Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Workshop Abstracts 21
marsupials. Indeed, further examination revealed that paired sperm
migrate almost exclusively to the isthmus of the oviduct shortly after
mating. This is the first in vivo evidence of an adaptive advantage
conferred by sperm cooperation in any mammal.
In summary, the tammar, opossum and other marsupials provide
important examples of how metatherians have evolved alternative
reproductive adaptations in the face of selective pressures.
WS14-2
Non-invasive assessment of female reproductive
physiology in the Pygmy Hippopotamus (Choeropsis
liberiensis)
Paris, M 1,2 *; Millar, R 1,3 ; Colenbrander, B 4 ; Schwarzenberger, F 5
1Institute for Breeding Rare and Endangered African Mammals (IBREAM),
United Kingdom; 2 Dept. Equine Sciences, Faculty of Veterinary Medicine,
University of Utrecht, The Netherlands; 3 MRC Human Reproductive Sciences
Unit, United Kingdom; 4 Dept. Farm Animal Health, Faculty of Veterinary
Medicine, University of Utrecht, The Netherlands; 5 Dept. of Natural Sciences
– Biochemistry, Vet. Med., Austria
The pygmy hippopotamus (Choeropsis liberiensis) is listed by IUCN
as vulnerable, with 2000-3000 individuals remaining. In Nigeria it is
already listed as being critically endangered (IUCN Red list, 2006).
Thus, maintaining a viable population in zoological institutions is of
utmost importance. As a consequence of their cryptic behavior, there
is little information about this species collected in the wild. Current
information on reproduction is derived from studbook data and
behavioral descriptions, but hormonal characterization of the estrus
cycle had not yet been undertaken. The lack of basic reproductive
knowledge currently compromises the breeding management of the
pygmy hippopotamus in zoos.
The aim of this study was to obtain basic information on female
reproductive physiology by monitoring faecal reproductive hormones
in juvenile, sexually mature, and old-age females. Fecal sample
collections were collected for at least 1 year from 8 zoological
institutions throughout Europe. Currently collections are continuing
with 15 zoological institutions in total. The fecal samples were stored
at -20°C until transported frozen to the laboratory. After methanol
extraction, the analysis used group specific EIAs with antibodies
against 5ß-pregnane-3α-ol-20-one 3HS: (20 oxo-pregnanes), 5βpregnane-3α-20α-diol
3HS:BSA (Pg-diol) and estradiol-17ß-OH
17HS:BSA (total estrogens).
The data presented here are collected from 7 sexually mature females
(of which 4 have hormonal data obtained during pregnancy and/or
lactation), and 1 possible post-reproductive cow. The total estrogens
measured suggested monthly cycling patterns (33 ± 3 days). In
addition, estrogen rises were also recognized at several occasions
during pregnancy and also during lactation. Data obtained measuring
20 oxo-pregnanes were not interpretable. Pg-diol levels were elevated
significantly during pregnancy, but not during cycling.
In conclusion, data obtained so far indicate that the pygmy
hippopotamus can cycle year round, and that estrogens can reliably
monitor reproductive patterns. The absence of progesterone
metabolite rises during cycling indicate that either a) the assay needs
further refinement, or b) that this species is possibly an induced
ovulator, which would be in accordance with its solitary existence in
the wild. Further work is done to confirm or refute this hypothesis.
The data also show that follicular development takes place during
pregnancy and during lactation.
WS14-3
Faecal steroid hormone metabolites – a parameter for the
free (non protein bound) hormone fraction
Moestl, E
Department of Basic Sciences, Veterinary University, Vienna, Austria
In plasma steroid hormones are transported mainly in a protein bound
form and are excreted after extensive metabolism via urine and faeces.
Faecal sampling is easier than blood or urine collection and in case of
glucocorticoid determinations it does not cause a disturbance of the
animals.
The quantification of these steroid hormone metabolites is used as
surrogate parameter for the plasma concentration but the correlation
between steroid hormone values in plasma and faeces it is an open
question as there are no data if the metabolites in faeces represent the
“total” or the non-protein bound fraction of the plasma hormones.
Therefore, an artificial binding protein was raised in sheep by
immunising 12 sheep against cortisol-CMO linked to three different
carrier proteins (1 mg/animal, booster injections 0.5 mg using
Gerbu®-adjuvant in weeks 8 and 15).
A stimulation test using 0.25 mg ACTH i.v. (Synacthen®, Fa.
Novartis) was performed three times (two weeks before immunisation
and 11 and 17 weeks later). Heparinized blood samples were taken
from the jugular vein after 0, 30, 60, 120, 360 and 540 min. Faecal
samples were collected after 0, 4, 6, 8, 10, 12 and 14 hours.
In plasma, the titer against cortisol was determined using tritiumlabelled
cortisol and the concentrations of cortisol (plasma) and the
cortisol metabolites in faeces (immunoreactive 11-
oxoetiocholanolone) were measured using enzyme immunoasssays.
Only steroid concentrations from animals with a titer higher than 10%
at a dilution of 1:500 of the plasma were considered in the analysis (n
= 10).
The plasma concentrations of cortisol increased with increasing titer
(baseline values: 14 ± 7, 35 ± 22 and 47 ± 33 ng/ml plasma) whereas
in faeces the concentrations of the immunoreactive metabolites were
not significantly elevated by the higher cortisol concentrations in
plasma (285 ± 144, 180 ± 97 and 454 ± 393 ng/g faeces). This result
may lead to the assumption that the hormone metabolites excreted
derive from the “free” (non-protein bound) hormone fraction of the
plasma.
WS14-4
Deep intra-uterine insemination with sex-sorted semen in
elephants
Hildebrandt, TB 1 *, Goeritz, F 1 , Rath, D 2 , Behr, B 1 , Schnorrenberg, A 3 ,
Knieriem, A 4 , Sieg, B 2 , Hermes, R 1
1Reproduction Management, Leibniz Institute for Zoo & Wildlife Research,
Germany; 2 Institute for Animal Breeding, Federal Agricultural Research
Centre, Germany; 3 Schnorrenberg Surgical Equipment Development Inc.,
Germany; 4 Hannover Zoo, Germany
Since the introduction of the non-surgical artificial insemination (AI)
technique in 1995 (Hildebrandt and Schnorrenberg, 1996; Hildebrandt
et al. 1998) in captive elephant breeding programs the reproduction
rate has been increased dramatically especially in African elephants in
North America. However, this optimistic development is still not
sufficient to stop the global trend of aging of the captive populations
in African and Asian elephants. Historically, the in-situ populations
in Asia and Africa have always served as a source of new individuals
for ex-situ populations. However, the in-situ population are critically
endangered due to conversion of habitat for agriculture and the
encroachment of urban civilization resulting in fragmentation and
disturbed sex ratio. Due to this disappointing situation, in-situ
populations are no longer available to import wild-caught elephants
for re-populating the aged captive populations and, more importantly,
the captive populations have become paramount for the conservation
of the all elephant species and sub-species.
The positive impact of AI programs on the demographic situation of
the captive elephant populations can be accelerated by using X
chromosome–enriched sperm samples which would produce a higher
percentage of female offspring and future number of potential
breeding cows. In result the captive breeding management of
elephants can be improved.
This strategy, however, has to overcome two major obstacles. First
step was the successful development of an efficient staining and
sorting protocol for elephant spermatozoa using a flow cytometry
technology (Behr et al. in press). Second step was the development of
a non-invasive, intra-uterine insemination technique allowing the
semen deposition approx. 2.5 m inside the reproductive tract in a nonsedated
female due to the general low sperm count of sorted samples
and so called “sorting injuries” of the spermatozoa used. The sperm