Clinic for Horses - Unit for Reproductive Medicine of Clinics

University of Veterinary Medicine, Hannover

___________________________________________________________________

Investigations on genital blood flow and embryo recovery

after superovulation with eFSH® and on laparoscopic techniques for flushing

the oviduct in the mare.

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by

Melanie Carola Witt

from Braunschweig

Hannover 2013

Supervisor: Prof. Dr. H. Sieme

Supervision Group: Prof. Dr. H. Sieme

Prof. Dr. S. Meinecke-Tillmann

Prof. Dr. W. Kanitz

1st Evaluation: Prof. Dr. H. Sieme

Clinic for Horses-Unit for Reproductive

Medicine of Clinics

University of Veterinary Medicine Hannover

Prof. Dr. S. Meinecke-Tillmann

Department of Reproductive Biology

University of Veterinary Medicine Hannover

Prof. Dr. W. Kanitz

FBN Leibniz-Institut für Nutztierbiologie

Dummerstorf, Germany

2nd Evaluation: Prof. Dr. Heiner Niemann

FLI Institut für Nutztiergenetik

Mariensee, Germany

Date of final exam: 28.10.2013

Für

Lasse, Lena und Max

und in Erinnerung an

Prof. Erich Klug

„Am Anfang jeder Forschung steht das Staunen.

Plötzlich fällt einem etwas auf.“

Wolfgang Wickler (*1931)

Parts of this thesis have already been published: I Journal articles Witt, M., H. Bollwein, J. Probst, C. Baackmann, E.L. Squires and H. Sieme (2012):

Doppler sonography of the uterine and ovarian arteries during a superovulatory program in horses, Theriogenology 77, 1406–1414

Köllmann, M., A. Rötting, A. Heberling and H. Sieme (2011):

Laparoscopic techniques for investigating the equine oviduct. Equine Vet. J. 43 (1), 106-111

Köllmann, M., J. Probst, C. Baackmann, J. Klewitz, E.S. Squires u. H. Sieme (2008):

Embryogewinnungsrate nach Superovulation mit equinem Hypophysenextrakt (eFSH®) bei der Stute. Pferdeheilkunde 24 (3), 397– 405

II Abstracts and posters Köllmann, M., A. Rötting, A. Heberling u. H. Sieme (2010):

Laparoskopische Technik zur Untersuchung des equinen Eileiters. 21. Arbeitstagung der DVG- Fachgruppe Pferdekrankheiten, 12.-13.03, Hannover ISBN 978-3-939902-62-1

Köllmann, M., A. Rötting, A. Heberling and H. Sieme (2010): Laparoscopic techniques to investigate the equine oviduct. 6th International Conference on Equine Reproduction - What´s New in Equine Reproduction? - Proceedings 5. Leipziger Tierärztekongresss, Leipzig, 21.-23.01.2010, S. 275-277 ISBN 978-3-86583-441-6

Köllmann, M., C. Baackmann, J. Probst, E.L. Squires and H. Sieme (2009):

Luteal and genital blood flow in mares in a superovulation program. 3th Annual Conference of the European Society for Domestic Animal Reproduction – ESDAR, 10.-12.09. 2009, Ghent, Belgium, In: Reproduction in Domestic Animals 44, Suppl.3 ISSN 0936-6768

Köllmann, M., J. Probst, C. Baackmann, J. Klewitz, E.L. Squires and H. Sieme (2008): Embryo recovery rate in mares after treatment with equine follicle stimulating hormone (eFSH®). 41. Jahrestagung Physiologie und Pathologie der Fortpflanzung, 28. - 29.2. 2008, Gießen Reprod. Dom. Anim. 43 (Suppl.), 21

Köllmann, M., J. Probst, C. Baackmann, J. Klewitz, E.L. Squires, u. H. Sieme (2008):

Embryogewinnungsrate nach Superovulation mit equinem Hypophysenextrakt (eFSH®) bei der Stute. 20. Arbeitstagung der DVG- Fachgruppe Pferdekrankheiten, 29.02.-01.03. Hannover Proc. S. 15, ISBN 978-3-939902-62-1

Köllmann, M., J. Probst, C. Baackmann, E.L. Squires and H. Sieme (2008): Embryo recovery after AI with cooled semen 12 and 36 hours after hCG administration in spontaneously ovulating mares and superovulating mares treated with equine pituitary extract (eFSH). 7th Intn. Equine Embryo Transfer Symposium, 09.-11.07.2008, Cambridge, UK, Havemeyer Foundation Abstract book pp.100

Köllmann, M., J. Probst, C. Baackmann, J. Klewitz, E.L. Squires, u. H. Sieme (2008):

Embryogewinnungsrate nach Superovulation mit equinem Hypophysenextrakt (eFSH®) bei der Stute. 35. Jahrestagung der Arbeitsgemeinschaft Embryotransfer Deutschland AET-de, 19./20.06. Dipperz / Friesenhausen

III. Theses Heberling, A. (2010):

Untersuchungen zur Etablierung eines minimal-invasiven chirurgischen Zugangs zum Eileiter der Stute. Hannover, Tierärztl. Hochsch. Diss.

Probst, J. (2009): Untersuchungen zur Superovulation bei der Stute: Einfluss von eFSH® auf Follikelentwicklung und –durchblutung. Hannover, Tierärztl. Hochsch. Diss.

Baackmann, C. (2008): Untersuchungen zur Superovulation bei der Stute: Einfluss von equinem FSH (eFSH®) auf die genitale Durchblutung unter besonderer Berücksichtigung der lutealen Durchblutung. Hannover, Tierärztl. Hochsch. Diss.

This project was supported by the Mehl-Mülhens-Stiftung, Germany

Contents I

Contents

Chapter

1 Introduction……………………………………………………………………… 1

1.1 Study background- embryo transfer in the mare…………… 2

1.2 Introduction in the theme complex of superovulation……… 4

1.2.1 Reproductive cycle in the mare ……………………………… 4

1.2.2 Endocrine regulation of estrus ……………...………….…….. 4

1.2.3 Follicular development..…………………………………….…. 6

1.2.4 Ovulation………………………………………………………… 9

1.2.5 Double ovulations……………………………………………… 10

1.3 Superovulation in the mare……………………………………. 11

1.4 Genital blood flow in the mare………………..…………….. 15

1.4.1 Doppler sonography …………………………………………… 15

1.4.2 Genital blood supply in the mare……………………………… 17

1.4.3 Uterine blood flow during estrus cycle in the mare………… 18

1.4.4 Ovarian blood flow during estrus cycle in the mare……….....18

1.5 The equine oviduct……………………………………………… 19

1.5.1 Fallopian tubes………………………………………………… 19

1.5.2 Equine embryo development in the oviduct………………….. 20

1.5.3 Methods of oviductal flushing or embryo recovery

from the oviduct………………………………………………… 21

1.5.4 Laparoscopic evaluation of the Fallopian tubes in

women…………………………………………….……………… 23

1.6 Aims of the study……………………………………………… 25

II Contents

Chapter

2 Embryo recovery rate following superovulation with equine pituitary

extract (eFSH®) in mares……………………………………………………… 28

3 Doppler sonography of the uterine and ovarian arteries during a

superovulatory program in horses …………………………………………… 32

4 Laparoscopic techniques for investigating the equine oviduct ..……..…. 36

5 General Discussion………………………………………………………….…. 39

5.1 Superovulation………………………………………………………… 40

5.2 Doppler Ultrasonography……………………………………………… 42

5.3 Laparoscopy of the equine oviduct…………………………………… 44

6 Summary……………………………………………………………….……….. 48

7 Zusammenfassung…………………………………………………………….. 51

8 References………………………………………………………………….…… 54

9 Appendix………………………………………………………………………….83

9.1 Material and methods study I and II………………………………….. 83

9.1.1 Animals…………………………………………………………………. 83

9.1.2 Mare management……………………………………………………. 83

9.1.3 Study design…………………………………………………………… 83

9.1.4 Superovulation treatment…………………………………………….. 84

9.1.5 Embryo collection………………………………………………………. 85

9.1.6 Transrectal Doppler sonography……………………………………… 86

9.1.7 Blood collection, progesterone and estrogen analysis……………. 88

Contents III

9.1.8 Statistical analysis……………………………………………………… 88

9.2 Material and methods study III……………………………………….. 89

9.2.1 Animals………………………………………………………………… 89

9.2.2 Preoperative management…………………………………………… 89

9.2.3 Instruments used in experiment I……………………………………. 90

9.2.4 Surgery experiment I…………………………………………………… 91

9.2.5 Preoperative management experiment II…………………………….. 92

9.2.6 Instruments used in experiment II……………………………………. 92

9.2.7 Surgery experiment II………………………………………………….. 94

9.2.8 Postoperative care in both experiments…………………………….. 101

IV Abbreviations

Abbreviations A./ Aa. Arteria/ Arteriae

a.m. ante meridiem

B-mode brightness modulation

BFV blood flow volume

bwt body weight

c cycle

CF continuous-wave doppler

Ch. Charrière

CL corpus luteum

cm centimeter

CO2 carbon dioxide

°C degree Celsius

D day

eCG equine chorionic gonadotropin

EDTA ethylenediaminetetraacetic acid

eFSH equine follicle stimulating hormone

IGF insulin-like growth factor

EIA enzyme immunoassay

EPE equine pituitary extract

et al. et alii

ET embryo transfer

E estradiol

Etot total estrogens

FDP follicular development phase

Fig. figure

FSH follicle stimulating hormone

g gram

g acceleration of gravity

GIFT gamete intra-fallopian tube transfer

GnRH gonadotropin-releasing hormone

Abbreviations V

h hour(s)

hCG human chorionic gonadotropin

hMG human menopausal gonadotropin

ICSI intra-cytoplasmic sperm injection

IGF insulin-like growth factor

IU international units

im intramuscular

iv intravenous

IVF in vitro fertilization

Kg kilogram

L liter

LH luteinizing hormone

mg milligram

MHz megahertz

min minute

mL milliliter

mmHg mm mercury

μL microliter

μm micrometer

Ø mean value

OV ovulation

ov ovarian

ovBFV ovarian blood flow volume

ovPI ovarian pulsatility index

P level of significance

P4 progesterone

pFSH porcine follicle stimulating hormone

PGE2 prostaglandin E2

PGF2α prostaglandin F2α

PI pulsatility index

p.m. post peridiem

VI Abbreviations

POP preovulatory phase

PW pulsed-wave doppler

R./ Rr. ramus/ rami

S.E.M. standard error of mean

SC stimulated cycles

sid semel in die; single a day

TAMV time averaged maximum velocity

USC unstimulated cycles

USP United States Pharmacopeia System

ut uterine

utBFV uterine blood flow volume

utPI uterine pulsatility index

UTJ utero-tubal junction

V./ Vv. vena/ venae

List of figures and tables VII

List of figures and tables

Fig. 1-1 Concentrations of FSH and estradiol in peripheral circulation and size of the largest follicle and concentrations of LH, progesterone and prostaglandinF2α in the peripheral circulation throughout the equine cycle (AURICH 2011)…………… 5

Fig. 1-2 Illustration of the hormonal aspects of deviation using a-two follicle model (GINTHER et al. 2001)…………………………. 8

Fig. 1-3 Follicular development during oestrus cycle of the mare; size of largest follicle, concentrations of hormones in peripheral circulation and occurrence within the largest follicle (AURICH, 2011)……………………………………….. 9

Fig. 1-4 Pulsatile arterial flow over one cardiac cycle………………………. 16

Fig. 1-5 Lateral view of arterial blood supply of the mare´s genital tract (GINTHER 2007)……………………………………………………….. 18

Fig. 1-6 Drawing of lateral view of ovary and associated structures of the mare……………………………………………………………… 20

Fig. 1-7 Scanning electron micrographs of the mare oviduct at the estrus phase……………………………………………………….. 21

Fig. 9-1 eFSH® treatment protocol in cycle 2 and 4………………………….. 85

Fig. 9-2 Ultrasound investigation of the left A. uterina of a mare one day before ovulation……………………………………………………. 87

Fig. 9-3 Ultrasound investigation of the right A. ovarica of a mare two days before ovulation…………………………………………… 87

Fig. 9-4 Trocar set used for experiment I……………………………………… 90

Fig. 9-5 Picture of the flushing catheter……………………………………… 93

VIII List of figures and tables

Fig. 9-6 Picture of the 1) plastic guide sleeve and 2) metal guide sleeve for guidance of the catheter …………………………………………… 94

Fig. 9-7 Drawing of the left flanc region and portal sites…………………….. 95

Fig. 9-8 Laparoscopic view of the right ovary (star), infundibulum (large arrow), oviduct (arrowheads) and tip of the uterine horn (circle) before oviductal flushing……………………………… 96

Fig. 9-9 Picture of the left flanc laparoscopy surgery site for oviductal

flushing in the standing sedated mare………………………………. 97

Fig. 9-10 Laparoscopic view of the left part of the infundibulum „opened up“ by a Babcock forceps; the balloon catheter is directed into the abdominal ostium (large arrow)…………………… 98

Fig. 9-11 The catheter (large arrow) is introduced approximately 2 cm

into the ampulla and the balloon (arrowhead) is insufflated with 2 ml of air, the Babcock forceps is positioned around the catheter and the abdominal ostium to prevent back-flow, methylene blue fluid in the proximal ampulla (asterisks)…… ….. 99

Fig. 9-12 Picture showing the oviductal ampulla filled with methylene

blue solution (arrowheads) following the oviductal flushing, the isthmus (stars) is not visibly filled or distended………………… 100

Tab. 9-1 Overview of the five investigated estrus cycles and the

according treatments (eFSH®-treatment, induction of ovulation with hCG, insemination and embryo collection 6.5 days post OV respectively)…….…………………………… 84

Tab. 9-2 Pain score system…………………………………………………… 101

Tab. 9-3 Clinical details of 10 mares of experiment I and II……………….. 102

Introduction 1

Chapter 1

General Introduction

2 Introduction

1. Introduction

1.1 Study background- embryo transfer in the mare

Embryo transfer (ET) has been part of cattle breeding for more than 35 years

(SCHERZER et al. 2008) and has also gained remarkable interest from the equine

industry after several breeds allowed registration of more than one foal per year.

However, success rates after superovulation and cryopreservation of embryos in

horses are still lagging behind those of cattle (SCHERZER et al. 2008). During the

last 20 years the number of equine ETs performed annually worldwide has grown

enormously. This is illustrated by the International Embryo Transfer Society’s (IETS)

annual statistics for equine ET which document 475, 11672, 27594 and 28661

commercial equine ETs worldwide in 1999, 2004, 2010 and 2011, respectively

(THIBIER 2000, 2005; STROUD 2011, 2012).

The most important breakthroughs were the development of techniques for non-

surgical transfer of embryos that yielded pregnancy rates >80%, not only for freshly

transferred embryos (VOGELSANG et al. 1985; RIERA and MCDONOUGH 1993;

MCKINNON et al. 1998), but also for embryos transported at 5°C for up to 24 h

(CARNEVALE et al. 1987; CARNEY et al. 1991).

The majority of embryos collected from donor mares are from spontaneously

ovulating mares with single ovulations. They are generally recovered 7 or 8 days

after ovulation (SQUIRES et al. 2003). Following fertilization at the ampulla-isthmus

junction, transport through the oviductal isthmus occurs rapidly and the late morula or

early blastocyst enters the uterus through the utero-tubal junction 144 - 156 h after

ovulation (OGURI and TSUTSUMI 1972; WEBER et al. 1996; BATTUT et al. 1997).

Unfertilized eggs on the other hand are retained in the oviduct (VAN NIEKERK and

GERNEKE 1966).

The day of recovery, number of ovulations, age of donor mare and the quality of

semen are factors that affect embryo recovery (SQUIRES 1996). Mean embryo

recovery per cycle from spontaneously ovulating mares with single ovulations is

approximately 50%. An ability to consistently induce multiple follicles > 30 mm and

multiple ovulations in mares would enhance embryo recovery from donor mares,

Introduction 3

provide multiple follicles for collection of oocytes, and improve pregnancy rates from

subfertile mares (SQUIRES 2006).

At present, however, the vast majority (>95%) of horse embryos are transferred fresh

or after cooled storage for up to 24 h, whereas cryopreservation or vitrification is

rarely employed in clinical use (STOUT 2012). A major impediment to the

implementation of embryo cryopreservation in the field is that acceptable pregnancy

rates (>55%) are, at present, achievable only with embryos recovered at an early

developmental stage (day 6–6.5; morula to early blastocyst) when they are <300 µm

in diameter (CZLONKOWSKA et al. 1985; SLADE et al. 1985; ELDRIDGE-

PANUSKA et al. 2005). The influence of size appears to be even more absolute for

vitrification, because embryos >300 µm show a reduced ability to re-expand during

post-warming incubation (HOCHI et al. 1995) and very rarely result in pregnancy

after vitrification and warming (ELDRIDGE-PANUSKA et al. 2005; CARNEVALE

2006; SCHERZER et al. 2011), whereas larger embryos cryopreserved by slow-

freezing do yield normal pregnancies, albeit at a lower rate (<20%) than for small

embryos (SLADE et al. 1985; BARFIELD et al. 2009). The embryonic capsule and

the amount of blastocoel fluid in embryos of larger diameter were hypothesized as

reasons for the low success of vitrification or freezing (MACLELLAN et al. 2002;

BASS et al. 2004; BARFIELD et al. 2009). But nevertheless, in latest works from

laboratories using micromanipulation capabilities, first positive attempts in vitrification

of expanded blastocytes were made (CHOI et al. 2010). It was found that collapsed

(by embryo biopsy) equine blastocysts (initial diameters 407-565 µm) could be

efficiently vitrified and resulted in pregnancy rates of 46% (6/13) (CHOI et al. 2011;

HINRICHS and CHOI 2012).

Moreover, the exact time of the passage into the uterus and rate of embryo

development appear to vary, depending for example on the time of year, type of

semen used (fresh vs. frozen) and age of the donor mare (STOUT 2006). The scale

of the variation in developmental rate was demonstrated by COLCHEN et al. (2000)

who recorded ranges in diameter and cell number, respectively, of 159–365 µm and

272–2217 cells for embryos collected at 168 ± 0.5 h, and 162–245 µm and 117–417

cells for embryos collected at 156 ± 0.5 h after ovulation (COLCHEN et al. 2000).

4 Introduction

A number of inter-related factors have contributed to the slow development and

implementation of equine embryo cryopreservation, and these include the following:

- the absence of commercially available products for reliably stimulating

superovulation

- very poor pregnancy rates following cryopreservation of embryos >300 µm in

diameter

- difficulty in recovering embryos at early developmental stages amenable to

cryopreservation (BATTUT et al. 1997; STOUT 2012)

In the following section an introduction into the fields of superovulation and follicle

development in the mare will be given, and the oviduct as the early storage of

embryos will be introduced in detail.

1.2 Introduction in the theme complex of superovulation 1.2.1 Reproductive cycle in the mare

Mares are a seasonally polyestrous species with ovulatory activity being related to

long days and light. They will experience reproductive activity during the spring and

summer month, between May and October. During the breeding season, average

estrus cycle length is about 21 to 22 days in length. An estrus during the follicular

phase lasts 5–7 days characterized of behavioural signs like increased interest in

stallions and proceptive behaviour in response to the sexual attractivity of a stallion

(CROWELL-DAVIS 2007). This is followed by the luteal phase, or diestrus, and lasts

14 to 16 days (NIE 2007; AURICH 2011). The cycle length is also affected by time of

breeding season or reproductive stage (HEIDLER et al. 2004).

1.2.2 Endocrine regulation of estrus

The endocrinological control of the estrus cycle is governed by the hypothalamic-

pituitary-gonad axis. In the mare, the gonadotropins LH and FSH are considered to

be under the control of GnRH alone. So far there is no evidence that a specific FSH-

releasing factor exists in the horse (AURICH 2011). Hypothalamic GnRH release is

modulated by steroid feedback mechanisms (IRVINE and ALEXANDER 1993). An

early periovulatory rise in peripheral concentrations of LH is accompanied by a

Introduction 5

modest increase in FSH subsequently declining to its nadir concentration while LH is

reaching its maximum (BERGFELT et al. 1991). In the mid-luteal phase, a second

and robust FSH rise occurs with no concomitant increase in LH. This second FSH

surge occurs on different days of the cycle among individual mares (GINTHER et al.

2005). In contrast to other domestic animal species which exhibit a short and

pronounced preovulatory LH surge, no distinct periovulatory LH peak exists in the

mare. However, during estrus the period of elevated concentrations of LH lasts for

several days. A simplified overview of concentrations of FSH, estradiol, LH and

progesterone in relation to the largest follicle is shown in Fig.1-1.

Fig. 1-1: Concentrations of FSH and estradiol in peripheral circulation and size of the largest follicle and concentrations of LH, progesterone and prostaglandinF2α in the peripheral circulation throughout the equine cycle (AURICH 2011)

Estradiol

Estrus

Estrus

Day of estrus cycle

6 Introduction

1.2.3 Follicular development

Mares are a monovulatory species: typically one follicle becomes dominant and

several subordinate follicles regress during the primary follicular wave of the estrus

cycle before ovulation (GINTHER and BERGFELT 1992). Compared to other

domestic animal species, the mares´ ovary has a unique structure characterized by a

large size and weight (35–120 cm3 in volume; 40–80 g in weight) (KIMURA et al.

2005), the presence of an ovulation fossa and an inverted location of its cortex and

medulla (KAINER 1993).

Although scientific interest in equine follicles existed already since the 1920s

(SEABORNE 1925), detailed studies on equine follicle dynamics did start by the

pioneering lead of OJ Ginther at the University of Wisconsin (reviewed in GINTHER

1979, DONADEU and PEDERSEN 2008).

As in other farm animal species and humans, the development of antral follicles in

the horse is characterized by the periodic growth of cohorts of follicles (SIROIS et al.

1989; BERGFELT and GINTHER 1993). Follicular waves are generally divided into

primary and secondary waves during the estrus cycle of the mare. Only the primary

wave appears to produce the dominant follicle that goes on to ovulate during estrus

(GINTHER et al. 2004). The primary wave emerges during midluteal diestrus from

the stimulation of a FSH surge, the dominant follicle becomes preovulatory and

results in ovulation at the end of estrus. The secondary wave emerges during late

estrus of the previous estrus cycle or early diestrus where the dominant follicle is

either anovulatory and regresses, forming an anovulatory hemorrhagic follicle, or

results in secondary ovulation during mid-diestrus (BEG and GINTHER 2006).

In the mare the primary follicular wave emergence is characterized by a follicle

diameter of 6 mm in the largest follicle (GASTAL et al. 1997). A mean number of 7 -

11 follicles emerge over several days and enter a common growth phase of about 3

mm per day (GASTAL et al. 2004; GINTHER et al. 2004). The emergence of each

follicular wave is temporally associated with an FSH surge. FSH reaches a plateau

when the largest follicle reaches a size of about 13 mm in diameter (GASTAL et al.

1997; DONADEU and GINTHER 2001). Subsequently, FSH declines to a

concentration that does not support pronounced further growth of subordinate

Introduction 7

follicles but is sufficient for continuing growth of the dominant follicle. The inhibition

of growth of the smaller follicles does not depend on follicle-to-follicle inhibitory

mechanisms, but follicle deviation involves important changes in the largest follicle

(AURICH 2011). These are characterized by an increased sensitivity to circulating

concentrations of FSH. Dramatic changes in the insulin-like growth factor (IGF)

system (IGF-I and -II, IGF binding protein, IGF binding protein proteases) in the

largest follicle before the beginning of size deviation play a crucial role (BEG and

GINTHER 2006). Simultaneously, the future dominant follicle suppresses circulating

concentrations of FSH, most probably due to follicular synthesis and release of

estrogens (GASTAL et al. 1999; DONADEU and GINTHER 2001) and Inhibin

(WATSON and AL-ZI'ABI 2002).

The low FSH concentration, yet, does not restrict the growth of the dominant follicle

which by that time has acquired the ability to more efficiently use circulating

gonadotropins for growth (GINTHER et al. 2003) and to produce high levels of inhibin

and estradiol. These declining FSH concentrations continue to support growth of the

follicles of the wave until the appearance of the two largest follicles at the time of

deviation: generally 22.5 and 19 mm in diameter, 16 days postovulation or about 8

days before ovulation (GINTHER et al. 2004; JACOB et al. 2009).

Follicular deviation is an abrupt event recognized by the sudden decrease in the

growth rate of the second largest follicle by about 2 days after the beginning of

deviation (ROSER and MEYERS-BROWN 2012). This is thought to be a key

component of the follicular selection process in monovulatoy species such that

usually one follicle becomes the preovulatory follicle, whereas the others regress

owing to an interplay between circulating gonadotropins and follicular factors within

the ovary (BEG and GINTHER 2006; GINTHER 2012). During these 2 days,

depending on the size difference of the two follicles and the subordinate follicles,

treatment with exogenous gonadotropins could enhance the potential for multiple

dominant follicles by rescuing those follicles that start to regress. Mechanisms of

follicle development and deviation are shown in Fig. 1-2 and 1-3.

The critical role of low FSH levels in the deviation mechanism in mares is illustrated

by the disruption of the deviation mechanism after administration of FSH (SQUIRES

8 Introduction

2006) or immunization against inhibin (MCCUE et al. 1992) leading to the

development of multiple ovulatory follicles.

Fig. 1-2: Illustration of the hormonal aspects of deviation using a two- follicle model (GINTHER et al. 2001).

0= dominant and subordinate follicle When the follicles reach about 13 mm, they both secrete increasing concentrations of inhibin during the common-growth phase (before deviation). About a day before deviation, increased estradiol is secreted by the largest follicle under the influence of increased concentrations of LH. Apparently, the increasing estradiol acts in conjunction with Inhibin to continue the reduction in FSH concentrations after deviation. The elevated LH continues to stimulate the production of estradiol by the developing dominant follicle and has a positive diameter effect on the dominant follicle within 2 days after the beginning of deviation.

Fo

llic

le

Introduction 9

Fig. 1-3 Follicular development during the estrus cycle of the mare; size of follicles, hormone changes in peripheral circulation and occurrence within the largest follicle; =follicle = ovulation (AURICH 2011)

1.2.4 Ovulation

The preovulatory follicular development and ovulation in horses differ from other

animal species. The preovulatory follicle is much bigger in size and ovulates at the

ovulation fossa - a specific region of the mare´s ovary (AURICH 2011). The

preovulatory follicle grows at an average rate of 3 mm per day and reaches a

diameter of approximately 35 mm four days before ovulation. Continued growth

occurs up to 2 days before ovulation when follicular size reaches a plateau of

Follicle size

(primary wave)

10 Introduction

approximately 40 mm (GINTHER et al. 2008b). During ovulation, the oocyte enters

the oviduct, while most of the follicular fluid passes into the peritoneal cavity

(TOWNSON and GINTHER 1989) and only a small volume of follicular fluid appears

to accompany the oocyte/cumulus complex into the oviduct (TOWNSON and

GINTHER 1989). Hormones from this fluid are rapidly absorbed into the circulation

leading to a pronounced increase in concentrations of inhibin on the day of ovulation

(BERGFELT et al. 1991). The ovulatory process of the equine follicle involves a

specific and unique pattern of gene regulation in theca and mural granulosa cells.

This includes differences in the expression of a variety of factors among them

prostaglandins and prostaglandin metabolizing enzymes (SAYASITH et al. 2009).

1.2.5 Double ovulations

Spontaneous double ovulations may occur in horses. The double ovulation rate is

affected by various factors such as breed, reproductive status, age and

pharmacological manipulation of the estrus cycle (STABENFELDT et al. 1972;

GINTHER et al. 1982; SIEME and KLUG 1996). The incidence of spontaneous

double ovulation varies between approximately 2% in ponies and 25% in

thoroughbreds, respectively. When two dominant follicles (two follicles >28 mm)

develop in the same follicular wave, double ovulations occur in about 40% of mares

(GINTHER et al. 2008a). These may occur synchronously (within 12 h), but intervals

up to two days and more have been reported between ovulations (GINTHER et al.

2008a). During the 2.5 immediately days before ovulation, the rate of dominant

follicle growth in double ovulating mares is less pronounced than in single ovulating

mares resulting in a lower preovulatory follicle diameter in twin ovulating mares

(GINTHER and BERGFELT 1992). The reduced follicular growth is related to lower

FSH concentrations, most probably due to higher estradiol concentrations from the

two preovulatory follicles (GINTHER et al. 2008a). The peak of FSH levels that

occurs 3 days before deviation is responsible for the development of the dominant

follicle(s), and the decline of FSH causes the regression of subordinate follicles

during the primary follicular wave. But the role of LH, inhibin, and estradiol still needs

to be elucidated in the further development and maturation of the preovulatory follicle

Introduction 11

to ovulation (ROSER and MEYERS-BROWN 2012). Taken together, it is difficult to

discern the role of FSH, LH, and estradiol in inducing one ovulation or double

ovulations during the pre- and periovulatory period. It is conceivable that the effects

of systemic hormones on the intrafollicular factors and their receptors in dominant

and subordinate follicles during the primary follicular wave and the periovulatory

period play a major role in determining whether mares are multiple ovulators

(ROSER and MEYERS-BROWN 2012). Mares that tend to have multiple ovulations

continue to do so in a superovulatory regimen (LOGAN et al. 2007).

1.3 Superovulation in the mare

The percentage of double ovulations in mares is low. The success of advanced

reproductive technologies in the mare would be enhanced by effective superovulation

to provide multiple oocytes and multiple embryos for such techniques as embryo

transfer, gamete intra-fallopian tube transfer (GIFT) and intra-cytoplasmic sperm

injection (ICSI). Superovulation can increase pregnancy rates in normal and

subfertile mares as well as when using semen from subfertile stallions (SQUIRES

2006).

The basis of superovulation is manipulation of the hormones that control the

dominant follicle and inhibit the regression of subordinate follicles (SQUIRES and

MCCUE 2007). Superovulation has been attempted in the cycling mare during the

past 35 years beginning with studies by DOUGLAS et al. (1974). LAPIN and

GINTHER (1977) reported induction of ovulation and multiple ovulations in

seasonally anovulatory and ovulatory mares with an equine pituitary extract (EPE)

preparation. Since then, many other investigators have used various hormone

regimens to induce superovulation in the cycling mare (for reviews see: MCCUE

1996; SQUIRES and MCCUE 2007; SQUIRES and MCCUE 2011). Attempts to

superovulate cyclic mares using preparations of equine chorionic gonadotropin

(DINGER et al. 1982), GnRH (BECKER and JOHNSON 1992; DIPPERT et al. 1992),

porcine FSH (FORTUNE and KIMMICH 1993; CULLINGFORD et al. 2010; RAZ et al.

2010) and active immunization against inhibin (MCCUE et al. 1992; NAMBO et al.

12 Introduction

1998; DERAR et al. 2004) have demonstrated great variability in the results in most

cases.

EPE lead to an increase in the number of smaller follicles. Some of the earlier studies

in pony mares showed an increase in the number of ovulations during anestrus

(DOUGLAS 1979). During the natural breeding season, treatment before a 25- mm

follicle was present resulted in increased ovulations, whereas treatment of mares

with a follicle over 25 mm did not change ovulation rates or increase the number of

ovulations and embryos recovered (DIPPERT et al. 1992). These data suggest that

treatment initiated after the dominant follicle is established, usually around day 15,

may not be effective in rescuing subordinate follicles and increasing ovulation rate. It

was suggested that the reason for this finding was that because of the FSH within the

preparation follicles were rescued from atresia. The variability in the response in

these studies may be due to the variability in the size of the cohort of follicles present

at the time of initial administration of EPE, as the standard time of initial treatment

was 5-6 days postovulation and not based on the size of the follicles present.

Therefore, administration of EPE, before the dominance is established, was found to

be the treatment of choice (PIERSON 1990). Of 170 mares treated with EPE at

Colorado State University, an average of 3.2 ovulations was detected and 1.96

embryos were recovered per mare compared to 0.65 embryo recovered from

untreated control mares (SQUIRES and MCCUE 2007).

In addition, purity of EPE is a problem, as the ratio of FSH to LH does not remain

constant between preparations (ROSER and MEYERS-BROWN 2012).

Equine FSH

In the past decade, a semipurified EPE (eFSH; Bioniche Animal Health, Bogart, GA)

became commercially available. Based on radioimmunoassay, this preparation

contained 110 mg of FSH/mg and 10 mg of LH/mg, an FSH to LH ratio of 10:1

compared with an EPE preparation that had a 5:1 ratio measured by

radioimmunoassay (WELCH et al. 2006). Although eFSH was commercially available,

there was still variability of responses between mares (ALLEN 2005). Factors that

affect the response of mares include day of initial treatment, size of follicles at

initiation, and frequency of treatment injection (ALLEN 2005). To design an optimal

Introduction 13

treatment regimen using eFSH for the present study the following aspects of earlier

studies were considered:

Dose

NISWENDER et al. (2003) first investigated the use of 12 mg (twice-daily

intramuscular injections- total 25 mg/day) or 25 mg of eFSH given in twice-daily

intramuscular injections (total 50 mg/day) to mares during the ovulatory season.

Treatment was initiated 5-6 days postovulation to ensure stimulation to occur during

the active growth phase of follicular waves. For both treatment groups luteolysis was

induced on the second day of treatment and was used to remove the effect of

progesterone. When a majority of follicles measured 35 mm in diameter, ovulation

was induced with either deslorelin or human chorionic gonadotropin. Treatment with

twice daily 12 mg of eFSH increased the number of follicles >35 mm. Ovulations

were also increased to 3.6 versus 1.0 in control animals. Embryos retrieved

increased from 0.5 to 1.9 in mares given the 12-mg-dose twice a day. Treatment with

25 mg of eFSH twice daily resulted in an increased number of follicles but not

ovulation rates. Treatment with 12 mg (twice-daily intramuscular injections- total 25

mg/day) of eFSH was determined as an optimal dose (NISWENDER et al. 2003).

Treatment start

MCCUE et al. (2006, 2007) evaluated different times for treatment start with eFSH

and reached the best results when treatment start was 5–7 days after ovulation when

a cohort of follicles 20–25mm in diameter was present.

Pretreatment

Different protocols for pretreatments before eFSH application to increase embryo

recovery rates have also been reported. The basis for these studies was to induce a

follicular wave with progesterone and estradiol, simulating the mare’s physiological

follicular waves and timing of follicular development and deviation so as to more

accurately time treatment with eFSH. In a study of RAZ et al. (2005) there was no

advantage with a progesterone and estradiol treatment, LOGAN et al. (2007)

reported that pretreatment with progesterone and estradiol-17ß plus 12.5 mg of

eFSH, decreased the number of ovulations compared with administration of eFSH

alone. The number of embryos recovered was 0.7 and 1.5 embryos in the

14 Introduction

progesterone- and estradiol-17b-treated group compared with 2.6 embryos in the

eFSH-only group.

“Coasting”

“Coasting” can be defined as a certain time period of stopping the eFSH treatment

before induction of ovulation. In a study conducted by WELCH et al. (2006) the

authors found a higher embryo recovery rate by stopping the twice-daily treatment of

eFSH at the time of a 32-mm follicle for 42-50 hours before hCG then giving hCG

right after eFSH treatment when a follicle reached 35 mm in diameter. This idea was

adapted from studies in women where results of a continous stimulation program

also showed an ovarian hyperstimulation (FLUKER et al. 1999). An ovarian

hyperstimulation could be seen in studies in cattle (SIRARD et al. 1999) after a

continous stimulation treatment. According to SQUIRES and MCCUE (2011), the

benefits of coasting are to prevent hyperstimulation, which would result in a reduced

receptor response, limit the occurrence of anovulatory follicles, and shorten the

treatment regimen, thereby decreasing the cost of eFSH (SQUIRES and MCCUE

2011).

Recombinant FSH and LH

Given the problems in using EPE and eFSH, in part due to the variability of the ratio

of FSH:LH, it was hypothesized that development of recombinant equine

gonadotropins would provide pure and large quantities of eFSH from the laboratory

using molecular biology and cloning techniques (ROSER and MEYERS-BROWN

2012). Recombinant human FSH was reported to increase follicular activity in

humans, primates, rodents, and cattle (THARASANIT et al. 2006). When tested in

mares, there was no increase in ovulation rate or embryo recovery (ROSER and

MEYERS-BROWN 2012). This may have been due to the fact that the equine FSH

receptors show differences in their DNA sequence and structure compared with other

species (THARASANIT et al. 2006). But the development and efficacy of

recombinant equine gonadotropins (reFSH and reLH) have recently been reported

(JABLONKA-SHARIFF et al. 2007; JENNINGS et al. 2009; MEYERS-BROWN et al.

2010).

Introduction 15

1.4 Genital blood flow in the mare

In human medicine, color Doppler sonography has been used for more than two

decades to predict the outcome of assisted reproduction technologies (BROUSSIN

2007; LAMAZOU et al. 2009). For example, in women undergoing hormonal

treatment, transvaginal color Doppler sonography has been successfully used to

study ovarian blood flow during IVF cycles, and ovarian blood flow was found to be

related to ovarian response to stimulation (WEINER et al. 1993; ZAIDI et al. 1996).

Correlations between genital blood flow and ovarian response to hormonal treatment

have also been verified in cows (HONNENS et al. 2008; 2009). Furthermore, ovarian

blood flow has already been investigated in the mare by BOLLWEIN et al. (2002b).

Using transrectal color Doppler sonography, these authors found characteristic

changes in ovarian blood supply during the estrus cycle in mares, which were related

to alterations of sexual steroid hormone levels (BOLLWEIN et al. 2002b). HONNENS

et al. (2011) investigated the relationships between uterine blood flow, peripheral sex

steroids, expression of endometrial estrogen receptors and nitric oxide synthases

during the estrous cycle in the mare and concluded that the nitric oxide synthase

system plays a major role in regulation of uterine perfusion during the estrous cycle

in the mare. Currently there is no information about ovarian blood flow during

hormonal stimulation of superovulation in the mare.

1.4.1 Doppler sonography

In 1980, Palmer and Driancourt published the first report on the use of transrectal

ultrasound in equine gynaecology, which was rapidly followed by a widespread

utilization of ultrasound scanners for use in this area (GINTHER 1986). This

technology is used for both color-flow and power-flow imaging, and for spectrally

displaying on a viewing screen the blood velocity at a target point in a vessel

(GINTHER et al. 2007). The assessment of ovarian blood flow and ovarian structures

– topics of great interest to equine veterinarians – has received much research

interest in recent years (BOLLWEIN et al. 2002a; GASTAL et al. 2006; MIRO et al.

2010). Doppler ultrasound technology is based on Dopplershift, wherein the

ultrasound frequency of echoes from moving red blood cells is increased or

16 Introduction

decreased as the cells move toward or away from the transducer. In spectral mode,

the blood flow in a specific vessel can be assessed by placing a sample-gate cursor

on the image of the lumen of the vessel (GINTHER 2004). Arterial blood flow to the

reproductive tract is pulsatile in response to the heartbeats or pulsations of the left

ventricle. The red line in Fig. 1-4 shows relative velocity or pressure changes during

systole and diastole of the cardiac cycle or arterial pulse in a major artery.

Peak systolic, end diastolic, and time-averaged maximum velocities are calculated

and shown for a selected cardiac cycle. Doppler indices (resistance index, RI;

pulsatility index, PI) are ratios that are calculated from various points on the spectrum.

The indices correspond to the hemodynamics of the tissue supplied by the artery.

Increasing RI or PI values indicate increasing resistance and decreasing perfusion of

the distal tissues (GINTHER 2004).

Fig. 1-4 Pulsatile arterial flow over one cardiac cycle, red line: relative velocity or pressure changes during systole and diastole of the cardiac cycle; ultrasound pictures with transverse section of an external iliac artery and associated vein in colorflow mode showing different color spectra depending on the blood flow velocity (GINTHER 2007)

Introduction 17

1.4.2 Genital blood supply in the mare

Uterine artery

The uterus receives blood from the uterine branch of the ovarian artery, a main

supply from the uterine artery, and the uterine branch of the vaginal artery (Fig. 1-5).

The uterine artery originates from the external iliac artery in the mare. The aorta

continues as a common trunk of a few centimetres between the origins of the

external and internal iliac arteries. Following the mesometrium the uterine artery

forms a cranial and caudal branch (GINTHER 2007).

Ovarian artery

The ovarian artery leaves the aorta, as shown in Fig 1-5, runs dorsally along the

abdominal wall, and enters the mesovarium. The right artery crosses along the vena

cava ventrally. The ovarian artery passes along the cranial aspect of the

mesovarium. In mares the ovarian artery is relatively straight and located a few

centimetres caudal to the uteroovarian vein. The uterine branch of the ovarian artery

or uteroovarian anastomosis is highly variable among individuals and sides

(GINTHER 2007).

The detailed description of location of the uterine and ovarian arteries using Doppler

ultrasound are reviewed by GINTHER (2007).

18 Introduction

Fig.1-5 Lateral view of arterial blood supply of the mare´s genital tract

(GINTHER 2007) bua Rr. uterinae; cvc V. cava caudalis; dca A. circumflexa iliumprofunda; eia A. iliaca externa; iia A. iliaca interna; iia A. iliaca interna; ipa A. pudenda interna; oa A. ovarica; ov V. ovarica; ua A. uterina; uboa R. uterinus to A. ovarica; ubva R. uterinus to A. vaginalis; uma A. umbilicalis; va A. vaginalis

1.4.3 Uterine blood flow during estrus cycle in the mare

Uterine blood flow during estrus cycle shows a bimodal profile (BOLLWEIN et al.

1998; BOLLWEIN et al. 2002b). The uterine blood flow resistance, characterized by

the uterine pulsatility index (utPI) was highest during the early luteal phase and again

during late luteal phase and low during mid-luteal phase and before ovulation. The

uterine PI was highest on days 1 and 11 and lowest on days D5 and D-2 (D0= Day of

ovulation) (BOLLWEIN et al. 1998).

1.4.4 Ovarian blood flow during estrus cycle in the mare

During estrus cycle the ovarian blood flow changes. Values of the ovarian pulsatility

index (ovPI) are lower in the A. ovarica ipsilateral to the corpus luteum (CL)

Introduction 19

compared to the A. ovarica contralateral to the CL during diestrus (D0 - D15). During

D0 to D2 ovPI was highest in the A. ovarica ipsilateral to the CL, decreased until D6

and continuously increased until D15 (BOLLWEIN et al. 2002a). The PI was low on

the expected days of high progesterone concentrations and was attributable to

evaluated blood flow in the CL. During estrus (D-6 to D-1) there was a negative

correlation between the diameters of the largest follicle and the ovPI of the ipsilateral

A. ovarica (BOLLWEIN et al. 2002a).

Currently there is only limited information (PROBST 2009) about ovarian blood flow

during hormonal stimulation of superovulation in the mare.

1.5 The equine oviduct

1.5.1 Fallopian tubes

The first anatomical description of a mammalian oviduct was published by

FALLOPIO in 1561 (cited in BECK and BOOTS 1974). Each uterine tube consists of

an expansive infundibulum covering the ovary´s ovulation fossa, a highly tortuous

ampulla about 6 mm in diameter, and a less tortuous isthmus (about 3 mm in

diameter). The whole uterine tubes are 20-30 cm in length. The isthmus terminates in

a small uterine ostium on a papilla within the cranial end of a uterine horn. The

uterine ostium is about 2 - 3 mm in diameter. The inner circular muscle of the

oviductal musculature increases to form a sphincter at the utero-tubal junction. The

abdominal ostium in the centre of the infundibulum is about 6 mm in diameter. The

distal one third of the oviduct is extremely convoluted and has a well developed

Lamina muscularis (MENEZO and GUERIN 1997). The ovarian bursa of the mare is

a peritoneal pouch extending from the ovulation fossa caudal to the cranial aspect of

the uterine horn. Laterally it is bounded by the uterine tube and mesosalpinx. A fold

of broad ligament containing the proper ligament of the ovary forms the medial part of

the ovarian bursa (KAINER 1993).

20 Introduction

Fig. 1-6: Drawing of lateral view of the right ovary and associated

structures of the mare (GINTHER 1986)

amp=ampulla; inf=infundibulum; ist=isthmus; luh=left uterine horn; mo= mesovarium; ms=mesosalpinx; rl=round ligament; tm=tubal membrane; tuj=tubo-uterine junction left picture=lateral view of the right ovary and associated structures; right picture= lateral view of the right ovary and associated structures with lifted infundibulum and mesosalpinx and view of the mucosal side of the tip of the uterine horn with the tubo- uterine junction (uterine papilla) 1.5.2 Equine embryo development in the oviduct

The oviduct of the mare is the smallest component of the tubular genital tract but is

also the site of significant reproductive events - gamete transport and fertilisation. It is

considered as a reproductive organ having both transport and secretory functions

that are essential for early reproductive events. The equine embryo, in contrast with

embryos of most other domestic species, remains in the oviduct longer (FREEMAN

et al. 1991) and embryo development at the time of uterine entry is relatively

advanced in the horse versus the pig, cow or sheep (FREEMAN et al. 1991). Equine

embryos that enter the uterus are compact morulae to early blastocysts.

Following ovulation, the oocyte arrives in the ampulla of the oviduct still surrounded

by its protective coating of cumulus cells. At the ampullary-isthmic junction it lodges

Introduction 21

and, assuming mating/insemination has already taken place, the oocyte is fertilised

by one of the spermatozoa present (BOYLE et al. 1987; HUNTER 2005). The

developing embryo remains there during its subsequent cleavage divisions

(BETTERIDGE et al. 1982; WEBER et al. 1996). The developing conceptus, still

located at the ampullary-isthmic junction, contains approximately 4 blastomeres, and

the embryonic genome is activated (BETTERIDGE et al. 1982). Embryonic

development continues within the oviduct for another 4 days until the compact morula

begins to secrete PGE2 (WEBER et al. 1991) which induces relaxation of the

ampullary-isthmic ‘sphincter’ and enables the embryo to pass rapidly through the

isthmus and uterotubal junction to enter the uterine lumen at around day 6-6.5 after

ovulation (FREEMAN et al. 1991; BATTUT et al. 1997). At the time of uterine entry,

the embryo is at the late morula or early blastocyst stage of development

(BETTERIDGE et al. 1982; FREEMAN et al. 1991; BATTUT et al. 1997; RAMBAGS

et al. 2005). In contrary to embryos unfertilized eggs are retained in the oviduct (VAN

NIEKERK and GERNEKE 1966).

Fig.1-7: Scanning electron micrographs of the mare oviduct at the estrus phase. (b), ampulla; (c), isthmus.; l, lumen; m, muscle layer; mf, mucosal folds, arrow, mucosal fold in isthmus. Bar: b, 560 µm; c, 486 µm; (DESANTIS et al. 2011) 1.5.3 Methods of oviduct flushing or embryo recovery from the oviduct

The diagnostic and therapeutic options for oviduct disorders in the mare are limited.

Transrectal palpation and ultrasonographic evaluation of oviductal disorders can be

subjective and difficult to diagnose. For evaluation of tubal patency, desposition of

22 Introduction

fluorescent microspheres (LEY et al. 1998) and starch granules (ALLEN 1979) on the

surface of the ovary and fimbria have been described but neither test has received

wide acceptance (NEAL 2011).

A major advance in understanding oviducal function in the mare was achieved when

it was demonstrated that Day 5 equine embryos secrete significant quantities of

PGE2 (WEBER et al. 1991). This hormone binds to the oviductal musculature

(WEBER et al. 1992), and continuous infusion of small quantities of PGE2 onto the

surface of the ipsilateral oviduct in inseminated mares via a minipump surgically

implanted into the mesovarium hastens embryonic transport through the oviduct

(WEBER et al. 1991). WEBER et al. (1992) demonstrated marked inhibition by PGE2

of histamine-induced contractility of equine isthmic circular smooth muscle in vitro.

TROEDSSON et al. (2005) observed how PGE2 can also cause contraction of the

longitudinal smooth muscle of the oviduct in rabbits (BLAIR and BECK 1977) and

pigs (RODRIGUEZ-MARTINEZ et al. 1985). These important research findings on

the roles of PGE2 in oviducal transport were supported by the report that application

of a few drops of a PGE2-laced cervical gel onto the surface of the ipsilateral oviduct

of inseminated mares on Day 4 after ovulation hastened entry of the compact

morula-stage embryo into the uterus by 24 h (ROBINSON et al. 2000).

Catheterisation of the equine oviduct through the UTJ is an extremely difficult

procedure, unlike other mammalian species, since the distal third of the duct is

extremely convoluted and has a well-developed Lamina muscularis. This acts as a

sphincter (MENEZO and GUERIN 1997), making mechanical entry from the uterus

exceedingly difficult (KAINER 1993; BENNETT 2007).

Laparoscopy, via a lateral flank approach or a ventral abdominal approach under

general anaesthesia are other possibilities, allowing the upper tract to be viewed in

situ (BENNETT 2007). Zent et al. (1993) successfully restored fertility in 3 of 5

Thoroughbred mares with well documented histories of unexplained conception

failure by flushing saline through their oviducts (from infundibulum to uterine horn)

during surgical laparotomy performed under general anaesthesia.

Another method to collect tubal stage embryos was described by BESENFELDER

and BREM (1998) in the cow. They introduced a transvaginal approach to the oviduct

Introduction 23

and described the collection of tubal stage embryos via a laparoscopic guided

transvaginal oviductal flushing (BESENFELDER et al. 2001).

Currently, the best technique for diagnosis and treatment of oviductal disorders is

laparotomy and exploratory surgery under general anaesthesia and the

catheterisation of the infundibulum (BENNETT 2007), which is relatively invasive. A

simple technique which allows the accurate evaluation of oviducal function, in respect

of gamete transport, would be highly desirable in the investigation of the infertile

mare. Although such a technique remains elusive, considerable progress has been

made with the application of laparoscopic techniques for diagnosis and treatment

(NEAL 2011).

With the development of biotechnologies in the mare, the catheterisation of the

infundibulum was performed in context of oocyte transfer (OT) or gamete

intrafallopian transfer (GIFT) (CARNEVALE 2004; CARNEVALE et al. 1993;

HINRICHS et al. 2000, 2002; SCOTT 2001). Oocytes recovered from a valuable

donor mare are, after being matured in vitro, injected into the fimbria of the oviduct

which is ipsilateral to the ovary containing a maturing follicle in a recipient mare. This

recipient may have been mated or inseminated artificially with semen from the

desired sire immediately before intrafallopian transfer of the donor oocytes, or a low

number (0.5– 5 · 106) of washed spermatozoa may be injected into the oviduct

simultaneously with the M-II stage oocytes (ALLEN 2005). In both techniques the

intrafallopian transfer is performed via an invasive lateral incision and the manual

extorsion of the oviduct without using laparoscopy.

Until now, however, laparoscopic evaluation of the oviduct in the standing sedated

mare has allowed visualisation but not catheterisation and therefore had only limited

diagnostic and therapeutic potential (BENNETT 2007).

1.5.4 Laparoscopic evaluation of the Fallopian tubes in women

Laparoscopy is widely accepted as the ‘‘gold standard’’ method for evaluating tubal

patency. At present, it is considered the most accurate diagnostic test available for

evaluating tubal-related subfertility (SAUNDERS et al. 2011). Its advantages include

24 Introduction

an ability to simultaneously evaluate the abdominal cavity and other pelvic structures

for an enhanced diagnostic evaluation of other etiologies of subfertility. The

procedure also allows for therapeutic excision of endometriotic lesions and, usually,

restoration of abnormal pelvic findings. Laparoscopy incurs, however, operative risks,

costs, and a period of postoperative recovery. As an invasive and expensive

procedure, it is not an ideal first-line screening test for subfertility when suitable

alternative office procedures are available. When clinical history, laboratory, or these

office procedures suggest tubal-related pathology, laparoscopy may disclose a

definitive diagnosis and offer a treatment option (SAUNDERS et al. 2011).

Introduction 25

1.6 Aims of the study

Reasons for the study

Beside the fact that during the last 20 years the number of equine ETs performed

annually worldwide has grown enormously, success rates after superovulation and

cryopreservation of embryos in horses are still lagging behind those of cattle

(SCHERZER et al. 2008).

An ability to consistently induce multiple follicles and ovulations in estrus cycling

mares would enhance embryo recovery from donor mares, provide multiple follicles

for collection of oocytes, and improve pregnancy rates from subfertile mares

(SQUIRES 2006). The basis of superovulation is manipulation of the hormones that

control the dominant follicle and inhibit the regression of subordinate follicles

(SQUIRES and MCCUE 2007).

The most effective drug to induce multiple ovulations so far is eFSH or recombinant

equine FSH. Although results are encouraging, between-mare variability is

considerable (SQUIRES 2006). Being able to identify donor mares that respond

favourably to eFSH based on follicular development, ovulation, and embryo recovery

would be a great advantage. In human medicine, color Doppler sonography has been

used for more than two decades to predict the outcome of assisted reproduction

technologies (BROUSSIN 2007; LAMAZOU et al. 2009). Correlations between

genital blood flow and ovarian response to hormonal treatment have also been found

in cows (HONNENS et al. 2008; HONNENS et al. 2009). Although ovarian blood flow

has already been investigated in the mare by BOLLWEIN et al. (2002) using

transrectal color Doppler sonography, currently there is no information about ovarian

blood flow during hormonal stimulation of superovulation in the mare.

At present, the vast majority (>95%) of horse embryos are transferred fresh or after

chilled storage for up to 24 h, whereas cryopreservation is rarely employed (STOUT

2012). The collection of embryos from the oviducts would be a great advantage, as in

concern of freezing or vitrification acceptable pregnancy rates (>55%) are achievable

only when embryos recovered at an early developmental stage (day 6 to 6.5; morula

to early blastocyst <300 µm in diameter) are transferred (CZLONKOWSKA et al.

26 Introduction

1985; SLADE et al. 1985). Although in recent studies it was also possible to vitrify

expanded blastocysts (CHOI et al. 2011; HINRICHS and CHOI 2012) using an

embryo biopsy technique for blastocoel fluid aspiration in order to shrinken (collapse)

the embryos, this technique requires micromanipulation capabilities and is, at the

moment, no technique for the widespread clinical use.

Thus, a laparoscopic minimally invasive technique for the catheterisation of the

equine oviduct might offer the opportunity for oviductal flushing and thereby the

collection of early stage embryos.

Hypothesis

It was hypothesised that superovulation with eFSH increases ovulation and embryo

recovery rate in the mare, and affects genital blood flow as well as steroid hormone

levels. Changes in genital blood flow might serve as useful parameters in order to

predict mares that respond favourably to a superovulation treatment.

To further optimize efficiency of equine embryo transfer, we hypothesize that entering

the infundibulum and subsequent orthograde flushing of the oviduct is possible by

surgical minimal-invasive laparoscopic techniques in the standing sedated mare.

Aims of the study

Therefore the aim of the present study was to induce superovulation in mares using

eFSH® and to study the effects of stimulation on genital blood flow using color

Doppler ultrasonography and to develop a minimal invasive laparoscopic method for

flushing the oviduct in the standing sedated mare.

In the first part of the study we compared follicle development and ovulation rates in

mares (6 mares in 5 cycles) after spontaneous ovulation and superovulation with

equine pituitary extract (eFSH®) when treatment start was restricted to follicle

diameter, and compared the embryo recovery rate when AI was performed 12h and

36h after hCG application with cooled-stored semen of a fertile stallion (Chapter 2).

Next, it was assessed if uterine and ovarian blood flow in mares during this

superovulation program differs from untreated controls using transrectal Doppler

Introduction 27

sonography. We further investigated if there were relationships between genital blood

flow, steroid hormone levels, and ovarian response (Chapter 3).

In Chapter 4 the development of a minimal invasive laparoscopic technique will be

described in two experiments: The first involved a transvaginal laparoscopic

approach (n=8), the second a laparoscopic flank approach (n=12). Passage of fluid

into the uterus was visualized by post operative hysteroscopy.

Embryo recovery rate following superovulation in mares 28

Chapter 2:

Embryo recovery rate following superovulation with equine pituitary extract

(eFSH®) in mares

Embryogewinnungsrate nach Superovulation mit equinem Hypophysenextrakt (eFSH®) bei der Stute

Melanie Köllmann, Jeanette Probst, Christine Baackmann, Jutta Klewitz,

Edward S. Squires1, Harald Sieme

Klinik für Pferde und Reproduktionsmedizinische Einheit der Kliniken der Stiftung

Tierärztliche Hochschule Hannover, 1Animal Reproduction and Biotechnology

Laboratory, Colorado State University, Fort Collins, USA

Pferdeheilkunde 24 (2008) 3 (Mai/Juni) 397-405

http://www.hippiatrika.com/download.htm?id=20080310

Embryo recovery rate following superovulation in mares 29

The extent of Melanie Witt´s (formerly M. Köllmann) contribution to the article is

evaluated according to the following scale:

A. has contributed to collaboration (0-33%)

B. has contributed significantly (34-66%)

C. has essentially performed this study independently (67-100%)

1. Design of the project including design of individual experiments: C

2. Performing of the experimental part of the study: B

3. Analysis of the experiments: C

4. Presentation and discussion of the study in article form: C

30 Embryo recovery rate following superovulation in mares

2.1 Abstract

Embryo recovery rate following superovulation with equine pituitary extract

(eFSH®) in mares.

Embryo recovery from single ovulating mares is approximately 50% per estrus cycle,

leading to a non-economical state of embryo transfer in the mare. An ability to

consistently induce multiple follicles and ovulations in estrus cycling mares would

enhance embryo recovery from donor mares, provide multiple follicles for collection

of oocytes, and improve pregnancy rates from subfertile mares. There have been

numerous approaches to superovulation of the mare. Injections of porcine FSH,

inhibin vaccines, equine chorionic gonadotropin (eCG) and GnRH have been of

limited success in stimulating multiple ovulations in the mare. Numerous studies have

shown that injection of equine pituitary extract (EPE) will result in three to four

ovulations per estrus cycle and two embryos. Recently, a commercial purified equine

pituitary extract product (eFSH®) has been available. In the present study six

normally cycling mares were investigated over five cycles and ovulation rate and

embryo recovery rate were compared between control cycles and stimulated cycles.

Cycle one and three were designed as control cycles without stimulation and

insemination. In cycles 2 and 4 mares were treated with 12.5 mg eFSH®

intramuscularly twice daily beginning when the diameter of the largest follicle was 20

to 25 mm. Prostaglandin was administered on the second day of eFSH® therapy.

Treatment with eFSH® was continued until follicle(s) were 32-35 mm in diameter. The

mares were subsequently allowed to ‘coast’ for 36 h, after which 2500 IU human

chorionic gonadotropin were administered to induce ovulation. Mares were

inseminated with 750 Mio. progressive motile sperms of a fertile warmblood stallion.

Embryo recovery was performed 6.5 days following ovulation. In the last cycle (5)

mares were treated and inseminated in the same way as in cycle 2 and 4, but without

eFSH® stimulation. Ovulation rate in control cycles was lower (1.3 ovulations) than in

eFSH® treated cycles (4.4 ovulations). The number of days of eFSH® treatment

required for reaching a follicle size of 32-35 mm was on average 4.0 days. Embryo

recovery rate in control mares was 1.2 per cycle, whereas in eFSH® treated mares ø

Embryo recovery rate following superovulation in mares 31

2.9 embryos could be flushed. The eFSH® protocol used in this study was efficient to

induce multiple ovulations and increase embryo recovery rate in mares. Albeit the

number of embryos obtained is quite encouraging, individual mare variation is

considerable. Being able to identify donor mares that respond favourably to eFSH

based on follicular development, ovulation and embryo recovery would be a great

advantage. In current studies a possible influence of follicle development and genital

blood flow on embryo recovery rate is investigated to identify „good donor mares“ in a

superovulation program.

Keywords: mare, reproduction, embryo transfer, superovulation, eFSH

32 Doppler sonography of the uterine and ovarian arteries during superovulation

Chapter 3

Doppler sonography of the uterine and ovarian arteries

during a superovulatory program in horses

M. C. Witt, H. Bollweina, J. Probst, C. Baackmann, E.L. Squiresb, H. Sieme

Clinic for Horses and Unit for Reproductive Medicine, aClinic for Cattle, University of

Veterinary Medicine Hanover Foundation, Buenteweg 9, 30559 Hanover, Germany;

bAnimal Reproduction and Biotechnology Laboratory, Colorado State University, Fort

Collins, Colorado, USA

Theriogenology 77 (2012) 1406–1414

doi:10.1016/j.theriogenology.2011.11.005

Doppler sonography of the uterine and ovarian arteries during superovulation 33

34 Doppler sonography of the uterine and ovarian arteries during superovulation

The extent of Melanie Witt´s contribution to the article is evaluated according to the

following scale:

A. has contributed to collaboration (0-33%)

B. has contributed significantly (34-66%)

C. has essentially performed this study independently (67-100%)

1. Design of the project including design of individual experiments: C

2. Performing of the experimental part of the study: B

3. Analysis of the experiments: B

4. Presentation and discussion of the study in article form: C

Doppler sonography of the uterine and ovarian arteries during superovulation 35

3.1 Abstract

Doppler sonography of the uterine and ovarian arteries during a

superovulatory program in horses

The aim of the present study was to investigate the effects of a gonadotropin

treatment to induce superovulation on ovarian and uterine blood flow and its

relationship with steroid hormone levels and ovarian response in mares, using color

Doppler sonography. Mares were examined sonographically in five consecutive

cycles for three days (t1 to t3) each during the follicular development phase (FDP)

beginning at a follicle size of ≥22 mm, and for four days (D-4 to D-1; D0 = Ovulation)

in the preovulatory phase (POP). After each examination, total estrogens (Etot) and

progesterone (P4) levels were determined in peripheral plasma. Cycles 1, 3, and 5

(c1, c3, c5) were unstimulated cycles (USC); in c2 and c4, the mares were stimulated

(SC) with eFSH and inseminated when in estrus at 12 and 24 h after hCG

administration. Embryo recovery was performed 6.5 days post ovulation. Cycle 5 (c5)

was an unstimulated cycle with hCG treatment, insemination, and embryo recovery.

Ovarian and uterine blood flow was quantified by the blood flow volume (BFV) and

the pulsatility index (PI) in ovarian and uterine arteries. The mean number of

ovulations and developing CL was 1.3 + 0.4 in USC and 4.4 + 3.1 in SC with no

difference (p≥0.05) between the ovaries within mares. No difference (p>0.05) was

observed in utBFV and utPI during FDP between USC and SC, but during POP, utPI

was lower (p<0.05) and utBFV higher (p<0.001) in SC than USC. The ovBFV was

higher (p<0.01) and ovPI lower (p<0.05) in SC compared to USC. All uterine and

ovarian blood flow parameters were related to the number of developing follicles in

SC. Parameters utPI (r=-0.67;p<0.001) and ovPI (r=-0.53; p<0.001) were negatively

correlated with the number of ovulations on t3, and with the number of collected

embryos on t3 (utPI:r=-0.81; p<0.001), D-4 (utPI:r=-0.64; p<0.0001), and D-1

(ovPI:r=-0.41; p<0.01). P4 levels were not positively correlated with utBFV (p>0.05),

but Etot concentrations (D-4: r=0.790; D-3: r=0.639; p<0.001; D-1: r=0.48; p<0.001)

and ovBFV from D-4 to D-1 (r= 0.64; p<0.001) in SC were. The results of the present

study show that in mares treatment with gonadotropins to induce superovulation is

36 Doppler sonography of the uterine and ovarian arteries during superovulation

associated with a marked increase in uterine and ovarian perfusion, concurrent with

the development of multiple follicles and an increase in Etot levels. The increased

blood flow seems to be related to the effectiveness of ovarian response to stimulation.

37 Laparoscopic techniques for investigating the equine oviduct

Chapter 4

Laparoscopic techniques for investigating the equine oviduct

M. Köllmann, A. Rötting, A. Heberling, H. Sieme

Clinic for Horses and Unit for Reproductive Medicine,

University of Veterinary Medicine Hannover Foundation

Equine Vet. J.

(2011) 43 (1) 106-111

doi: 10.1111/j.2042-3306.2010.00143.x

Laparoscopic techniques for investigating the equine oviduct 38

The extent of Melanie Witt´s (formerly M. Köllmann) contribution to the article is

evaluated according to the following scale:

A. has contributed to collaboration (0-33%)

B. has contributed significantly (34-66%)

C. has essentially performed this study independently (67-100%)

1. Design of the project including design of individual experiments: C

2. Performing of the experimental part of the study: C

3. Analysis of the experiments: C

4. Presentation and discussion of the study in article form: C

39 Laparoscopic techniques for investigating the equine oviduct

4.1 Abstract

Laparoscopic techniques for investigating the equine oviduct

Reasons for performing study: The diagnostic and therapeutic options for oviduct

disorders in the mare are limited. The current best techniques require exploratory

surgery under general anaesthesia or flank laparotomy.

Hypothesis: The orthograde flushing of the oviduct for diagnostic or therapeutic

options is possible using laparoscopic techniques in the standing sedated mare.

Methods: Development of a laparoscopic technique for catheterization of the

infundibulum and flushing of the oviduct (sterile methylene blue solution) in the

standing sedated mare was examined in two experiments. The first involved a

transvaginal laparoscopic approach, the second a laparoscopic flank approach.

Passage of fluid into the uterus was assessed by postoperative hysteroscopy.

Results: In experiment I, visualisation of the infundibulum was possible (left side 7/8

cases, right side in 6/8 cases). The beginning of the oviductal ampulla could be seen

in 3 of 8 cases on the left side. An adequate opening of the infundibulum and

visualisation or catheterisation of the abdominal ostium were not possible. In

experiment II, catheterisation of the ampulla was successful in 7 of 11 cases, and in

5 of these 7 cases the injected fluid could be identified in the uterus by postoperative

hysteroscopy.

Conclusion: A transvaginal laparoscopic approach to the oviduct is not appropriate

for oviductal flushing in the mare. However, a laparoscopic flank-approach permits

investigation and flushing of the oviduct.

Potential relevance: Laparoscopic flushing could become a practical method for

diagnosis and therapy of oviduct disorders and a minimally invasive technique for

collection of young embryos or the transfer of gametes (GIFT).

Discussion 40

Chapter 5

Discussion

41 Discussion

5 Discussion

During the last 20 years the number of equine ET´s performed annually worldwide

has grown enormously (International Embryo Transfer Society, IETS). Mean embryo

recovery per cycle from single ovulation mares is approximately 50%. An ability to

consistently induce multiple follicles and ovulations in estrus cycling mares would

enhance embryo recovery from donor mares (SQUIRES 2006). However, success

rates after superovulation and cryopreservation of embryos in horses are still lagging

behind those of cattle (SCHERZER et al. 2008).

At present, the vast majority (>95%) of horse embryos are transferred fresh or after

chilled storage for up to 24 h, whereas cryopreservation is rarely employed. Freezing

and vitrification of embryos <300 µm has been effective (SLADE et al. 1985;

ELDRIDGE-PANUSKA et al. 2005), but at the moment it requires recovery of

embryos on Day 6 after ovulation in clinical praxis.

Therefore the aim of the present study was to induce superovulation in mares using

eFSH® and to study the effects of stimulation on genital blood flow using color

Doppler ultrasonography and to develop a minimal invasive laparoscopic method for

entering the infundibulum and subsequent orthograde flushing of the oviduct in the

standing sedated mare.

5.1 Superovulation

The eFSH® protocol used in this study was efficient to induce multiple ovulations in

mares. The mean number of ovulations and developing CL was 1.3 + 0.4 in

unstimulated cycles (USC) and 4.4 + 3.1 in stimulated cycles (SC) with no

differences (p ≥ 0.05) between the left and right ovaries of both sides. The protocol

was further efficient to increase embryo recovery rate in mares similar to the studys

of SQUIRES and MCUE (2007), LOGAN et al. (2007) and RAZ et al. (2009a). The

total embryo recovery rate was higher (p<0.05) in stimulated cycles (2.9 ± 1.7)

compared to control cycles (1.2 + 0.4).

Discussion 42

But embryo recovery rates per ovulation were lower in SC compared to c5

(unstimulated cycle with hCG treatment, insemination, and embryo recovery) cycles

(66% vs. 87.5%). Several earlier studies noted that with increased number of

ovulations, embryo recovery per ovulation remained the same or decreased,

following repeated injections of either EPE or eFSH (ROSAS et al. 1998;

ALVARENGA et al. 2001; SCOGGIN et al. 2002; SQUIRES 2006; WELCH et al.

2006; LOGAN et al. 2007; ARAUJO et al. 2009; RAZ et al. 2009b). Several factors

can affect the embryo/ovulation ratio, such as oocyte quality (GEARY et al. 1989;

WOODS 1998; CARNEVALE et al. 1999) and the capability of the oocyte to enter the

oviduct via the ovulation fossa (CARMO et al. 2006). According to CARMO et al.

(2006), there was no significant difference in the number of oocytes that appeared in

the oviduct from superovulated mares compared to control mares supporting the

concept that oocyte development and maturation may be a critical factor affecting the

embryo per ovulation ratio in superovulated mares (MEYERS-BROWN et al. 2011).

The findings that an increased number of anovulatory follicles are present after

superovulation regimens (ALVARENGA et al. 2001; SQUIRES 2006; WELCH et al.

2006; RAZ et al. 2009b) can also support the concept that a less than optimum

embryo/ovulation ratio may be a function of oocyte dysfunction.

The between-mare variability in the present study was considerable, as it was

observed in earlier eFSH studies (SQUIRES 2006). This suggests that no one

treatment will work in a similar fashion in all the mares. To further work on the

problems of superovulation in the mare, ROSER et al. (2012) considered if follicular

waves in individual mares need to be evaluated before treatment. They further

assumed that mares then can be subjected to different protocols. Furthermore, they

considered if endogenous levels of FSH, LH, estradiol, inhibin, activin, IGF-I, and

perhaps other hormones or paracrine/autocrine factors in individual mares need to be

measured during the cycle before treatment so as to control the response to

treatment at any one time.

However, understanding the physiology and endocrinology of each reproductive

stage of the mare, will be the key in determining the optimum treatment regimen for

43 Discussion

mares in general. Each mare will have to be approached with the idea in mind that

she may need a different combination of drugs to initiate the desired response.

Beside these considerations, the development and efficacy of recombinant equine

gonadotropins (reFSH and reLH) have recently been reported (JABLONKA-SHARIFF

et al. 2007; JENNINGS et al. 2009; MEYERS-BROWN et al. 2010). The single chain

recombinant products are not contaminated with other hormones or pathogens and

are biologically active in stimulating follicular development and ovulation in the mare.

So more conststant superovulation results may be expected with recombinant

products in future.

But nevertheless, being able to identify donor mares that respond favorably to a

superovulation treatment before the hormone therapy, like demonstrated in the cow

(HONNENS et al. 2008, 2009) or in women, (WEINER et al. 1993; ZAIDI et al. 1996)

would be a great advantage.

In human medicine, color Doppler sonography has been used for more than two

decades to predict the outcome of assisted reproduction technologies (BROUSSIN

2007; LAMAZOU et al. 2009). For example, in women undergoing hormonal

treatment, transvaginal color Doppler sonography has been successfully used to

study ovarian blood flow during IVF cycles, and it was found to be related to ovarian

response to stimulation (WEINER et al. 1993; ZAIDI et al. 1996). Correlations

between genital blood flow and ovarian response to hormonal treatment have also

been found in cows (HONNENS et al. 2008, 2009). Therefore, the aim of this study

was to investigate and compare uterine and ovarian blood flow in mares during a

superovulation program with untreated controls using transrectal Doppler

sonography, and to investigate if there are relationships between genital blood flow,

steroid hormone levels, and ovarian response.

5.2 Doppler Ultrasonography

The hormonal superovulatory treatment with eFSH led to an increase in uterine and

ovarian blood flow and estradiol levels concurrent with the development of multiple

follicles.

Discussion 44

The results of the present study show that mares treatment with gonadotropins to

induce superovulation is associated with a marked increase in uterine and ovarian

perfusion, concurrent with the development of multiple follicles and an increase in Etot

levels. The increased blood flow seems to be related to the effectiveness of ovarian

response to stimulation. The marked increase in ovarian blood flow volume and the

decrease in ovarian PI in SC in the present study were also reported in cows

undergoing hormonal treatment to induce superovulation (HONNENS et al. 2009).

In the present study, we did not detect any differences in uterine or ovarian blood

flow in the presence of one or two follicles, but with increasing numbers of follicles,

the uterine/ovarian BFV increased and uterine/ovarian PI decreased in eFSH cycles.

These findings correspond to a study in human medicine where the uterine PI was

correlated with the number of follicles and collected oocytes in IVF patients

(CACCIATORE et al. 1996).

In the present study, correlations were observed between the uterine and ovarian

BFV and the number of further ovulations in SC. Both parameters were correlated in

the preovulatory phase but also in the follicle developing phase (t2, t3) so that the

BFV might be a parameter that is useful for predicting ovarian response to

superovulatory program in mares.

In contrary to a study in cows, where no significant correlations between the ovBFV,

ovPI, and the number of follicles that developed in response to an eCG hormonal

treatment were detected (HONNENS et al. 2009) we found relationships between the

ovarian BFV (t3, D-4 to D-1), ovPI (t3, D-1) as well as the uterine BFV and utPI and

the number of developing follicles.

Several studies have shown that the superovulatory response in cattle is positively

related to the pools of primordial and growing follicles in the bovine ovary

(MONNIAUX et al. 1983; SINGH et al. 2004). The superstimulatory response can be

predicted by the number of follicles ≥2 mm at wave emergence. The number of

follicles ≥5 mm after ovarian superstimulation can be expected to be approximately

71% of the number of follicles ≥2 mm at the time of wave. A study with color Doppler

ultrasonography revealed that small follicles with blood flow become larger around

the follicle selection period compared with small follicles without blood flow (ACOSTA

45 Discussion

et al. 2005). Since blood supply to individual follicles will provide gonadotropins and

nutrients that support follicular growth, follicle cohorts which include many follicles

with detectable blood flow will show a good response to superovulatory treatment.

Therefore, examination of the number of small follicles which have detectable blood

flow at the start of gonadotrophin treatment may be a useful index as a predictor of

superovulatory response.

In the present study, there were no correlations between the ovarian blood flow

parameters during t1 to t3 and D-4 to D-1 and the embryo recovery rate, which was

found before in studies in cows (HONNENS et al. 2009). But the uterine PI was

correlated with the number of collected embryos on t3 and D-4. Therefore the color

Doppler sonography of the uterine arteries might be useful in predicting the success

of a superovulatory regime in the mares as it has been used in women in IVF

treatments (CACCIATORE et al. 1996; OZTURK et al. 2004). Nevertheless this

correlation should be verified in further studies.

5.3 Laparoscopy of the equine oviduct

Currently, the best techniques for diagnosis and therapy of tubal disorders are

exploratory surgery under general anaesthesia or lateral flank laparotomy with

flushing of the oviduct (BENNETT 2007).

In the present study a minimal invasive laparoscopic technique for oviductal flushing

was successfully established. With the lateral flank approach, an excellent

visualisation of the ovary, infundibulum and the whole oviduct from the side was

possible in all cases. After “opening” the infundibulum, the abdominal ostium could

be seen in 8 cases (72.7%). Catheterisation of the ampulla was successful in 7 of 11

cases (63.6%), and in 5 of these 7 cases the injected fluid could be identified in the

uterine lumen by postoperative hysteroscopy. The laparoscopic flushing of the

oviduct is possible with the lateral flank approach and might be used for the collection

of tubal stage embryos in future.

Laparoscopic techniques for a variety of procedures in horses have been described

in recent years (COLBERN and REAGAN 1987; HANSON and GALUPPO 1999;

Discussion 46

WALMSLEY 1999). Standing female urogenital endoscopic surgery is facilitated by

the more dorsal location of the organs of the female reproduction tract. The most

common reason for laparoscopic surgery on the female urogenital system is

ovariectomy; however, the technique has been used to diagnose periparturient or

reproductive diseases and to perform surgical embryo transfer. Standing surgical

approaches avoid the risk and expense of general anesthesia, but these techniques

are limited by the temperament and size of the patient and the availability of facilities

for restraint (DECHANT and HENDRICKSON 2000). The reported advantages

include increased intraoperative visualisation, the use of smaller incisions, less

extensive manipulation of the abdominal viscera, reduced post operative pain and

reduced periods of convalescence compared with corresponding procedures

performed using conventional methods (HANSON and GALUPPO 1999;

WALMSLEY 1999). The disadvantages of laparoscopy might be the financial

investment associated with the equipment and consumables and the relative

technical difficulty of certain procedures. We assume that the procedure has no

negative influence on fertility because all mares were used for further research and

ET programmes and continued to have good fertility. Negative effects might be

inflammation or adhesions of the fimbria or adhesions in the oviduct which might lead

to an obstruction.

Earlier, the laparoscopically guided administration of PGE2-laced triacetin gel onto

the surface of the oviducts was used to hasten embryonic transport (ROBINSON et

al. 2000) and as a therapeutic option in mares that were presumed to have tubal

obstructions (ALLEN et al. 2006).

The transvaginal approach used in Experiment I was unlike the situation in cattle

(BESENFELDER et al. 2001) inadequate for an investigation or catheterisation of the

oviduct. The visualisation of the medial side of both ovaries was possible and, after

turning the ovary to the mid side, visualisation of the infundibulum was possible in 7

of 8 cases (87.5%) on the left side and in 6 of 8 cases (75.0%) on the right side. The

beginning of the oviductal ampulla could be seen in only 3 of 8 cases (37.5%) on the

left side. An adequate opening of the infundibulum and thereby a visualisation or

catheterisation of the abdominal ostium was not possible with the transvaginal

47 Discussion

laparoscopic approach. Due to the anatomical characteristics of the equine ovary and

infundibulum, catheterisation of the abdominal ostium is only possible following the

180° turn of the ovary in cranio-medial direction and a lifting of the infundibulum

which was not possible using this approach. Nevertheless, the technique used in

Experiment I could be of benefit for the application of a PGE-gel onto the outer

surface of the ovary and ampulla because both sites could be reached in one session.

The techniques of natural orifice transluminal endoscopic surgery (NOTES) using

laparoscopic and endoscopic instrumentation transvaginally into the mare’s abdomen

is seemingly well tolerated and safe and provides adequate observation of most

structures within the dorsal caudal region of the abdomen on the side of endoscope

or laparoscope insertion (ALFORD and HANSON 2010). The transvaginal approach,

to the equine abdomen is not novel. Transvaginal ovariectomy, by colpotomy, is well

established in mares (WALKER and VAUGHAN 1980; COLBERN and REAGAN

1987; EMBERTSON 2006).

The transvaginal laparoscopic approach to the oviduct is not appropriate for oviductal

flushing in the mare. However, a laparoscopic flank-approach permits investigation

and flushing of the oviduct. Laparoscopic flushing could become a practical method

for the diagnosis and therapy of oviduct disorders.

Recently modification of vitrification methods allowed the establishment of a

vitrification technique that supports normal pregnancy rates for expanded blastocysts

up to 650 µm in diameter. This technique uses blastocyst collapse, vitrification

medium containing ethylene glycol and galactose, in combination with warming

medium containing sucrose, and a low-volume vitrification device (HINRICHS and

CHOI 2012). Vitrification of expanded equine blastocysts might be one method to

make the embryo transfer management of equine reproduction more efficient. On the

other hand the laparoscopic technique used in this study has the further potential to

function as a minimally invasive technique for collection of young embryos <300 µm,

which are then available for cryopreservation. This would give potential for simplifying

recipient mare management and facilitating long-term storage and international

transport of embryos.

Discussion 48

The results of the present study show that in mares treatment with gonadotropins to

induce superovulation is associated with a marked increase in uterine and ovarian

perfusion, concurrent with the development of multiple follicles and an increase in Etot

levels. The uterine PI was correlated with the number of collected embryos on t3 and

D-4. The transrectal color Doppler sonography of the uterine arteries might be useful

in predicting the success of a superovulatory regime in the mares if this correlation

can be verified in further studies. The laparoscopic flank-approach permits

investigation and flushing of the oviduct and could become a practical method for

diagnosis and therapy of oviduct disorders and a minimally invasive technique for

collection of young embryos or the transfer of gametes (GIFT).

Summary 49

Chapter 6

Summary

50 Summary

6 Summary Melanie Witt - Investigations on genital blood flow and embryo recovery after

superovulation with eFSH® and on laparoscopic techniques for flushing the oviduct in

the mare.

The aim of the present study was to investigate the effects of a gonadotropin

treatment to induce superovulation on ovarian and uterine blood flow and its

relationship with steroid hormone levels and ovarian response in mares, using color

Doppler sonography and to develop a minimal invasive laparoscopic method for

flushing the oviduct in the standing sedated mare.

Methods: In the present study six mares were investigated over five cycles and

ovulation rate and embryo recovery rate were compared between unstimulated

cycles (USC= c1, c3, c5) and stimulated cycles (SC= c2, c4). Cycle 5 (c5) was an

unstimulated cycle with hCG treatment, insemination, and embryo recovery. The

mares were examined by transrectal color Dopler sonography for three days (t1 to t3)

during the follicular development phase (FDP) beginning at a follicle size of ≥22 mm,

and for four days (D-4 to D-1; D0 = Ovulation) in the preovulatory phase (POP).

Ovarian and uterine blood flow was quantified by the blood flow volume (BFV) and

the pulsatility index (PI) in ovarian and uterine arteries. Total estrogens (Etot) and

progesterone (P4) levels were determined in peripheral plasma each day. In c2 and

c4, the mares were stimulated with 12.5 mg eFSH® (Bioniche-Animal Health, Athens,

GA, USA) im twice daily beginning when the diameter of follicles was 20 to 25 mm.

Prostaglandin F2α was administered on the second day of eFSH® therapy. The eFSH

treatment was continued until follicle(s) were 32-35 mm in diameter. Following a 36 h

lasting coasting period 2500 IU hCG (Ovogest®, Intervet, Unterschleißheim) was

administered iv to induce ovulation. Mares were inseminated with cooled-stored

semen (750 Mill. progressively motile spermatozoa) 12 and 36 h post hCG. Embryo

recovery was performed 6.5 days after ovulation (D0).

The development of a laparoscopic technique for catheterisation of the infundibulum

and flushing of the oviduct in the standing sedated mare was examined using 10

mares in two different experiments. The first involved a transvaginal laparoscopic

Summary 51

approach (n=8), the second a laparoscopic flank approach (n=12). Passage of fluid

into the uterus was assessed by post operative hysteroscopy.

Results: The mean number of ovulations and developing CL was 1.3 + 0.4 in USC

and 4.4 + 3.1 in SC. The mean number of collected embryos was 1.2 vs 2.9. No

difference (p>0.05) was observed in utBFV and utPI during FDP between USC and

SC, but during POP, utPI was lower (p<0.05) and utBFV higher (p<0.001) in SC than

USC. The ovBFV was higher (p<0.01) and ovPI lower (p<0.05) in SC compared to

USC. All uterine and ovarian blood flow parameters were related to the number of

developing follicles in SC. Parameters utPI (r=-0.67;p<0.001) and ovPI (r=-0.53;

p<0.001) were negatively correlated with the number of ovulations on t3, and with the

number of collected embryos on t3 (utPI:r=-0.81; p<0.001), D-4 (utPI:r=-0.64;

p<0.0001), and D-1 (ovPI:r=-0.41; p<0.01).

By transvaginal approach (Experiment I) visualisation of the infundibulum was

possible (left side 7/8 cases, right side in 6/8 cases). Access to the oviductal ampulla

could be achieved in 3 of 8 cases on the left side. Stretching of the infundibulum and

visualisation or catheterisation of the abdominal ostium was not possible. The

laparoscopic approach (Experiment II) enables catheterisation of the ampulla (7/11),

and in 5/7 the injected fluid could be visualized in the uterus by postoperative

hysteroscopy.

Potential relevance: The results of the present study show that in mares treatment

with gonadotropins to induce superovulation is associated with a marked increase in

uterine and ovarian perfusion, concurrent with the development of multiple follicles

and an increase in Etot levels. The uterine PI on t3 and D-4 was correlated with the

number of collected embryos. Transrectal color Doppler sonography of the uterine

arteries might be useful in predicting the success of a superovulatory regime in the

mare, however, this correlation needs to be verified by further studies. The

laparoscopic flank-approach permits visualization, entering and orthograde flushing

of the oviduct. This procedure bears the potential to aid in diagnosis and therapy of

oviductal disorders and represents a minimal invasive technique in the standing

sedated mare for collection of early stage embryos as well as intraoviductal transfer

of gametes (GIFT).

52 Zusammenfassung

Chapter 7

Zusammenfassung

Zusammenfassung 53

7 Zusammenfassung

Melanie Witt - Investigations on genital blood flow and embryo recovery after

superovulation with eFSH® and on laparoscopic techniques for flushing the oviduct in

the mare.

Ziel der Arbeit war es Zusammenhänge zwischen der ovariellen Stimulationsantwort

nach einer Superovulationsbehandlung bei Stuten und dem mittels transrektaler

Dopplersonographie gemessenen genitalen Blutfluss sowie den

Steroidhormonwerten im Blut zu untersuchen. Des Weiteren war es Ziel dieser

Studie eine minimal-invasive laparoskopische Technik zur Katheterisierung des

Infundibulums und Spülung des Eileiters an der stehenden, sedierten Stute zu

entwickeln.

Methode: Sechs Stuten wurden über 5 Zyklen untersucht und die Anzahl der

Ovulationen und gewonnenen Embryonen aus den stimulierten Zyklen mit denen der

Kontrollzyklen verglichen. Zyklus 1 und 3 wurden als Kontrollzyklen ohne Stimulation

und Besamung definiert. Im zweiten und vierten Zyklus wurden die Stuten jeweils ab

einer Follikelgröße von 22-25 mm mit einer zweimal täglichen Gabe von 12,5 mg

eFSH® (Fa. Bioniche-Animalhealth, Athens, GA, USA) bis zu einer durchschnittlichen

Follikelgröße von 32-35 mm stimuliert. Am zweiten Tag der eFSH® Behandlung

wurden 250 μg Cloprostenol (Estrumate®) intramuskulär injiziert. Nach einer 36

Stunden dauernden coasting“-Periode wurde 2500 IE humanes Choriogonadotropin

(Ovogest® Intervet, Unterschleißheim) i.v. appliziert und die Stuten 12 und 36

Stunden später mit gekühltem Frischsperma (750 Mio. progressiv motile Spermien)

inseminiert. Die Embryonengewinnung erfolgte an Tag 6,5 nach Ovulation durch

Uterusspülung. Im fünften Zyklus wurde eine Embryonengewinnung nach

Ovulationsinduktion und zweimaliger Besamung wie in den Zyklen 2 und 4, jedoch

ohne vorherige Stimulation durchgeführt. Zusätzlich wurden der uterine und ovarielle

Blutfluss täglich ab einer Follikelgröße von 22 mm bis zur Ovulation hin mittels

transrektaler Dopplersonographie dokumentiert und mithilfe der Parameter Pulsatility

Index (PI) und Blutflussvolumen (BFV) definiert. Die Entwicklung einer

laparoskopischen Technik zur Katheterisierung des Infundibulums und der Spülung

54 Zusammenfassung

des Eileiters erfolgte an 10 Stuten in zwei Experimenten. Im Ersten wurde ein

transvaginaler Zugang (n=8), im Zweiten ein lateraler Flankenzugang (n=12) gewählt.

Die Flüssigkeitspassage in den Uterus wurde mittels Hysteroskopie nachgewiesen.

Ergebnisse: Im Rahmen der in dieser Studie durchgeführten

Superovulationsbehandlung mit eFSH war die durchschnittlichen Ovulationen in den

Kontrollzyklen und stimulierten Zyklen (1,3 versus 4,4 OV), die

Embryonengewinnungsrate 1,2 vs 2,9 Embryonen. Mittels Dopplersonographie

konnte gezeigt werden, dass es im Rahmen einer Superovulationsbehandlung zu

einem gesteigerten uterinen und ovariellen Blutfluss kommt, der mit der Entwicklung

multipler Follikel und einer Erhöhung des Östradiolwertes im Blut einhergeht. Die

utPI (r=-0,67; p<0,001) und ovPI (r=-0,53; p<0,001) waren an t3 negativ mit der

Anzahl an OV korreliert sowie mit der Anzahl an gewonnenen Embryonen an t3

(utPI:r=-0,81; p<0,001), D-4 (utPI:r=-0,64; p<0,0001), and D-1 (ovPI:r=-0,41; p<0,01).

Der transvaginale Zugang ermöglichte eine Visualisierung der medialen Seite beider

Ovarien und nach Drehung des Ovars nach median konnte der Beginn der Ampulla

in 3/8 (37.5%) Fällen am linken Ovar gesehen werden. Eine Öffnung des

Infundibulums und Katheterisierung der Ampulla war mit dem transvaginalen Zugang

nicht möglich. Der laterale Flankenzugang machte eine Visualisierung des Ovars,

Infundibulums und des gesamten Oviducts in allen Fällen möglich. Nach der

Auffaltung des Infundibulums war das Ostium abdominale in 8 (72.7%) Fällen

sichtbar und eine Katheterisierung der Ampulla gelang in 7/11 (63.6%) Fällen. In 5

der 7 Spülungen konnte die Spülflüssigkeit intrauterine mittels Hysteroskopie

nachgewiesen warden.

Ausblick: In der vorliegenden Arbeit korrelierte der uterine PI mit der Anzahl

gewonnener Embryonen an t3 und D-4, so dass die transrektale Dopplersonographie

der uterinen Arterien hilfreich sein könnte den Erfolg einer

Superovulationsbehandlung vorherzusagen. Jedoch sollten in weiteren Studien diese

Ergebnisse bestätigt werden. Die laparoskopische Eileiterspülung ist mittels eines

lateralen Flankenzugangs möglich und könnte für die Gewinnung tubaler

Embryonalstadien und den intratubalen Transfer von Gameten (GIFT) zur Verfügung

stehen.

References 55

Chapter 8

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84 Appendix

9 Appendix

9.1 Material and methods study I and II

9.1.1 Animals

Six clinically healthy and normally cycling mares (3 Trotter, 2 Warmblood, 1

Thoroughbred) between 4 to 14 years of age were examined at the Unit for

Reproductive Medicine of the University of Veterinary Medicine Hannover, Germany,

during five estrus cycles in the breeding season 2007. Four mares were nulli-, one

uni-, and one pluripar. They were kept in barns and on pasture and were fed with hay

and oats. This animal study was approved by the Hannover Regional Administration

(reference number: 33.12-42502-04-07/1235) and conducted in compliance with the

German Protection of Animals Act (TierschG).

9.1.2 Mare management

Uterine swabs were collected transcervically from all mares at the start of the study

for bacteriological and cytological assessments. Mares were teased daily using a

stallion and were examined daily using B-mode sonography. Estrus was defined as

the time period during which mares showed behavioral signs of estrus and

endometrial edema on sonographic B-mode images. The day of ovulation (Day 0)

was defined as the day of disappearance of the preovulatory follicle or, if there were

multiple follicles, as the day of disappearance of the first follicle.

9.1.3 Study design

The mares were examined during five estrus cycles (Table 9-1). Each mare received

no treatment in the first cycle (c1); treatment with equine FSH (eFSH®, Bioniche-

Animal Health, Athens, GA, USA), insemination, and embryo flushing in the second

cycle (c2); no treatment in the third cycle (c3); and treatment with eFSH®,

insemination, and embryo flushing in the fourth cycle (c4). In the fifth cycle (c5),

mares were also inseminated and embryo flushing was performed, but without any

stimulation of multiple ovulations. Mares were treated with hCG to induce ovulation.

Appendix 85

Following embryo recovery after stimulated cycles (SC), the mares received

prostaglandin on D 6.5 (250 μg of cloprostenol, Estrumate®, Essex-Tierarznei,

Munich, Germany) to induce luteolysis. The short cycles following luteolysis were not

used for examination, and after the next ovulation c3 and c5 were continued.

Color Doppler assessments of uterine and ovarian blood flow were performed on

every cycle day starting at a follicular size of 22 mm until ovulation. Blood samples

were taken after each examination. For better comparison of data, results were

presented for the first three days of the follicular development phase (FDP) t1 to t3

(t1=starting with a follicle diameter of 22 mm, and after the first eFSH treatment in

c2/c4, which were on different days of the cycle in each mare). For comparison of

genital blood flow in the preovulatory phase (POP), data of cycle days -4 to -1 (D-4 to

D-1) were compared.

Tab. 9-1: Overview of the five investigated estrus cycles and the according treatments (eFSH®-treatment, induction of ovulation with hCG, insemination and embryo collection 6.5 days post OV respectively)

cycle eFSH®

treatment

Induction of

ovulation with hCG

insemination embryo collection 6.5

days post OV

1 - - - -

2 + + + +

3 - - - -

4 + + + +

5 - + + +

9.1.4 Superovulation treatment

In stimulation cycles, mares were treated with 12.5 mg of eFSH® im (lateral neck

musculature) twice daily (7 a.m. and 7 p.m.), starting when follicular diameter was

>22 mm. On the second day of eFSH® therapy (following the third eFSH injection),

prostaglandin (250 μg of cloprostenol, Estrumate®, Essex-Tierarznei, Munich,

Germany) was administered im (lateral neck musculature). Treatment with eFSH®

was continued until follicle(s) were 32-35 mm in diameter (duration between 2.5 - 8.5

days; Ø 4.0 ± 1.6 days of FSH® treatment; Ø 100 ± 40 mg eFSH® in total ). The

mares were subsequently allowed to “coast” for 36 h, and were then injected with

86 Appendix

2500 IU of iv hCG (Ovogest®, Intervet, Unterschleissheim, Germany) to induce

ovulation. Mares were inseminated with cooled-stored semen (750 x 106

progressively motile spermatozoa, 20 ml in skin-milk extender INRA 82 collected

from one fertile warmblood stallion on the same day) 12 and 36 h after hCG injection

(Fig. 9-1). Insemination was performed using a sterile lubricated gloved arm guiding

the insemination pipette (Besamungspipette Universal für Pferde, Minitüb GmbH,

Tiefenbach, Germany) transcervically into the uterine body. The plunger of the

syringe was slowly depressed, introducing the semen.

D4-5 D6.5

OV foll.-Ø until foll.-Ø 36h “coasting“ OV

≥22-25mm ≥32-35mm ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ eFSH

® (2xdaily 12.5mg) 2500IU 1.ins. 2.ins. embryo

PGF2α at 2. treatment day hCG iv +12h +36h collection

Fig. 9-1: eFSH® treatment protocol in cycle 2 and 4 (D= day, foll= follicle, OV=

ovulation, ins.= insemination)

9.1.5 Embryo collection

Transcervical embryo collection in cycles 2, 4 and 5 was performed by two persons

(JP, JK) 6.5 days after ovulation using a standard non-surgical procedure (IMEL et al.

1981). A balloon catheter (Ø 32 Ch., Embryospülkatheter, Minitüb, Tiefenbach,

Germany) was introduced into the uterus and the uterus was flushed four times with

1 L of 37 °C warm Dulbecco´s phosphate buffered saline (PBS) supplemented with

0.1 g/L bovine serum albumin fraction V (Sigma-Aldrich, Taufkirchen). The solution

was collected in a Petri dish (Miniflush Schale®, Minitüb, Tiefenbach, Germany) after

being filtered through a filter (Miniflush Filter®, pore size 73 µm Minitüb, Tiefenbach,

Germany). The collected fluid was inspected under a transmitted-light microscope

(Wild Hamburg, Germany) with 20 to 115 fold magnification. If not all expectable

embryos were found in the flush a second flush with four times of 1 l Dulbecco´s PBS

solution was performed directly following injection of 20 IU oxytocin iv (Oxytocin® Fa.

WDT, Garbsen, Germany). Oocytes were not included in the research.

Appendix 87

9.1.6 Transrectal Doppler sonography

In all cycles, examinations were carried out daily (starting at a follicle diameter of

22 mm) at the same time of day (09.00 to 11.00 a.m.). Doppler measurements were

always performed by the same operator (JP) using an ultrasound device (Logic P5,

GE Healthcare, Solingen, Germany) equipped with a 4 to 10 MHz linear probe (I 739,

GE Healthcare, Solingen, Germany). Collected data were digitized on DVDs

(Verbatim, 4.7 GB, Eschborn, Germany) and saved with e-Film Workstation 1.5.3 (E-

Film Medical Inc. Toronto, Canada) using the software program Pixelflux Scientific

(Chameleon-Software, Leipzig, Germany).

To measure uterine blood flow by transrectal ultrasonography, uterine arteries were

located in B-mode (brightness modulation). At the division from the aorta the A. iliaca

externa was followed latero-ventrally until the A. circumflexa iliumprofunda and the A.

uterina separated from the A. iliaca externa. The A. uterina was measured in

diameter shortly after the origin of the A. uterina.

The Aa. ovaricae were found following the ovarian vessel convolute in the

mesovarium. The A. ovarica was followed retrograde, and proximal of the separation

of the Ramus uterinus of the A. ovarica the diameter of the A. uterina was measured.

Following the measurement in B-mode, the CF-mode (continuous-wave Doppler)

was activated and the probe was positioned that the maximum blood flow was

visible. Using the PW-mode (pulsed-wave Doppler) several Doppler waves were

documented. For analysis of uterine and ovarian blood flow, the values of two similar

uniform consecutive pulse waves were averaged using the software program

PixelFlux Scientific (Chameleon-Software, Leipzig, Germany). Ovarian and uterine

blood flow were characterized by parameters pulsatility index (PI) and blood flow

volume (BFV), which were described in detail in earlier studies for ovarian

(HONNENS et al. 2009; BOLLWEIN et al. 2004) and uterine blood flow (HONNENS

et al. 2009; BOLLWEIN et al. 1998, 2000). The program PixelFlux Scientific

calculates the peak systolic velocity, the end diastolic velocity and the time averaged

maximum velocity over the time of a cardiac cycle (TAMV). From these data the

pulsatility index (PI) is calculated. Using the value of TAMV and the vessels diameter

the blood flow volume (BFV) can be calculated.

88 Appendix

Fig. 9-2: Ultrasound investigation of the left A. uterina of a mare one day before ovulation 1: A. uterina identification in B-mode, 2: measurement of the arteries diameter, 2: uterina blood flow using CF-mode, 4: measurement in PW-mode

Fig. 9-3: Ultrasound investigation of the right A. ovarica of a mare two days before ovulation 1: ovarian vessels convolute in the mesovarium in CF-mode, 2: measurement of the arteries diameter, 2: ovarian blood flow using CF-mode, 4: measurement in PW-mode

vessels convolute A. ovarica

Appendix 89

9.1.7 Blood collection, progesterone and estrogen analysis

EDTA-blood samples were obtained from the jugular vein using a vacutainer system

(Becton Dickinson Vacutainer System containing EDTA, Belliver Industrial Estate,

Plymouth, UK) after each scan. Plasma was separated after 2 h (centrifugation for

10 min at 1800 x g) and frozen in Sarstedt-tubes (Sarstedt AG & Co/ Nümbrecht) at -

20°C until analysis of plasma progesterone (P4) and total estrogen (Etot) levels.

Samples were assayed in the endocrinologic laboratory of the Clinic for Cattle,

School of Veterinary Medicine Hannover, Germany. They were analysed in duplicate,

and a separate assay was performed for each trial.

P4 was estimated by enzyme immunoassay (EIA) as published earlier (PRAKASH et

al. 2000). The intra- and inter-assay coefficients of variation were 2.7% and 3.8%,

respectively.

Plasma levels of Etot were estimated following hydrolysis and extraction by EIA as

described previously (MEYER et al. 2000). The intra- and inter-assay coefficients of

variation were 5.4% and 9.6%, respectively.

9.1.8 Statistical analysis

Statistical analysis was carried out using SPSS for Windows, version 15.0 (SPSS

Inc., USA). The Shapiro-Wilk test was used to test for normal distribution of data. All

data were presented as mean ± S.E.M. for better comparison. Student`s t-test

(normally distributed data) and the Mann-Whitney U-test (not normally distributed

data) were used to determine differences in measurements between groups and

days.

Correlations between BFV, PI values and number of follicles, ovulations, embryos

and plasma hormone levels were determined by the Pearson (normally distributed

data) or Spearman (not normally distributed data) correlation coefficient (r).

Differences and correlations with p<0.05 were considered to be statistically

significant.

90 Appendix

9.2 Material and methods study III

The study was performed in the Clinic for Horses and Unit for Reproductive

Medicine, University of Veterinary Medicine Hannover, Germany. Experiment I was

conducted between July 2007 and September 2007 and experiment II between

November 2008 and June 2009. This research was conducted in accordance with

Animal Welfare (No. 33.12-42502-04-07/1235).

9.2.1 Animals

Ten mares (3-16 years old) were examined and randomized by replicate into two

groups for experiment I (8 surgeries) and II (12 surgeries). Two mares underwent

both experiments (Table 9-3). For both procedures feed was withheld for 24 hours

preoperatively. Animals had full access to water. The mares were judged to be in

good physical health on the basis of an initial physical examination. Gynaecologic

examinations were performed before surgery to assess the day of the oestrus cycle.

The time interval between surgeries in the same mare was between 3 and 102 days

(Ø 45 days).

9.2.2 Preoperative management

For both experiments, mares received a central venous catheter (Vygonüle T®,

Vygon, Aachen, Germany) in the jugular vein for the duration of the preoperative

preparations and surgery time (experiment I: 81.25 min, experiment II Ø 26.8 min).

Antimicrobial therapy was provided with sodium benzylpenicillin (Penicillin

„Grünenthal“ 10 Mega®, Grünenthal, Aachen, Germany, 22.000 IU/kg iv) and

gentamicin (Genta 100®, cp-pharma, Burgdorf, Germany, 6.6 mg/kg sid, iv)

administered as a single dose immediately preoperatively. Flunixin meglumine

(Flunidol®, cp-pharma, Burgdorf, Germany, 1.1 mg/kg bwt, iv) was given to provide

perioperative analgesia. Post operative analgesia was maintained with flunixin

meglumine (Flunidolgel®, cp-pharma, Burgdorf, Germany, 1.1 mg/kg bwt, per os) for

48 hours or longer as required.

Appendix 91

The mares were restrained in stocks and sedated with a combination of detomidine

hydrochloride (Domosedan®, Pfizer, Karlsruhe, Germany, 10 μg/kg) and butorphanol

tartrate (Torbugesic®, Fort Dodge, Würselen, Germany, 10 μg/kg) administered

intravenously. Detomidine and butorphanol (0.5–1 μg/kg, iv) were administered

repeatedly as needed during surgery.

9.2.3 Instruments used in experiment I

1) Universal tube (Ø 12.5 mm, length 52.0 cm STORZ, Tuttlingen, 66016 AB)

2) mandrin with truncated tip (Ø 10.0 mm, length 52.0 cm, STORZ, Tuttlingen)

3) sharp mandrin (Ø 10.0 mm, length 52.0 cm, STORZ, Tuttlingen)

4) tubing system with two channels for catheter and laparoscope (STORZ, Tuttlingen)

5) laparoscope (Ø 10.0 mm, length 57.0 cm, 30° forward Hopkins optic, STORZ,

Tuttlingen, 6015 BV) All instruments are shown in Fig. 9-4.

Fig. 9-4: Trocar set used for experiment I, 1) Universal tube, mandrin with truncated tip, 3) sharp mandarin, 4) tubing system with two channels, 5) laparoscope

Furthermore were used a

KARL STORZ Endovision TELECAM, color system PAL (STORZ) containing of:

- TELECAM pal camera head (STORZ 20210030)

- TELECAM pal camera-control unit (STORZ 20210020)

- Digivideo picture processing system (STORZ 20202020)

ø 12,5 mm 52 cm length

30 °

1)

2)

3)

4)

5)

92 Appendix

- cold light source Xeno n 300 (STORZ 20133020)

- glass fibre-light cable (STORZ 495NE, Ø 3.5 mm, lenght3 m)

- 36 cm color monitor (Sony, Trinitr on color video Monitor, PVM-2053MD)

- Video-recorder (Panasonic, Viddeo Cassette Recorder NVFS88 HQ)

9.2.4 Surgery experiment I

For experiment I (n=8), mares were fixed in a stock, the tail of the mares were fixed

and secured with a bandage and the stock was covered with a sterile sheet (Buster®-

OP-Cover, 90 cm x 120 cm). Following manual evacuation of the rectum, the outer

genital were prepared antiseptically using a soap (Lifosan soft®, B. Braun, Melsungen,

Germany) and iodine (Braunol®, Braun, Melsungen, Germany). The vestibulum and

vagina were flushed with 500 ml of an iodine solution (NACL 0.9%, Ecotainer® ,

Braun, Melsungen, Germany) and (0.5 % iodine solution (Braunol® , B. Braun,

Melsungen, Germany) by inserting the left hand with a sterile glove and a flushing

tube into the vagina.

In the first two surgeries, the mares received an epidural anaesthesia with

lidocainhydrochloride 2% (Lidocain®, belapharm, Vechta, Germany).

The trocar set (Storz, Tuttlingen, Germany) consisting of a universal tube (12.5 mm x

52 cm) and a mandrin with a truncated tip were hold in the operators right hand,

covered with a sterile glove an were inserted and positioned middorsally in the fornix

of the vagina. The trocar was fixed by the operator’s left hand and the right hand was

inserted into the rectum. Following a transrectal manual control of the correct position

of the trocar and that no intestine was in front of the tip of the trocar, the atraumatic

mandrin was replaced by a sharp mandarin. The trocar set was introduced through

the vaginal wall into the peritoneal cavity, which was confirmed by passive influx of

air. The mandrin was replaced by a tubing system, containing the laparoscope

(Storz, Tuttlingen, Germany, Ø 10 mm, 30° forward Hopkins optic) and a 6 Ch.

flushing catheter (ERU®, Willy RÜSCH GmbH, Kernen i. R., Germany) in the working

channel. The ovaries were fixed by manipulation per rectum, and were turned 180° in

cranio-medial direction so that the infundibulum and the adjacent part of the ampulla

could be clearly identified. An attempt was then made to introduce the catheter into

Appendix 93

the abdominal ostium. The instruments were removed and the mare was fastened in

the stable for 2 hours to prevent aspiration due to the sedation.

9.2.5 Preoperative management experiment II

For experiment II (n=12) the paralumbar fossa was clipped and prepared aseptically

(Hibiscrub®, Chlorhexidine gluconate, Regent Medical, irlam, Manchester, UK). After

the infiltration of skin and muscle at the surgery side with 80 - 100 ml lidocaine

(Lidocain®, belapharm, Vechta, Germany) the mare and stock were covered with

sterile sheets (Buster®-OP-Cover, 90 cm x 120 cm).

9.2.6 Instruments used in experiment II

The following instruments were needed or developed for experiment two:

Endoscope-set (KARL STORZ, Tuttlingen, Germany) containing:

- 4 trocar sleeves, one with automatic valve for CO2–insufflation (Ø12.5 mm,

3 x lengths 20 cm, 1x length 15 cm, STORZ, 62103H3)

- trocar with truncated tip (Ø 10.0 mm, length 28 cm, STORZ, 62103 AL)

- laparoscope with a 30° viewing angle (Ø 10 mm, length 57 cm, Hopkins® optic,

62032 BP, STORZ)

KARL STORZ Endovision TELECAM, color system PAL (STORZ) containing:

- TELECAM pal camera head (STORZ, 20210030)

- TELECAM pal camera-control unit (STORZ, 20210020)

- Digivideo picture processing system (STORZ, 20202020)

- cold light source Xeno n 300 (STORZ, 20133020)

- glass fibre-light cable (STORZ, 495NE, Ø 3.5 mm, length 3 m)

- 36 cm color monitor (Sony, Trinitron color video Monitor, PVM-2053MD)

- video-recorder (Panasonic, Video Cassette Recorder NVFS88 HQ)

- CO2-insufflator with insufflation-tube (DVM-medizintechnik GmbH Ludwigsfelde

95C8032)

- sterile filter (mtp medical technical promotion GmbH, Neuhausen, RGF 031122-02)

- 2 Babcock forceps (atraumatic, Ø 10 mm, length 43 cm, Click’line ®, STORZ)

94 Appendix

- 2 scalpel handles with blades (size 22 and 10)

- injection filter (Sterifit 0.2 Ym, Braun, Melsung, Germany)

- suture material (Difalon 1 USP, Braun, Tuttlingen, Germany)

- surgical instrument set (needle holder, clamps, scissors)

- methylene blue solution:

(2 g of methylene blue powder (Microscopy Certistain Methylenblau, MERCK

KGaG, Darmstadt, Germany) were given in 1 L 0.9% NaCl solution (NaCl 0.9%

solution Ecotainer 1 L, Braun, Melsungen, Germany)

40 ml of the solution were filtered through an injection filter (Sterifit 0.2 Ym®, Braun,

Melsung, Germany) and aspirated into a 10 ml syringe.

- balloon catheter (Fig. 9-5):

ureter catheter with balloon after Steffens and Vahlensiek, Ch. 7, 75 cm length,

PVC material (26 30 00, Willy RÜSCH GmbH, Kernen i. R., Germany) with a two

channel system (one channel for balloon insufflation of 5 ml air and one for fluid

application, “flute-tip” with three “eyes”, the one in front of the balloon was closed by

a 2 cm x 15 mm piece of PVC tape (Coronaplast®, Conmetall, Celle, Germany)

Fig. 9-5: Picture of the flushing catheter a: catheter with three openings, one closed by a PVC tape, b: 5 ml syringe, c: balloon insufflated with 3 ml of air

Appendix 95

- guide sleeve (Fig. 9-6):

a plastic guide sleeve was designed from a guide sleeve of a gynaecologic swab

for mares (Stutentupfer®, Equi-Vet, Ø 5,0 mm 75 cm length, Eichemenger,

Denmark), the tip was formed in a 30° angle using a hot air gun and tested in the

first two surgeries. Then a similar guide sleeve of metal was constructed of the fine

mechanics workshop the Institute of Physiology of the School of Veterinary

Medicine, Hannover, Germany)

Fig. 9-6: Picture of the 1) plastic guide sleeve and 2) metal guide sleeve for guidance

of the catheter

Before usage the catheter and guide sleeve were placed in a 5 % disinfection

solution (Descoton Forte®, Dr. Schumacher GmbH, Melsungen, Germany) for one

hour.

9.2.7 Surgery experiment II

A standard laparoscopic approach was used. Briefly, an 12 mm overall diameter

trocar and cannula (STORZ, Tuttlingen, Germany) was passed through a 1 cm skin

incision made at the ventral level of the tuber coxae, midway between the tuber

1)

2)

96 Appendix

coxae and the last rib (Fig. 9-7) and then inserted through the locally anaesthetised

abdominal muscles into the peritoneal cavity.

Fig. 9-7: Drawing of the left flanc region and portal sites for the 1) laparoscope, 2)/3) Babcock forceps and 4) guide sleeve and catheter

This allowed the insertion of a rigid laparoscope with a 30° viewing angle (10 mm,

length 57 cm, Hopkins optic, 62032 BP, STORZ,) into the peritoneal cavity. The

abdomen was then distended by controlled insufflation with 5-10 l/min CO2 (CO2-

insufflator with insufflation-tube (DVM-medizintechnik GmbH Ludwigsfelde 95C8032)

and a sterile filter (mtp medical technical promotion GmbH, Neuhausen, RGF

031122-02) until an intraabdominal pressure of 10-15 mmHG was reached. Then the

the ovary, infundibulum, oviduct and tip of the ipsilateral uterine horn were visible

(Fig. 9-8).

Appendix 97

Fig. 9-8: Laparoscopic view of the right ovary (star), infundibulum (large arrow), oviduct (arrowheads) and tip of the uterine horn (circle) before oviductal flushing

Three subsequent instrument portals were created, one located 10 cm ventral and

two located 5 cm ventral and 5 cm cranial or caudal of the laparoscope portal (Fig.9-

7 and Fig. 9-9). Two laparoscopic Babcock forceps (atraumatic, Ø 10 mm, length 43

cm, Click’line®, STORZ) were introduced through two of the three instrument portals

and the infundibulum was grasped at each two opposite locations and “opened up”,

so that the inner side of the infundibulum and the 6 mm abdominal ostium in the

centre of the infundibulum could be visualized (Fig. 9-10).

a)

�

�

98 Appendix

Fig. 9-9: Picture of the left flanc laparoscopy surgery site for oviductal flushing in the standing sedated mare 1: trocar sleeve with inserted laparoscope and gas cable 2: trocar sleeve for guide sleeve and flushing catheter 3, 4: trocar sleeve for the two Babcock forceps

Appendix 99

Fig. 9-10: Laparoscopic view of the left part of the infundibulum „opened up“ by a Babcock forceps; the balloon catheter is directed into the abdominal ostium (large arrow)

A 7 Ch. balloon catheter (ureter catheter with balloon after Steffens and Vahlensiek,

Ch. 7, Art. Nr. 26 30 00, Willy RÜSCH GmbH, Kernen i. R., Germany) inserted in the

guide sleeve which was curved at the tip was positioned into the lower instrument

portal and directed into the abdominal ostium of the oviduct and approximately 2 cm

into the ampulla. The balloon was inflated with 2 ml of air. One of the Babcock

forceps holding the infundibulum was removed and positioned around the abdominal

ostium and the catheter to secure the catheter in place and to avoid a back pressure

of the catheter (Fig. 9-11).

a)

100 Appendix

Fig. 9-11: The catheter (large arrow) is introduced approximately 2 cm into the ampulla and the balloon (arrowhead) is insufflated with 2 ml of air, the Babcock forceps is positioned around the catheter and the abdominal ostium to prevent back-flow, methylene blue fluid in the proximal ampulla (asterisks)

The oviduct was then flushed with 20 ml of sterile methylene blue solution (Fig. 9-12).

Following the flushing, the balloon was opened and following the efflux of gas all

instruments were removed from the abdominal cavity and the portals were closed in

a simple interrupted pattern in the subcutaneous tissues and the skin with Dafilon 1

USP (Braun, Tuttlingen, Germany).

b)

Appendix 101

Fig. 9-12: Picture showing the oviductal ampulla filled with methylene blue solution (arrowheads) following the oviductal flushing, the isthmus (stars) is not visibly filled or distended To assess the passage of fluid through the oviduct, a hysteroscopy was performed

15 min post operation. Following antiseptic preparation of the outer genitalia, a

flexible video endoscope of 200 cm in length (SIF 100, Olympus, Hamburg,

Germany) was passed under digital control through the cervix into the uterine body.

An average intrauterine pressure of 25±5 mmHg (BARTMANN and SCHIEMANN

2003) was adjusted for distension of the uterine lumen by insufflation of filtered

atmospheric air using the endoscopic pump (cold light source: CLV-U20, Olympus,

Hamburg, Germany). A complete exploration of the uterine cavity was performed.

Following hysteroscopy, the uterus was flushed with 5 L of 37°C warm, sterile

Ringer´s solution (Ecobac®click, Braun, Melsungen, Germany) by manually guided

flushig tube (Irrigator, Eickmeyer Medizintechnik, Tuttlingen, Germany) which was

inserted into the uterus transcervically.

b)

�

�

�

102 Appendix

9.2.8 Postoperative care in both experiments

The central venous catheter was removed and mares were examined in their stable

6, 12 and 18 hours post surgery (day 0) for signs of postoperative pain. Using a pain-

score system (see Table 9-2) the sum of scores were used. A total score of 0-4 was

defined as not painful, 5-8 as slight painful, 9-17 as moderate painful and 18-24 as

highly painful. Additionally, a clinical examination containing behaviour, appetite,

pulse (beats/min), respiration (breaths/min), rectal temperature, gut sounds, capillary

refill time, mucous membranes was performed daily from days 0-5. At days 1, 3 and

5 evaluations of an EDTA-blood sample was performed. The values in brackets were

defined as normal. Haematocrit (25-45%), total protein (55-75 g/l), leucocytes (5-10

G/l). Administration of flunixin meglumine (Flunidolgel 1.1 mg/kg bwt, per os) was

continued for 48 hours.

Tab. 9-2 Pain score system parameter/points 0 1 2 3

behaviour alert calm dull colic, apathy

appetite good not eating

defecation existing not existing,

diarrhoea pulse (beats/min)

28-40 >40 >52 >60

respiration (breaths/min)

<16 >16 >32 >40

abdominal wall tension

relaxed slightly tense moderately tense

highly tense

pressure pain of wound

insensible slightly painful moderately painful

highly painful

Appendix 103

Tab. 9-3: Clinical details of 10 mares of experiment I and II Mare Age at

surgery

(years)

Breed Body weight

(kg)

Surgery no.

Cycle

dioestrus oestrus

D1-5 D6-10 D11-16 D16-21 (24)

A 12 trotter 530 I.1 x

I.2 x

B 12 trotter 470 I.6 x

C 12 trotter 540 I.7 x

D 12 trotter 580 I.8 x

E 12 Hannov. 650 I.3 x

I.4 x

14 II.1 x

II.2 x

F 6 German R. 600 I.5 x

8 II.6 x

II.9 x

G 11 trotter 470 II.3 x

II.7 x

H 16 trotter 480 II.4 x

II.5 x

I 8 Trakehner 430 II.8 x

II.10 x

J 3 Trakehner 390 II.11 x

II.12 x

I = experiment I, II = experiment II * surgery excluded from results because of a right dorsal displacement of the colon ascendens D = days, Hannov. = Hannoveranian, German R. = German riding horse

104 Appendix

Composition of skin-milk extender INRA 82 (page 85) 25 g glucose 1.5 g lactose 1.5 g raffinose 0.4 g potassium-citrate 0.3 g tri-sodium-citrate-dihydrat 4.76 g HEPES 0.5 g penicillin 0.5 g gentamicin ad 0.5 l aqua bidest. ad 1000 ml skin milk (0.3%)

Composition of phosphate buffered saline, PBS (page 85) Solution 1: 64 g NaCl 1.6 g KCl 0.8 g MgCl26H2O 0.8 g CaCl2 in aqua bidest ad 1000 ml Solution 2: 1.2 g NaH2PO4H2O 10 g Na2HPO42H2O 1.6 g KH2PO4 9.8 g glucoseH2O 0.288 g pyruvat 0.8 g bovine sermalbumine fraktion V (Sigma Aldrich,Taufkirchen, Germany) in auqa bidest ad 7000 ml Mixture of solution 1 and 2 (8 L) , 37-38°C warm

Appendix 105

Erklärung

Hiermit erkläre ich, dass ich die Dissertation “Investigations on genital blood flow and

embryo recovery after superovulation with eFSH® and on laparoscopic techniques for

flushing the oviduct in the mare” selbständig verfasst habe.

Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten

(Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat

von mir unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die

im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

Ich habe die Dissertation an folgender Institution angefertigt:

Klinik für Pferde und Reproduktionsmedizinische Einheit der Kliniken der Stiftung

Tierärztliche Hochschule Hannover.

Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen

ähnlichen Zweck zur Beurteilung eingereicht.

Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig

und der Wahrheit entsprechend gemacht habe.

Hannover, den Melanie Witt

106 Appendix

Acknowledgements

I express my gratitude to my supervisor Prof. Dr. Harald Sieme for entrusting me with

this interesting work and for his continuous academic guidance, confidence,

motivation, patience and good humor.

I thank Prof. Dr. Sabine Meinecke-Tillmann and Prof. Dr. Wilhelm Kanitz, from my

tutorial group, for thoughtful discussions, comments and suggestions.

I also thank the Mehl-Mülhens-Stiftung, Germany, who supported this project.

Many thanks to Dr. Anna Rötting, for the interesting discussions and qualified help in

many issues concerning surgery and laparoscopy and for the fact that she never lost

the belief those laparoscopies would be successful…

I thank Antje Heberling, Christine Baackmann and Jeannette Probst for working

together on the interesting projects.

I also thank to the National Stud Celle for semen collection any day of the week.

Thanks to the laboratory of the Clinic for Cattle, University of Veterinary Medicine

Hannover and JProf. Marion Piechotta for the help in hormone analysis.

Special thanks to Joachim König and Juliane Marx and all the Veterinarian

Technicians of the Clinic for horses for technical assistance and patience during

laparoscopies.

Thanks to Katharina Höffmann for demonstrating the technique of transvaginal tubal

flushing in the cow.

I thank all colleagues from the Clinic for horses and the Reproductive Unit of Clinics,

University of Veterinary Medicine Hannover for the good times we have had together.

I thank Astrid Bienert-Zeit, Jessica Cavalleri, Florian Geburek, Rolf Wagels, Anna

Rötting, Miriam v. Borstel, Jutta Klewitz and Frauke Uhlendorf for the great time in

the Clinic for horses, for their general help and fun and friendship!

Special thanks to my family, Lars, Lasse, Lena and Max, I love you!