IntroductionThere is increasing current interest in the minipig as an animal mod-el for embryofetal development (EFD) studies. The large litter size and rapid sexual maturation of the minipig make it a convenient species for these studies when traditional choices such as the rat, rabbit or mouse are inappropriate. CiToxLAB Scantox has many years of experience with this study type including studies intended for regulatory submission. For stud-ies of this nature a reliable study design producing robust data is imperative. In this poster data from studies conducted within the last 5 years (9 preliminary and 4 main embryofetal studies) are pre-sented. The poster will focus on the use of Regumate® (a synthetic progestomimetic) for synchronizing the gilts prior to start of mat-ing, and discuss the various advantages of this approach. Further-more the efficiency of monitoring pregnancy in the early phase of the study will be documented.

Material and MethodsAnimalsThe animals we use are purchased from Ellegaard Göttingen Mini-pigs ApS (EGM), Dalmose, Denmark.

Estrus synchronisation and matingEstrus is synchronised with Regumate® (11 mg Altrenogest/animal) added to the morning diet for at least 18 days. Two days after ces-sation of Regumate® treatment the females are introduced to the males and are mated over 1-3 days. On mating days, the female is introduced to the male twice daily. A maximum of 3 successful matings is allowed for each female. The day of the first successful mating is defined as Gestation Day 0 (GD0).

flad

saag

rafi

sk.d

k

Regulatory embryofetal development studies in minipigs: optimisation of techniques Thomas K. Andreassen, Christina Skytte, Jeanet Løgsted, Andrew MakinCiToxLAB Scantox, Ejby, Denmark, E-mail: contract.scantox@citoxlab.com

As far as possible, the females in each study treatment group are mated with different males. Brother to sister matings are strictly avoided. After 3 successful matings or when the gilt repeatedly refuses to mate on introduction to the boar, the females are allo-cated to the study.

Results and DiscussionRegumate® treatment efficiently synchronizes the estrus resulting in 99% of the gilts having GD0 within 4 days of each other and 83% within 2 days of each other (see Figure 1). The main part of the ani-mals have their GD0 on the 6th day after cessation of Regumate® treatment (53%).

The pregnancy rate among control animals of the most recent stud-ies is 97.9% (n=96 animals, N=13 studies) and control litter sizes of 5.5 (at C-section on GD110) and 5.4 (at C-section on GD70) are recorded (see Table 3-6). These litter sizes are higher than the lit-ter sizes obtained in the breeding facility of Ellegaard Göttingen Minipigs ApS (EGM) (see Table 7) where Regumate® is not used. At EGM total litter sizes of 5.1 (incl stillborn piglets) or 4.8 (excluding stillborns) are obtained in primiparous females (Data from 2010). Our litter size data thus indicates that treatment with Regumate® does not affect litter size negatively but might even improve it.

The efficient synchronization of pregnancies permits manageable necropsy planning for large embryofetal toxicity studies. By taking advantage of the fact that estrus has a length of 3 days in the mini-pig, synchronization can be even more efficient. If successful mat-ing is hindered prior to the 6th day after cessation of Regumate® treatment an even tighter synchronisation of pregnancies can be obtained. By using this “forced synchronisation” method 97 % (n= 116) of the documented GD0 are synchronized to the 6th day after cessation of Regumate® treatment (see Figure 2). The mating suc-cess in such a “forced synchronisation” protocol is still very good (97%) and the obtained fetal litter size of 5.6 does not differ signi-

Background information (Göttingen minipig) for embryo fetal studies

CiToxLAB Scantox Historical control data generated 2008 to 2012

Litter data (n=22, N=6)Preliminary study (GD70)

CiToxLAB Scantox Historical control data generated 2010 to 2012

Litter data (n=74, N=4)Main study (GD 110)

CiToxLAB Scantox Historical control data generated 2008 to 2012

Fetal data (n=22, N=6)Preliminary study (GD70)

CiToxLAB Scantox Historical control data generated 2010 to 2012

Fetal data (n=74, N=4)Preliminary study (GD70)

Embryo fetal toxicity studies in minipigs at CiToxLAB Scantox

• Sexual maturity: Approx. 5 months • Polyestrus

• Cycle length: 21-22 days • Estrus length: 3 days

• Implantation: GD 11 • Closure of the hard palate: GD 35

• Gestation length: 112-114 days • Diffuse epitheliochorial placentation

Pregnancy Uterus Littersize Dead Early Late Implantations Corpora Pre- Post- rate (%) weight fetuses/ resorptions resorptions lutea implanation implanation (g) litter loss (%) loss (%)

100 2471 5.5 0.00 0.44 0.26 6.24 6.8 7.41 12.1 (100) (1955- (4.7-6.4) (0.00) (0.2-0.8) (0.0-0.7) (5.3-7.0) (5.3-7.7) (0.0-14.3) (3.3-22.9) 3214)

Pregnancy Uterus Littersize Dead Early Late Implantations Corpora Pre- Post-rate (%) weight fetuses/ resorptions resorptions lutea implanation implanation (g) litter loss (%) loss (%)

94.7 3873 5.41 0.01 0.66 0.11 6.18 6.71 7.61 13.0 (94.4-100) (3380- (4.4-6.0) (0.00- (0.35-1.06) (0.11-0.12) (5.61-6.89) (6.3-7.4) (6.4-10.4) (8.7-21.3) 4356) 0.06)

Fetal body Placenta Nose to tail head Jaw length Male/female weight weight length (cm) distribution (g) (g) (cm) (%)

101 53.0 13.8 2.93 50.5 Male (30.8-61.1) (94-107) (30.9-80.0) (13.5-14.2) (2.93-3.10) 49.5 Female (38.9-69.2)

Fetal body Placenta Nose to tail head Jaw length Male/female weight weight length (cm) distribution (g) (g) (cm) (%)

372 116 22.2 4.7 Male 49.2 (44.4-57.5) (344-385) (103-130) (21.7-22.6) (4.5-4.9) Female 50.8 (42.5-55.6)

Ellegaard Göttingen Minipigs Aps database generated July 2009 to June 2010

Littersize at Ellegaard Göttingen Minipigs ApsPrimiparous females (gilts)

Litter size (total) Litter size (viable)

5.1 4.8Group size

Treatment

Dose administration

Pregnancy check

Caesareansectioning

Fetalexamination

Historicalcontrol data

Historicalbackground data

Preliminary study

3-5 (5 is recommended)

GD11-GD36 (organogenesis)

Oral gavage, capsule dosing, subcutaneous injection, intravenous (VAP) injection

Ultra-sound scanning between GD25 and GD30

Estrus cycle monitoring between GD20 and GD70

GD70

External malformations

Main study

18 (excl. kinetic satellite groups)

GD11 to GD 36 (organogenesis)

Oral gavage, capsule dosing, subcutaneous injection, intravenous (VAP) injection

Ultra-sound scanning between GD25 and GD30

Estrus cycle monitoring between GD20 and GD70

GD110

External malformationsVisceral malformations (fresh dissection)Head abnormalities (bouins)Skeletal examination (alizarin staining)

Control databases on external, visceral and skeletal malformations Databases on external and visceral malformations in stillborn piglets from supplier (Ellegaard Göttingen Minipigs ApS) colony

ficantly from the litter size obtained using the standard protocol. Such study setups can be advantageous in situations where tightly controlled treatment prior to mating is desired, ie. when looking at effects on the unfertilised egg or on other pre-mating parameters.

Synchronisation of mating is also useful for optimal planning and conduct of juvenile toxicology studies in minipigs for the timely provision of adequate numbers of neonatal piglets on specific days.

To monitor mating success (pregnancy) in the minipigs ultrasound scanning is performed. This procedure can be employed from around GD25. The scanning procedure is a 99% accurate test for pregnancy (n=164). In one study (n=72) we tried to estimate the number of fetuses using the scanning procedure. In 86% of the lit-ters the scanning result was within +/- 2 fetuses of the actual lit-ter size (See Figure 3). The mean group litter size of the scanned result compared with the actual result at GD110 showed deviations of 0% to 17% per group. The discrepancies might be partly related to presence of early and late resorptions recognised as viable fe-tuses on the day of scanning and partly due to lack of accuracy of the method.

ConclusionThe Göttingen minipig is a valuable non-rodent animal model for embryofetal development studies. CiToxLAB Scantox has a large updated historical control database of fetal findings and continues to develop new tools for optimizing the management and monitor-ing of these studies. The data presented here show that synchro-nisation by Regumate® can be “fine-tuned” to meet specific needs and that pregnancy status can be efficiently monitored early in the study.

0

10

20

30

40

50

60

2nd 3rd 4th 5th 6th 7th 8th% o

f g

ilts

wit

h G

D0

Days after cessation of Regumate treatment

Synchronization of Heat in minipigs (gilts)

0

20

40

60

80

100

120

6th 7th 8th

% o

f g

ilts

wit

h G

D0

Days after cessation of Regumate treatment

"Forced" synchronization of Heat in minipigs (gilts)

10,0

20,0

30,0

40,0

50,0

% w

ith

in c

ate

go

ry

Accuracy of ultra sound scanning for pregnancy testing

0,0

10,0

exact +/- 1 +/- 2 >2

% w

ith

in c

ate

go

ry

Scanning result (category)

Table 1.

Table 2.

Table 3.

Table 4. Table 7.

Figure 1.

Table 5.

Table 6.

Figure 2.

Figure 3.

www.citoxlab.com