Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
Workshop Abstracts 21<br />
marsupials. In<strong>de</strong>ed, further exam<strong>in</strong>ation revealed that paired sperm<br />
migrate almost exclusively to the isthmus of the oviduct shortly after<br />
mat<strong>in</strong>g. This is the first <strong>in</strong> vivo evi<strong>de</strong>nce of an adaptive advantage<br />
conferred by sperm cooperation <strong>in</strong> any mammal.<br />
In summary, the tammar, opossum and other marsupials provi<strong>de</strong><br />
important examples of how metatherians have evolved alternative<br />
reproductive adaptations <strong>in</strong> the face of selective pressures.<br />
WS14-2<br />
Non-<strong>in</strong>vasive assessment of female reproductive<br />
physiology <strong>in</strong> the Pygmy Hippopotamus (Choeropsis<br />
liberiensis)<br />
Paris, M 1,2 *; Millar, R 1,3 ; Colenbran<strong>de</strong>r, B 4 ; Schwarzenberger, F 5<br />
1Institute for Breed<strong>in</strong>g Rare and Endangered African Mammals (IBREAM),<br />
United K<strong>in</strong>gdom; 2 Dept. Equ<strong>in</strong>e Sciences, Faculty of Veter<strong>in</strong>ary Medic<strong>in</strong>e,<br />
University of Utrecht, The Netherlands; 3 MRC Human Reproductive Sciences<br />
Unit, United K<strong>in</strong>gdom; 4 Dept. Farm Animal Health, Faculty of Veter<strong>in</strong>ary<br />
Medic<strong>in</strong>e, University of Utrecht, The Netherlands; 5 Dept. of Natural Sciences<br />
– Biochemistry, Vet. Med., Austria<br />
The pygmy hippopotamus (Choeropsis liberiensis) is listed by IUCN<br />
as vulnerable, with 2000-3000 <strong>in</strong>dividuals rema<strong>in</strong><strong>in</strong>g. In Nigeria it is<br />
already listed as be<strong>in</strong>g critically endangered (IUCN Red list, 2006).<br />
Thus, ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g a viable population <strong>in</strong> zoological <strong>in</strong>stitutions is of<br />
utmost importance. As a consequence of their cryptic behavior, there<br />
is little <strong>in</strong>formation about this species collected <strong>in</strong> the wild. Current<br />
<strong>in</strong>formation on reproduction is <strong>de</strong>rived from studbook data and<br />
behavioral <strong>de</strong>scriptions, but hormonal characterization of the estrus<br />
cycle had not yet been un<strong>de</strong>rtaken. The lack of basic reproductive<br />
knowledge currently compromises the breed<strong>in</strong>g management of the<br />
pygmy hippopotamus <strong>in</strong> zoos.<br />
The aim of this study was to obta<strong>in</strong> basic <strong>in</strong>formation on female<br />
reproductive physiology by monitor<strong>in</strong>g faecal reproductive hormones<br />
<strong>in</strong> juvenile, sexually mature, and old-age females. Fecal sample<br />
collections were collected for at least 1 year from 8 zoological<br />
<strong>in</strong>stitutions throughout Europe. Currently collections are cont<strong>in</strong>u<strong>in</strong>g<br />
with 15 zoological <strong>in</strong>stitutions <strong>in</strong> total. The fecal samples were stored<br />
at -20°C until transported frozen to the laboratory. After methanol<br />
extraction, the analysis used group specific EIAs with antibodies<br />
aga<strong>in</strong>st 5ß-pregnane-3α-ol-20-one 3HS: (20 oxo-pregnanes), 5βpregnane-3α-20α-diol<br />
3HS:BSA (Pg-diol) and estradiol-17ß-OH<br />
17HS:BSA (total estrogens).<br />
The data presented here are collected from 7 sexually mature females<br />
(of which 4 have hormonal data obta<strong>in</strong>ed dur<strong>in</strong>g pregnancy and/or<br />
lactation), and 1 possible post-reproductive cow. The total estrogens<br />
measured suggested monthly cycl<strong>in</strong>g patterns (33 ± 3 days). In<br />
addition, estrogen rises were also recognized at several occasions<br />
dur<strong>in</strong>g pregnancy and also dur<strong>in</strong>g lactation. Data obta<strong>in</strong>ed measur<strong>in</strong>g<br />
20 oxo-pregnanes were not <strong>in</strong>terpretable. Pg-diol levels were elevated<br />
significantly dur<strong>in</strong>g pregnancy, but not dur<strong>in</strong>g cycl<strong>in</strong>g.<br />
In conclusion, data obta<strong>in</strong>ed so far <strong>in</strong>dicate that the pygmy<br />
hippopotamus can cycle year round, and that estrogens can reliably<br />
monitor reproductive patterns. The absence of progesterone<br />
metabolite rises dur<strong>in</strong>g cycl<strong>in</strong>g <strong>in</strong>dicate that either a) the assay needs<br />
further ref<strong>in</strong>ement, or b) that this species is possibly an <strong>in</strong>duced<br />
ovulator, which would be <strong>in</strong> accordance with its solitary existence <strong>in</strong><br />
the wild. Further work is done to confirm or refute this hypothesis.<br />
The data also show that follicular <strong>de</strong>velopment takes place dur<strong>in</strong>g<br />
pregnancy and dur<strong>in</strong>g lactation.<br />
WS14-3<br />
Faecal steroid hormone metabolites – a parameter for the<br />
free (non prote<strong>in</strong> bound) hormone fraction<br />
Moestl, E<br />
Department of Basic Sciences, Veter<strong>in</strong>ary University, Vienna, Austria<br />
In plasma steroid hormones are transported ma<strong>in</strong>ly <strong>in</strong> a prote<strong>in</strong> bound<br />
form and are excreted after extensive metabolism via ur<strong>in</strong>e and faeces.<br />
Faecal sampl<strong>in</strong>g is easier than blood or ur<strong>in</strong>e collection and <strong>in</strong> case of<br />
glucocorticoid <strong>de</strong>term<strong>in</strong>ations it does not cause a disturbance of the<br />
animals.<br />
The quantification of these steroid hormone metabolites is used as<br />
surrogate parameter for the plasma concentration but the correlation<br />
between steroid hormone values <strong>in</strong> plasma and faeces it is an open<br />
question as there are no data if the metabolites <strong>in</strong> faeces represent the<br />
“total” or the non-prote<strong>in</strong> bound fraction of the plasma hormones.<br />
Therefore, an artificial b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> was raised <strong>in</strong> sheep by<br />
immunis<strong>in</strong>g 12 sheep aga<strong>in</strong>st cortisol-CMO l<strong>in</strong>ked to three different<br />
carrier prote<strong>in</strong>s (1 mg/animal, booster <strong>in</strong>jections 0.5 mg us<strong>in</strong>g<br />
Gerbu®-adjuvant <strong>in</strong> weeks 8 and 15).<br />
A stimulation test us<strong>in</strong>g 0.25 mg ACTH i.v. (Synacthen®, Fa.<br />
Novartis) was performed three times (two weeks before immunisation<br />
and 11 and 17 weeks later). Hepar<strong>in</strong>ized blood samples were taken<br />
from the jugular ve<strong>in</strong> after 0, 30, 60, 120, 360 and 540 m<strong>in</strong>. Faecal<br />
samples were collected after 0, 4, 6, 8, 10, 12 and 14 hours.<br />
In plasma, the titer aga<strong>in</strong>st cortisol was <strong>de</strong>term<strong>in</strong>ed us<strong>in</strong>g tritiumlabelled<br />
cortisol and the concentrations of cortisol (plasma) and the<br />
cortisol metabolites <strong>in</strong> faeces (immunoreactive 11-<br />
oxoetiocholanolone) were measured us<strong>in</strong>g enzyme immunoasssays.<br />
Only steroid concentrations from animals with a titer higher than 10%<br />
at a dilution of 1:500 of the plasma were consi<strong>de</strong>red <strong>in</strong> the analysis (n<br />
= 10).<br />
The plasma concentrations of cortisol <strong>in</strong>creased with <strong>in</strong>creas<strong>in</strong>g titer<br />
(basel<strong>in</strong>e values: 14 ± 7, 35 ± 22 and 47 ± 33 ng/ml plasma) whereas<br />
<strong>in</strong> faeces the concentrations of the immunoreactive metabolites were<br />
not significantly elevated by the higher cortisol concentrations <strong>in</strong><br />
plasma (285 ± 144, 180 ± 97 and 454 ± 393 ng/g faeces). This result<br />
may lead to the assumption that the hormone metabolites excreted<br />
<strong>de</strong>rive from the “free” (non-prote<strong>in</strong> bound) hormone fraction of the<br />
plasma.<br />
WS14-4<br />
Deep <strong>in</strong>tra-uter<strong>in</strong>e <strong>in</strong>sem<strong>in</strong>ation with sex-sorted semen <strong>in</strong><br />
elephants<br />
Hil<strong>de</strong>brandt, TB 1 *, Goeritz, F 1 , Rath, D 2 , Behr, B 1 , Schnorrenberg, A 3 ,<br />
Knieriem, A 4 , Sieg, B 2 , Hermes, R 1<br />
1<strong>Reproduction</strong> Management, Leibniz Institute for Zoo & Wildlife Research,<br />
Germany; 2 Institute for Animal Breed<strong>in</strong>g, Fe<strong>de</strong>ral Agricultural Research<br />
Centre, Germany; 3 Schnorrenberg Surgical Equipment Development Inc.,<br />
Germany; 4 Hannover Zoo, Germany<br />
S<strong>in</strong>ce the <strong>in</strong>troduction of the non-surgical artificial <strong>in</strong>sem<strong>in</strong>ation (AI)<br />
technique <strong>in</strong> 1995 (Hil<strong>de</strong>brandt and Schnorrenberg, 1996; Hil<strong>de</strong>brandt<br />
et al. 1998) <strong>in</strong> captive elephant breed<strong>in</strong>g programs the reproduction<br />
rate has been <strong>in</strong>creased dramatically especially <strong>in</strong> African elephants <strong>in</strong><br />
North America. However, this optimistic <strong>de</strong>velopment is still not<br />
sufficient to stop the global trend of ag<strong>in</strong>g of the captive populations<br />
<strong>in</strong> African and Asian elephants. Historically, the <strong>in</strong>-situ populations<br />
<strong>in</strong> Asia and Africa have always served as a source of new <strong>in</strong>dividuals<br />
for ex-situ populations. However, the <strong>in</strong>-situ population are critically<br />
endangered due to conversion of habitat for agriculture and the<br />
encroachment of urban civilization result<strong>in</strong>g <strong>in</strong> fragmentation and<br />
disturbed sex ratio. Due to this disappo<strong>in</strong>t<strong>in</strong>g situation, <strong>in</strong>-situ<br />
populations are no longer available to import wild-caught elephants<br />
for re-populat<strong>in</strong>g the aged captive populations and, more importantly,<br />
the captive populations have become paramount for the conservation<br />
of the all elephant species and sub-species.<br />
The positive impact of AI programs on the <strong>de</strong>mographic situation of<br />
the captive elephant populations can be accelerated by us<strong>in</strong>g X<br />
chromosome–enriched sperm samples which would produce a higher<br />
percentage of female offspr<strong>in</strong>g and future number of potential<br />
breed<strong>in</strong>g cows. In result the captive breed<strong>in</strong>g management of<br />
elephants can be improved.<br />
This strategy, however, has to overcome two major obstacles. First<br />
step was the successful <strong>de</strong>velopment of an efficient sta<strong>in</strong><strong>in</strong>g and<br />
sort<strong>in</strong>g protocol for elephant spermatozoa us<strong>in</strong>g a flow cytometry<br />
technology (Behr et al. <strong>in</strong> press). Second step was the <strong>de</strong>velopment of<br />
a non-<strong>in</strong>vasive, <strong>in</strong>tra-uter<strong>in</strong>e <strong>in</strong>sem<strong>in</strong>ation technique allow<strong>in</strong>g the<br />
semen <strong>de</strong>position approx. 2.5 m <strong>in</strong>si<strong>de</strong> the reproductive tract <strong>in</strong> a nonsedated<br />
female due to the general low sperm count of sorted samples<br />
and so called “sort<strong>in</strong>g <strong>in</strong>juries” of the spermatozoa used. The sperm