sperm motility compared with TRIS after

storage at 4° C for up to 72 h. Percentage of egg yolk included in extenders had no effect on

most of the sperm motility parameters. However, inclusion of 20% egg yolk in milk extender led

to a higher VCL and VAP followed by TRIS + 20% egg yolk, TRIS + 5% egg yolk, and milk +

5% egg yolk. At 72 h of storage, sperm cell motility declined sharply in extenders without EG.

Results indicate that milk extender supplemented with 1% EG, 20% egg yolk before storage, and

addition of PHE before analysis is beneficial for liquid storage of ram semen.

70

References

Acharya, M., J. M. Burke, E. Smyth, A. Davis, T. D. Lester, and R. Rorie. 2016. Effect of semen

extender and storage temperature on ram sperm motility over time. J. Anim. Sci.

94(suppl. 1): 53 (Abstr.).

Ball, B. A., A. T. Vo, and J. Baumber. 2001. Generation of reactive oxygen species by equine

spermatozoa. Am. J. Vet. Res. 62:508-515.

Bautista, J. A. N., Takahashi, Y., and H. Kanagawa. 1997. In vitro viability of mouse 8-cell

embryos vitrified in a simple solution of ethylene glycol. Jpn. J. Vet. Res. 45:67-73.

Camara, D. R., M. M. C. Mello-Pinto, L. C. Pinto, O. O. Brasil, J. F. Nunes, and M. M. P.

Guerra. 2011. Effect of reduced glutathione and catalase on the kinematics and

membrane functionality of sperm during liquid storage of ram semen. Small Rum. Res.

100: 44-49.

Desnoyers, L., and P. Manjunath. 1992. Major proteins of bovine seminal plasma exhibit novel

interactions with phospholipid. J. Biol. Chem. 267:10149-10155.

Gao D. Y., P. Mazur, and J. K. Critser. 1997. Fundamental cryobiology of mammalian

spermatozoa. In: Karow A. M., J. K. Critser (eds.) Reproductive tissue banking.

Academic press, San Diego pp 263-327.

Gil, J., S. Fierro, O. Bentancur, and J. Olivera‐Muzante. 2011. Chilled storage of ram semen

improves with the addition of egg yolk and glycerol to milk‐based extenders. Reprod.

Domest. Anim. 46:503-507.

Hammerstedt, R. H., and J. K. Graham. 1992. Cryopreservation of poultry sperm: the enigma of

glycerol. 29:26-38.

Hollinshead, F. K., J. K. O’Brien, L. Gillan, M. Meyers, W. Maxwell, and G. Evans. 2004.

Liquid storage of flow cytometrically sorted ram spermatozoa. Theriogenology. 62:587-

605.

Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger. 1996. SAS system for Mixed

Models. SAS Institute Inc., Cary, NC. 633 pp.


diluted and stored in three different extenders. Acta Vet. Scand. 40:1-9.

MacDonald, B., and J. Foulkes. 1981. A spectrofluorometric investigation, using 1-anilino-

naphthalene-8-sulphonate, of the interaction between washed bovine spermatozoa and

seminal plasma or egg-yolk lipoprotein. J. Reprod. Fertil. 63:407-414.

71

Manjunath, P., L. Baillargeon, Y. L. Marcel, N. G. Seidah, M. Chrétien, and A. Chapdelaine.

1988. Diversity of novel proteins in gonadal fluids. In M. Chrétien., K. W. McKerns

(Eds.), Molecular biology of brain and endocrine peptidergic system. Springer US. pp.

259-273.

Meizel, S. 1985. Molecules that initiate or help stimulate the acrosome reaction by their

interaction with the mammalian sperm surface. Dev. Dynam. 174:285-302.

Miller, G. F., D. W. Gliedt, J. M. Rakes, and R. W. Rorie. 1994. Addition of penicillamine,

hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or

in combination to bovine in vitro fertilization medium increases the subsequent embryo

cleavage rate. Theriogenology. 41:689-696.

Pace, M., and E. Graham. 1974. Components in egg yolk which protect bovine spermatozoa

during freezing. J. Anim. Sci. 39:1144-1149.

Person, C. N., T. D. Lester, M. D. Person, and R. W. Rorie. 2007. Effects of penicillamine,

hypotaurine and epinephrine (PHE) on post-thaw bovine sperm parameters, as measured

by computer-assisted sperm analysis. AAES Research Series. 553:47-50.

Quan, G. B., G. Q. Wu, Y. J. Wang, D. J. Li, Y. Ma, Q. H. Hong. 2016. Effects of the Tris, Tes,

or skim milk based extender on in vitro parameters of ram spermatozoa during liquid

storage. Small Rumin. Res. 134:14-21.

Rajabi-Toustani, R., M. Roostaei-Ali, and R. Motamedi-Mojdehi. 2014. Effect of different levels

of egg yolk on ram sperm coating and preserving at 5° C. Iran. J. Vet. Res. 15:168-171.

Ricker, J. V., J. J. Linfor, W. J. Delfino, P. Kysar, E. L. Scholtz, F. Tablin, J. H. Crowe, B. A.

Ball, and S. A. Meyers. 2006. Equine sperm membrane phase behavior: The effects of

lipid-based cryoprotectants 1. Biol. reprod. 74:359-365.

Salamon, S., and W. M. C. Maxwell. 1995. Frozen storage of ram semen. I. Processing, freezing,



111.

Susko-Parrish, J. L., M. B. Wheeler, R. L. Ax, N. L. First, and J. J. Parrish. 1990. The effect of

penicillamine, hypotaurine, epinephrine and sodium metabisulfite, on bovine in vitro

fertilization. Theriogenology. 33:333 (Abstr.).

Vásquez, J. H., V. H. Núñez, E. A. Florentini, J. M. Gonzales, L. A. Camargo, and M. E.

Valdivia. 2013. Effects of five cryoprotective agents on quality of sheep epididymal

spermatozoa during pre-freezing. Livest. Sci. 152: 94-9

72

Table 1. IVOS machine settings for ram semen motility analysis

Parameter Setting

Frames per second 60

Number of frames 30

Minimum contrast 60

Minimum size 5

Default cell size 5

Default cell intensity 55

Path velocity (VAP) 75.0

Straightness (STR) 80

Slow cells Static

VAP cut off 21.9

VSL cut off 6.0

Minimum intensity gate 0.25

Maximum intensity gate 1.50

Minimum size gate 0.60

Maximum size gate 8.00

Minimum elongation gate 0

Maximum elongation gate 95

UV fluorescent objective 10×

Target photometer 45-55

Field type Dark

Chamber depth 20

73

Table 2. CASA parameters

Total motility (%) Path velocity ≥ 30 µm/sec and progressive velocity ≥ 15

µm/sec

Progressive motility (%) Path velocity ≥ 50 µm/sec and straightness ≥ 70%

Track speed (VCL, µm/sec) Average velocity measured over the actual point-to-point

track.

Path velocity (VAP, µm/sec) Average velocity of the smoothed (best fit) sperm path.

Progressive velocity (VSL,

µm/sec)

Average velocity over straight line from the beginning to the

end of the sperm track.

Beat frequency (BCF, MHz) Frequency of sperm head crossing the sperm average path in

either direction.

Lateral amplitude (ALH, µm) Mean width of the head oscillation as the sperm swims.

Linearity (LIN, %) Departure of actual sperm path from straight line

(VSL/VCL).

Straightness (STR, %) Departure of average sperm path from a straight line

(VSL/VAP).

74

Table 3. Sperm motility parameters after extension using milk or TRIS and stored at 4° C for 72

h.

Results are expressed as least square means and standard error (SE).

Significant difference was declared at P < 0.05.

Extenders

Sperm characteristics Milk TRIS SE P-value

Total motility 49.7 43.6 1.3 0.0013

Progressive motility 24.5 16.6 0.7 < 0.0001

Track speed 185.6 182.5 4.2 0.60

Path velocity 104.7 95.1 1.9 0.0006

Progressive velocity 83.3 70.6 1.4 < 0.0001

Beat frequency 32.9 30.3 0.6 0.002

Lateral amplitude 6.8 7.4 0.2 0.007

Linearity 45.9 41.6 0.9 0.0009

Straightness 75.5 72.5 1.2 0.07

75

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.



Egg yolk percentage

Sperm characteristics 5 % 20 % SE P-value

Total motility 45.9 47.3 1.3 0.463

Progressive motility 20.3 20.7 0.7 0.721

Track speed 180.3 187.9 4.3 0.208

Path velocity 97.8 102.6 1.9 0.124

Progressive velocity 75.6 78.4 1.4 0.163

Beat frequency 31.4 31.8 0.6 0.641


Linearity 42.9 44.6 0.9 0.185

Straightness 73.1 75.1 1.2 0.231

76

Table 5. Sperm motility parameters after extending with or without ethylene glycol in milk or

TRIS and stored at 4° C for 72 h.



Treatments

Sperm characteristics Ethylene glycol Control SE P-value

Total motility 51.2 42.1 1.3 < 0.0001

Progressive motility 23.1 18.1 0.7 < 0.0001

Track speed 184.5 183.6 4.2 0.873

Path velocity 100.2 99.6 1.9 0.826


Beat frequency 32.5 30.6 0.6 0.029


Linearity 44.3 43.1 0.9 0.345

Straightness 75.5 72.5 1.17 0.06

77

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.



Treatments

Sperm characteristics PHE Control SE P-value

Total motility 48.4 44.8 1.3 0.058

Progressive motility 22.4 18.7 0.7 0.0007

Track speed 179.8 188.3 4.2 0.161

Path velocity 98.4 101.4 1.9 0.276


Beat frequency 32.9 30.2 0.6 0.001


Linearity 44.5 43.1 0.9 0.267

Straightness 75.5 72.5 1.1 0.078

78

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 are expressed.

Results are expressed as least square means and standard error (SE). abValues within the same row followed by different superscripts are different (P < 0.05). xyzValues within the same column with unlike superscripts differ (P < 0.05).

Parameters Hour Ethylene glycol Control SE

Total motility 24 55.4a,x 50.9a,x 2.3

48 48.6a,y 43.4a,y 2.3

72 49.6a,xy 31.7b,z 2.3

Progressive motility 24 26.1a,x 23.1a,x 1.3

48 21.5a,y 18.7a,y 1.3

72 21.5a,y 12.6b,z 1.3

Track speed 24 186a,x 197a,x 7.4

48 182a,x 191a,x 7.4

72 185a,x 162b,y 7.4

Path velocity 24 103.3a,x 109.1a,x 3.3

48 98.4a,x 104.5a,x 3.3

72 99.1a,x 85.2b,y 3.3

Progressive velocity 24 80.3a,x 84.1a,x 2.4

48 75.7a,x 80.2a,x 2.4

72 76.4a,x 65.1b,y 2.4

Linearity 24 46.5a,x 47.2a,x 1.5

48 43.8a,x 45.2a,x 1.5

72 42.8a,x 37.1b,y 1.5

Straightness 24 76.5a,x 77.1a,x 2.1

48 75.1a,x 74.6a,x 2.1

72 72.1a,x 65.7b,y 2.1

79

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 are expressed.

abcValues within the same row with unlike superscripts differ (P < 0.05).

Parameters Milk Milk + EG TRIS TRIS + EG SE

Progressive motility 23.8ab 25.9a 13.2c 20.1b 1.1

Progressive velocity 80.8a 85.7a 67.1c 74.1b 2.1

Beat frequency 33.1a 32.9a 28.3b 32.2a 0.8

Linearity 47.1a 44.7a 39.3b 44.1a 1.5

Straightness 76.7a 74.3a 68.2b 76.8a 2.3

80

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 are expressed.

abValues within the same row with unlike superscripts differ (P < 0.05).

Parameters Milk, 5% EY Milk, 20% EY TRIS, 5% EY TRIS 20% EY SE

Track speed 176.3b 195.1a 184.2ab 180.8ab 7.4

Path velocity 99.9b 109.4a 95.6b 94.6b 2.7

81

Chapter V: Correlation between anti-Mullerian hormone and estimated breeding values

for reproductive performance in ewes

Abstract

Anti-Mullerian hormone (AMH) in circulation may reflect the total pool of follicles

within the ovaries, and may be positively correlated with fertility in ewes. The objective of this

study was to investigate any correlation between serum AMH concentrations, number of lambs

born, and estimated breeding values determined through the 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, AMH quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4

the highest, pg/ml) were correlated with mean number of lambs born after fall versus summer

breeding. Blood samples were collected for serum analysis from 163 Katahdin ewes ranging

from 0.5 to 7-yr of age; their breeding and lambing records were obtained from flock data.

Correlation between AMH concentrations and EBVs were determined using PROC CORR and

means generated using least squares means procedure of SAS (SAS Inst. Inc., Cary, NC, USA).

For remaining data, continuous variables were analyzed by PROC GLM and categorical

variables were analyzed by chi-squared, using SAS. Mean serum AMH was 182 pg/ml and

ranged from 0 to 1112 pg/ml. There was no correlation between serum AMH and EBVs for NLB

(-0.04; P = 0.589), NLW (0.06; P = 0.401), MWWT (-0.01; P = 0.872), WWT (0.003; P =

0.964) and the Katahdin index (0.09; P = 0.251). Serum AMH concentration was similar in ewes

in different age category (less than 1, 1 to 2, 2 to 3, 3 to 4, and more than 4-yr of age; P > 0.05).

There was a correlation between EBV for NLB (0.29; P < 0.01) and NLW (0.19; P = 0.013)

with average number of lambs born. Ewes in a lowest AMH quartiles (Q1) had a lower mean

number of offspring born from summer breeding compared with ewes in other AMH quartiles

82

(Q2, Q3, and Q4; P < 0.05). Further study is needed to determine effectiveness of using serum

AMH for selecting ewes for summer (off-season) breeding.

Keywords: Anti-Mullerian hormone, ewes, estimated breeding values, reproductive performance

Introduction

Anti-Mullerian hormone (AMH) is a dimeric glycoprotein that belongs to a member of

the transforming growth factor-β (TGF-β) family (Cate et al. 1986). This hormone is produced

by granulosa cells of preantral and small antral follicles of 1-3 mm diameter (Bezard et al., 1987;

Monniaux et al. 2012). Anti-Mullerian hormone facilitates follicle recruitment and growth of

young follicles (Broer et al. 2014). However, when a follicle increases in size, AMH limits the

number of follicles developing from the gonadotropin responsive stage to the gonadotropin

dependent stage (Campbell, et al., 2012). Although AMH limits the number of ovulatory

follicles, studies have positively correlated plasma AMH with fertility in animals, including

sheep (Lahoz et al., 2012; Lahoz et al., 2016 a, b).

Individual ewes vary in reproductive performance. In the Katahdin breed, some ewe

lambs can lamb at 12-months of age, while others lamb later or may not lamb during their entire

life. Ewes also differ in their prolificacy ranging from singles to quadruplets or more

(Vanimisetti, et al., 2007). Similarly, their ability to conceive out-of-season also varies (Burke,

2005). Estimated breeding values (EBVs) are the mathematical predictions of the genetic merit

of an animal for specific traits. These values are calculated based on an individual animal’s

performance for a specific trait compared with animals within a contemporary group and animals

in other connected flocks. Estimated breeding values are useful to select for specific traits used in

breeding or commercial stock (www.nsip.org).

http://www.nsip.org/

83

Studies correlating serum AMH concentration with EBV 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 have not been reported

in ewes. Therefore, this study investigated any correlations between serum AMH, mean number

of lambs born, and EBV for NLB, NLW, MWWT, WWT, and the Katahdin index.


Location

The experiment was conducted at USDA, Agriculture Research Service (ARS), Dale

Bumpers Small Farms Research Center (DBSFRC) in Booneville, AR, USA. All animal


Animal management

Katahdin ewes were raised at USDA-ARS, DBSFRC in Booneville. A total of 163 ewes

of 0.5 to more than 7-yr of age were used in the study. All the ewes were maintained in a mixed

pasture of tall fescue (Schedonorus arundinacea) and bermudagrass (Cynodon dactylon) and

were supplemented with ewe ration (17% CP, 82% TDN on DM basis). Breeding and lambing

records of the flock were entered into National Sheep Improvement Program (NSIP) between

2001 and present to generate EBV. The EBVs of Katahdin hair sheep were obtained from NSIP

(www.nsip.org) and included were MWWT, WWT, NLB, NLW, and a Katahdin index.

Blood sampling and serum collection

Blood samples were collected July 2015 by jugular venipuncture into 10 ml BD

Vacutainer SS Plus Blood Collection Tubes (Becton, Dickson, Franklin Lakes, NJ). Blood tubes

were inverted to mix several times and allowed to clot for 30 min at room temperature. After

clotting, the tubes were held on ice until centrifuged (Beckman Coulter T J6 refrigerated

http://www.nsip.org/

84

centrifuge, Fullerton, CA) at 2500 × g for 25 min. Serum was collected and stored in 5 ml

polypropylene tubes at -20° C until analysis.

Anti-Mullerian hormone analysis

Equine and ovine AMH ELISA used in the study is a quantitative three-step sandwich

type of immunoassay (AnshLabs, Webster, TX). Analysis was performed as described by the

manufacturer (www.anshlabs.com). Before analysis, all the kit reagents were warmed to room

temperature and serum samples were thawed overnight at 4° C. Solutions were mixed as

recommended. Calibrators were serially diluted from 13.6 ng/ml to 0.05 ng/ml of AMH. Fifty

microliter of duplicate calibrators, control and serum samples were added to appropriate wells of

the 96-well assay plate. Fifty microliter of AMH assay buffer was added to each well using a

repeater pipette. Plate was shaken at 700 rpm on an orbital microplate shaker (Lab-Line

Instrument Inc. Melrose Park, IL) for 2 h at room temperature. Plate was removed and washed 5

times with wash solution in microplate strip washer (ELP40 Bio-Tek Instruments, New York,

NY). Antibody-Biotin Conjugate RTU (100 µl) was added using a repeater pipette, incubated at

700 rpm on orbital microplate shaker (Lab-Line Instrument Inc. Melrose Park, IL) for 1 h and

washed 5 times with wash solution using the microplate strip washer. Streptavidin-enzyme

conjugate-RTU (100 µl) was added after washing, incubated in the orbital microplate shaker at

700 rpm for 30 min, and washed 5 times using microplate washer. TBM chromogen (100 µl) was

added to each well using a repeated pipette and incubated in orbital microplate shaker at 700 rpm

for 20 min. Finally, 100 µl of stopping solution was added to each well and absorbance was read

using a model 1420-040 Victor3 Multilabel Counter (Perkin Elmer, USA) at 450 nm.

85

Description of animals and categories

A total of 163 ewes aged 0.7 to more than 7-years was categorized into 5 groups: less

than 1 (n = 17), 1 to 2 (n = 57), 2 to 3 (n = 23), 3 to 4 (n = 34), and more than 4-yr of age (n =

32). Lambing data from 2010 to 2015 for ewes used in the current study were obtained from

USDA-ARS sheep flock at Booneville, AR. Ewes were bred either during summer or during

winter. Ewes from first to sixth parity are included and any lambing record from seventh parity

or above are excluded.


Regression analysis (bivariate fit) was used to determine the relationship between

absorbance reading and standard concentration of AMH. The resulting regression equation was

used to calculate AMH concentration in each unknown sample within the appropriate assay

plate. Pearson correlation coefficients between AMH and EBV for NLB, NLW, MWWT, WWT,

and Katahdin index were analyzed using the PROC CORR procedure of SAS (version 9.4; SAS

Inst. Inc., Cary, NC, USA). For remaining data, continuous variables were analyzed by PROC

GLM and categorical variables were analyzed by chi-squared using SAS. For categorical data

AMH concentrations were divided into four quartiles: Q1 = 0 – 80 pg/ml, Q2 = 81 – 104 pg/ml,

Q3 = 105 – 248 pg/ml, and Q = 249 – 1112 pg/ml. Data were expressed as least squares means

and standard error. Means were compared using Tukey’s test.

Results

Three microplates were used to run 163 serum samples. The regression equation (R2 =

0.998) used to determine AMH concentration in the first plate was: AMH ng/ml = - 0.034853 +

0.5789461 × absorbance. For plate 2, the regression equation (R2 = 0.99) used to determine

AMH concentration ng/ml serum was = - 0.5929 + 3.9394 × absorbance, and for the third plate,

86

regression equation (R2 = 0.99) used to determine AMH concentration ng/ml was = - 0.059384 +

2.2414964 × absorbance. Duplicate samples within plates were used to calculate intra-assay

variability, while standard curves and control samples in each plates were used to calculate inter-

assay variability. Intra-assay variability of AMH assays ranged from 6.6 to 8.3%. Inter-assay

variability between standards was 4.3%. Serum AMH ranged from 0.00 to 1112 pg/ml, with a

means of 182 pg/ml.

Correlation of EBVs for reproductive performance with serum AMH

Mean NLB, NLW, MWWT, WWT, and Katahdin index values were 0.056 ± 0.09 %,

0.117 ± 0.07 %, 0.365 ± 0.60 lb, 0.641 ± 0.73 lb, and 105 ± 3.14 lb, respectively. There was no

correlation between serum AMH concentrations and EBV for NLB (-0.02; P = 0.793), NLW (-

0.09; P = 0.252), MWWT (-0.02; P = 0.712), WWT (-0.02; P = 0.757), and Katahdin index

(0.072; P = 0.355).

Correlation between EBVs with average number of lambs born

Average number of lambs born were significantly correlated with EBV for NLB (0.29; P

= 0.0002) and NLW (0.19; P = 0.013). However, EBV for WWT (-0.09; P = 0.242), MWWT (-

0.11; P = 0.138), and Katahdin index (0.04; P = 0.547) were not correlated with average number

of lambs born.

Relationship between AMH quartiles and mean number of lambs born from different summer and

fall breeding

The distribution of AMH concentrations was divided into quartiles (AMH Q1 though Q4,

with Q1 the lowest and Q4 the highest pg/ml) for analysis. Anti-Mullerian quartiles with their

concentration range and mean number of lambs born is given Table 1. There was no correlation

between AMH quartiles with mean number of lambs born from fall breeding. However, there

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was a significant correlation between mean number of lambs born from summer breeding with

AMH quartiles. Ewes in lowest quartile (Q1) had lower number of lambs born from summer

breeding than ewes in other quartiles (Q2, Q3, Q4; P < 0.05).

Influence of ewe age in serum AMH level

Mean AMH concentration in less than 1, 1 to 2, 2 to 3, 3 to 4, and more than 4-year age

was 129 ± 20, 148 ± 10, 159 ± 20, 195 ± 10, 166 ± 10 pg/ml respectively. There was no

difference in serum AMH concentration among age categories (P = 0.157).

Pregnancy and lambing rate of exposed ewes between breeding seasons

Overall pregnancy rate was higher (P < 0.01) in fall bred ewes (78.99%) compared with

summer bred (48.01%). Mean number of lambs born from fall breeding (1.44 ± 0.05) was higher

(P < 0.01) compared with mean number of lambs born from summer breeding (0.78 ± 0.05) per

ewes used for breeding.

Lambing rate per parity

Lambing rate was highest in third parity ewes and lowest in first parity ewes (P < 0.05).

Mean numbers of lambs born in first, second, third, fourth, fifth, and sixth parity are 0.81, 1.06,

1.52, 1.34, 1.09, and 1.33 respectively.

Discussion

Estimated breeding values (EBVs) are the mathematical predictions of the genetic merit

of an animal for specific traits. These values are calculated based on an individual animal’s

performance for a specific trait compared with animals within a contemporary group and animals

in other connected flocks, and also takes into account of progeny and relatives. An expected

progeny difference (EPD) on the other hand is predictor of genetic merit of an animal’s progeny.

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Mathematically EBV is twice the value of EPD. In the US cattle industry, EPD is more common,

whereas EBV are more common outside US.

The mathematical calculation process for EBV is a very complex process. Data from an

individual lamb is entered into a computerized database. At first, non-genetic and phenotypic

parameters such as number of lambs born, age of lamb, age of dam, weaning weight and more

are adjusted to remove non-genetic effect of performance. Individual performance thus obtained

is compared with average production of a group of contemporary animals. After this, probability

of gene sharing is calculated. For this, an individual’s measurement is compared to the

measurement taken from its relatives. All the records are expressed in relation to their

contemporary groups in order to remove non-genetic difference in average performance. At last,

the EBV calculated is adjusted for the heritability of each trait. The resulting EBV is the best

estimate of the true genetic merit of any individual for a specific genetic trait measured

(www.nsip.org). The accuracy of EBV increases with an increase in data. For example, accuracy

of EBV for a breeding ram with 300 offspring in 5 different flocks will be higher than a ram with

20 offspring in one flock. The EBVs also improves by using a ram in multiple flocks. It is

imperative that a flock use at least two sires within a lambing season and at least 20 off spring

per sire (fewer are accepted within NSIP).

The EBV for Number of lambs born measures genetic potential for prolificacy. The NLB

EBV are expressed in percentage. Average number of lambs born per parity in the Katahdin hair

sheep breed is 2.1. This value is adjusted from all flocks over the country that are enrolled in

NSIP. An ewe with an EBV NLB of 23% means she is expected to lamb 23% more than the

average. Thus, she would be expected to have 2.1 + 0.48 lambs. The 2.1 is the average and 0.48

is 2.1 × 0.23.

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Number of lambs weaned EBV measures the combined ewe effect on prolificacy and

lamb survival to weaning. The EBV NLW of -5% means a ewe is expected to wean 5% fewer

lambs than the average. If the average number of lambs weaned for Katahdins is 1.9, then she is

expected to wean 1.9 – 0.095 (1.9 × 0.05) lambs per parity or 1.805.

Maternal weaning weight EBV estimates genetic merit for mothering ability. The

MWWT EBV is derived by evaluating lambs that are heavier or lighter than expected based on

WWT EBV of the parents. This is mostly due to increased amount and quality of milk. Ewes

whose lamb grows faster than predicted are assumed to be better milk producers and vice-versa.

The MWWT EBV values are expressed in pound (in US).

Weaning weight EBV estimates preweaning growth potential. Preweaning weights are

taken at 45 to 90 d of age. Katahdin lambs are generally weaned at 90 d of age. The WWT EBV

values are expressed in pounds (in US). Suppose if average 90 d weaning weight of Katahdin

hair sheep is 60 lb; then WWT EBV of +1 of a lamb refers to potential weaning weight of 61 lb.

Katahdin index gives positive emphasis on maternal weaning weight, weaning weight,

and number of lambs weaned EBVs. Also gives small negative emphasis on number of lambs

born. Katahdin hair index = 0.246 × WWT EBV + 2.226 × MWWT EBV + 0.406 × NLW EBV

– 0.035 × NLB EBV. It predicts genetic value for total pounds of lamb marketed by a ewe (in

US). Katahdin hair index of 99 refers to 1 lb below the average whereas 101 refers to 1 lb above

the average.

In terms of link between AMH and EBVs, NLB is probably most directly related as it is

the closest estimate of pregnancy/lambing rate. The EBVs, NLW and MWWT are related to

mothering ability, which physiologically is related to functional hormones post-lambing such as

prolactin. However, EBVs for NLB and NWL are highly correlated. In the current study, EBVs

90

for NLB as well as NLW were correlated with number of lambs. Other EBVs, WWT, MWWT,

and Katahdin index were not correlated with number of lambs born.

In the current study, there was no correlation between serum AMH concentrations and

any of the EBVs. Ewes in the flock were selected for parasite resistance first, then maternal traits

(maternal milk and number of lambs weaned) for several years, but there was still a wide range

of EBV for maternal traits. Accuracy of some of these traits, especially in younger ewes may be

low enough to impact correlations. Other measures of fertility such as lifetime production after

accounting for environmental factors would be ideal but impractical.

Anti-Mullerian hormone is high during the fetal stage compared with postnatal

concentrations (Bezard et al., 1987). Similarly, plasma AMH concentrations in 40 d old ewes is

higher than 3.6-month old ewes (Lahoz et al., 2012; Torres-Rovira et al., 2014). Lahoz found a

vast difference between 1 and 5-month-old ewes, decreasing with age (Lahoz, et al., 2016b).

Serum AMH concentrations of ewes after puberty, at different reproductive age period has not

been reported. In the current study, serum AMH concentrations was similar in different age (less

than one, 1-2, 2-3, 3-4, and more than 4 yr) ewes. No difference in serum AMH concentrations in

different age mature ewes is similar to that reported in cattle (Rico et al., 2009; Ireland et al.,

2010).

Ovarian follicular population, an indicator for fertility is positively correlated with

plasma AMH concentration in young lambs (Lahoz et al., 2016b). Correlation between plasma

AMH concentrations during the prepubertal period and occurrence of ovulation in response to

eCG was reported (Lahoz et al., 2012). Ewes with a high plasma AMH (> 97 pg/ml) had higher

number of lambs born per ewe per year till 3.5 yr age compared with low (< 97 pg/ml) plasma

AMH concentrations measured at 3.5 months of age (Lahoz et al., 2016 a). When ewes were

91

compared by quartiles, based on serum AMH measurement after puberty, AMH hormone

concentration had no effect on mean number of lambs born. Similar results were obtained when

ewes were compared by quartiles, based on serum AMH measurement after puberty with mean

number of lambs born from fall breeding. However, ewes in lowest quartiles (Q1) had lower

number of lambs born from summer breeding than ewes in other quartiles. Low reproductive

performance of ewes in lowest quartiles (Q1) during summer in the current study is similar to the

result we recently reported in cattle (Rorie et al., 2016). In our previous study, we reported AMH

concentration at breeding, heifers in lowest quartiles (Q1) had lower AI pregnancy rate and

conceived at a later estrus cycle in breeding compared with heifers in other quartiles. Anti-

Mullerian hormone measurement after puberty could be an option for selection of ewes for

summer breeding in the Southern US.

In conclusion, serum AMH concentration were similar among age group. There was a

correlation between EBV for NLB and NLW with average number of lambs born. Ewes in the

lowest quartile (Q1) had lower number of offsprings born from summer breeding compared with

ewes in other AMH quartiles (Q2, Q3, and Q4). However, AMH was not useful in predicting

fertility or maternal traits based on EBV of the ewes.

92

References

Bézard, J., B. Vigier, D. Tran, P. Mauleon, and Nathalie Josso. 1988. Anti-Müllerian hormone in

sheep follicles. Reprod. Nutr. Dev. 28:1105-1112.

Broer, S. L., F. J. M. Broekmans, J. S. E. Laven, and B. C. J. M. Fauser. 2014. Anti-Müllerian

hormone: ovarian reserve testing and its potential clinical implications. Hum. Reprod.

Update. 20:688-701.

Burke, J. 2005. Lamb production of Dorper, Katahdin, and St. Croix bred in summer, winter, or

spring in the Southeastern US. Sheep Goat Res. J. 20:51-59.

Campbell, B. K., M. Clinton, and R Webb. 2012. The role of anti-Müllerian hormone (AMH)

during follicle development in a monovulatory species (sheep). Endocrinology 153:4533-

4543.

Cate, R. L., R. J. Mattaliano, C. Hession, R. Tizard, N. M. Farber, A. Cheung, E. G. Ninfa, A. Z.

Frey, D. J. Gash, E. P. Chow, R. A. Fisher, J. M. Bertonis, G. Torres, B. P. Wallner, K. L.

Ramachandran, R. C. Ragin, T. F. Manganaro, D. T. MacLaughlin, and P. K. Donahoe.

1986. Isolation of the bovine and human genes for müllerian inhibiting substance and

expression of the human gene in animal cells. Cell 45:685-698.

Fahmy, M. H. 1986. Preliminary results on fertility, prolificacy, lamb production and carcass

traits of Romanov sheep in Canada. Proc. 3rd World Congr. Genet. Appl. Livest. Prod.

IX:559-564. Lincoln, NE.

Ireland, J. J., G. W. Smith, D. Scheetz, F. Jimenez-Krassel, J. K. Folger, J. L. H. Ireland, F.

Mossa, P. Lonergan, and A. C. O. Evans. 2010. Does size matter in females? An

overview of the impact of the high variation in the ovarian reserve on ovarian function

and fertility, utility of Anti-Mullerian hormone as a diagnostic marker for fertility and

causes of variation in the ovarian reserve in cattle. Reprod. Fertil. dev. 23:1-14.

La Marca, A., V. Grisendi, and G. Griesinger. 2013. How much does AMH really vary in normal

women? Int. J. Endocrinol. 2013:1-8.

Lahoz, B., J. L. Alabart, and J. Folch. 2016a. Relationship between Anti-Müllerian hormone

(AMH) and the ovulatory response measured in three-month old ewe lambs with their

reproductive efficiency in adulthood. Asoc. Interprofes. Para el Des. Agr. 112:392-404.

Lahoz, B., J. L. Alabart, D. Monniaux, P. Mermillod, and J. Folch. 2012. Anti-Mullerian

hormone plasma concentration in prepubertal ewe lambs as a predictor of their fertility at

a young age. BMC Vet. Res. 8:2-9.

Lahoz, B., J. L. Alabart, M. J. Cocero, D. Monniaux, E. Echegoyen, P. Sánchez, and J. Folch.

2014. Anti-Müllerian hormone concentration in sheep and its dependence of age and

93

independence of BMP15 genotype: an endocrine predictor to select the best donors for

embryo biotechnologies. Theriogenology. 81:347-357.

Lahoz, B., S. Fabre, J. H. Calvo, L. Drouilhet, P. Sanchez, P. Sarto, E. Echegoyen, J. Folch, D.

Monniaux, J. L. Alabart. 2016b. Assessing the age-related relationship between plasma

Anti-Mullerian hormone (AMH) and ovarian follicle population in prepubertal ewes.

Published online https://core.ac.uk/download/pdf/71402615.pdf. Accessed on 4th July

2017.

Monniaux, D., L. Drouilhet, C. Rico, A. Estienne, P. Jarrier, J. Touzé, J. Sapa, F. Phocas, J.

Dupont, R. Dalbiès-Tran, and S. Fabre. 2012. Regulation of anti-Müllerian hormone

production in domestic animals. Reprod. Fertil. Dev. 25:1-16.

Rico, C., S. Fabre, C. Médigue, N. di Clemente, F. Clément, M. Bontoux, J. L. Touze, M.

Dupont, E. Briant, B. Remy, J. F. Beckers, and D. Monniaux. 2009. Anti-müllerian

hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can

help to predict superovulatory responses in the cow. Biol. Reprod. 80:50-59.

Rorie, R., H. Newberry, T. D. Lester, M. Acharya, and C. Hansen. 2016. Anti-müllerian

hormone at weaning and breeding as a predictor of beef heifer fertility. Reprod. Fertl.

Dev. 29:111-112 (Abstr.).

Scheetz, D. 2010. Regulation and role of Anti-Mullerian hormone in bovine reproduction

(Doctoral dissertation). Retrieved from

http://search.proquest.com/openview/9e2ae630eee668c494892306d4d53f0f/1?pq-

origsite=gscholar&cbl=18750&diss=y. Accessed on 4th July 2017.

The Baalands. 2017. Katahdin frequently asked question. Available online.

http://www.baalands.com/#!katahdinfaqs/cwoc. Accessed on 24th April 2017.

Torres-Rovira, L., A. Gonzalez-Bulnes, S. Succu, A. Spezzigu, M. E. Manca, G. G. Leoni, G. G.,

M. Sanna, S. Pirino, M. Gallus, S. Naitana, F. Berlinguer. 2014. Predictive value of antral

follicle count and anti-Müllerian hormone for follicle and oocyte developmental

competence during the early prepubertal period in a sheep model. Reprod. Fertil. Dev.

26:1094-1106.

Vanimisetti, H. B., D. R. Notter, and L. A. Kuehn. 2007. Genetic (co) variance components for

ewe productivity traits in Katahdin sheep. J. Anim. Sci. 85:60-80.

https://core.ac.uk/download/pdf/71402615.pdf

http://search.proquest.com/openview/9e2ae630eee668c494892306d4d53f0f/1?pq-origsite=gscholar&cbl=18750&diss=y

http://search.proquest.com/openview/9e2ae630eee668c494892306d4d53f0f/1?pq-origsite=gscholar&cbl=18750&diss=y

http://www.baalands.com/#!katahdinfaqs/cwoc

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Table 1. Relationship between AMH quartiles and mean number of lambs born from different

summer and fall breeding. Data expressed in mean ± standard error (SE).

AMH Quartiles AMH range (pg/ml) Fall Summer Overall

AMHQ 1 0 – 80 1.48 ± 0.11 0.41 ± 0.12b 0.65 ± 0.10

AMHQ 2 81 – 104 1.54 ± 0.11 0.92 ± 0.11a 0.81 ± 0.11

AMHQ 3 105 – 248 1.38 ± 0.11 0.89 ± 0.10a 0.87 ± 0.10

AMHQ 4 249 – 1112 1.43 ± 0.10 0.90 ± 0.10a 0.71 ± 0.11

Means with different superscripts in the same column differ (P < 0.05).

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Chapter VI: Conclusions

Most of the CASA motility parameters were significantly higher after extension and

storage at 4 compared with 15° C. Ram semen stored using milk or TRIS based extenders at 4° C

can maintain similar motility parameters but better compared with TEST or CJ-2 extenders after

storage for up to 3 d. Ram semen stored at 15° C for 3 d using TRIS extender had better sperm

motility parameters compared with milk, TEST or CJ-2 extender. Milk and TEST based

extenders had intermediate efficacy for maintaining motility at 15° C. In contrast, sperm cells

stored in CJ-2 extenders had drastic reduction of most motility parameters after storage at 15° C.

Combined results from both temperatures shows most of the motility parameters were similar for

up to 24 h of storage in all extenders but after that sperm motility parameters declined more

rapidly in milk or CJ-2 extenders compared with TEST or TRIS extender. Such decrease is due

to greater reduction after 24 h using milk and CJ-2 extenders and storage at 15° C. Considering

sperm progressive motility which is also a predictor for in vivo fertility shows milk and TRIS

extenders stored at 4° C has similar but higher progressive motility compared with ram sperm

extended in CJ-2 and TEST extenders and stored at 4° C. However, progressive motility of ram

sperm declined 75% when stored at 4° C for 24 h, and continued to decline over time irrespective

of extenders used. Further study can be done to supplement different supplements to milk or

TRIS based extenders for extension and storage of ram semen at 4° C.

Milk extender maintained better sperm motility compared with TRIS after storage at 4° C

for up to 72 h when different supplements were added. Addition of 5 or 20% egg yolk on

extenders had no difference on most of the sperm motility parameters. However, inclusion of

20% egg yolk in milk extender had highest track speed, and path velocity followed by TRIS +

20% egg yolk, TRIS + 5% egg yolk, and milk + 5% egg yolk, respectively. At 72 h of storage at

96

4° C, sperm cell motility declined sharply in extenders without ethylene glycol (EG) compared

with extenders supplemented with 1% EG. Results indicate that milk extender supplemented

with 1% EG, 20% egg yolk before storage, and addition of PHE before analysis is beneficial for

liquid storage of ram semen.

Serum AMH concentrations was similar in different aged ewes after puberty. There was a

correlation between EBV for NLB and NLW with average number of lambs born. Ewes in the

lowest quartile (Q1) had lower number of offsprings born from summer breeding compared with

ewes in other AMH quartiles (Q2, Q3, and Q4). However, AMH was not useful in predicting

fertility or maternal traits based on EBV of the ewes.

improvement in simple artificial insemination in sheep

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