Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
100 Poster Abstracts
endangered breeds at risk of extinction. This study reports on the
validation and use of a sperm DNA fragmentation test for domestic
stallion and donkey spermatozoa in which the sperm chromatin
dispersion test (SCD) was applied to both chilled and frozen semen
samples. The SCD test was conducted on spermatozoa that been
processed for routine chilled and frozen-thawed insemination. The
SCD test was applied to sperm that were subsequently incubated at
37ºC for 0, 4, 6, 24 and 48h in an attempt to try and emulate postinsemination
conditions within the mare's reproductive tract. The
results of this investigation revealed that there was no significant
difference in the sperm DNA fragmentation index (sDFI) of sperm
evaluated initially after collection compared to those tested
immediately after chilling or cryopreservation. However, within 1h of
incubation at 37ºC, both chilled and frozen-thawed spermatozoa
showed a significant increase in the proportion of sDFI; after 6 h the
sDFI had increased to over 50% and by 48 h almost 100% of the
spermatozoa exhibited DNA damage. While the sDFI of individual
stallions and donkeys at equivalent times of incubation was variable,
an analysis of the rate of change of sDFI revealed no significant
difference between animals or the way in which the semen was
preserved. In terms of sperm DNA fragmentation dynamics, the
highest intensity of sperm DNA damage occurred in the first 6 h of
incubation. SDF dynamics varied with respect to individual animals
and it was possible to separate animals on the basis of the rate of
DNA degradation. In the case of donkeys, the analysis of sperm DNA
fragmentation coupled with other classical parameters of semen
quality provided a useful measure of the fertility of each animal.
Additionally, the application of the SCD in this study leads us to
conclude that sperm chromatin organization is analogous in stallions
and donkeys, although they do differ with respect to the actual rate of
protein depletion after a standard lysing treatment. We conclude that
the SCD methodology originally developed for domestic stallions can
also be applied for the assessment sperm DNA fragmentation in the
donkey or in related wild Equid species such about which there is
limited information about sperm quality.
P218
Is there an effect of dose rate of Cloprostenol given in
dioestrus on interval from treatment to ovulation in
mares
Cuervo-Arango, J 1 *; Newcombe, JR 2
1Royal Veterinary College, Department of Veterinary Clinical Science,
University of London, UK; 2 Equine Fertility Unit, Warren house farm,
Brownhills, UK
Introduction Although the ovulatory effects of prostaglandins are
well documented in several domestic species included horses, there
has been little attention paid to the use of this drug for clinical
purposes. Mares often grow large follicles during the luteal phase
which may or may not ovulate before progesterone levels decline.
Clinical observations of administering prostaglandins in dioestrus
mares with large follicles suggest that there may be a negative
correlation between follicular diameter and interval from treatment to
ovulation (ITO). The aims of this study were two fold: a) to assess
the effect of different doses of Cloprostenol (a PGF 2 alpha analogue,
Estrumate®) when given to dioestrus mares with a dominant follicle
larger than 28mm on the ITO and b) to evaluate the effect of the
diameter of the dominant follicle at the time of treatment on ITO.
Materials and methods Data from 529 TB mares from several stud
farms and breeding seasons were analysed. Mares with a dominant
follicle > 28mm were given either 12.5µg (n=99), 75µg (n=203),
250µg (n=108) or 625µg (n=119) of Estrumate® (250µg
Cloprostenol/ml) while in dioestrus as identified by ultrasonographic
examination of a visible CL and absence of uterine oedema. For data
analysis mares were classified as having a dominant follicle of either
28-31mm (n=190), 32-35mm (n=163) or >36mm (n=176). Mares
were scanned every other day until ovulation was detected. A general
linear model of variance was used to test the effect of dose rate and
follicular diameter on ITO.
Results There was a significant effect of dose rate (P=.003) and
follicular diameter (P=.000) on ITO. Higher doses of Cloprostenol
induced ovulation faster than lower doses (4.5, 4.4, 3.8 and 3.2 days
for 12.5, 75, 250 and 625µg respectively) regardless of follicular
diameter. In the same way, mares with larger follicles at the time of
prostaglandin induction ovulated faster than those with smaller
follicles (4.5, 3.9 and 3.4 days for follicles of 28-31, 32-35 and
>36mm respectively) regardless of dose. The fastest ITO was induced
by 625µg of Cloprostenol in mares with a dominant follicle >36mm
(mean ITO 2.4 days).
Conclusion Prostaglandin dose and follicular diameter at the time of
induction have a significant effect on interval to ovulation and
therefore can be useful tools for the prediction of ovulation. Doses as
low as 12.5µg of Cloprostenol (0.05ml Estrumate®) are sufficient to
induce luteolysis, oestrus and ovulation when the CL is mature.
P219
Histological characterisation of mucus secreting cells in
the lower equine reproductive tract
Cummins, C*, Duggan, V, Fitzpatrick, E, Reid, C, Carrington, S
UCD Veterinary Sciences Centre, UCD, Belfield, Dublin 4, Ireland
Introduction Surface epithelial cells of the equine cervical and
vaginal mucosa secrete a mucus gel which fulfils a defensive function
by preventing colonisation of the epithelium by pathogens. The
physical characteristics of this gel vary at different stages of the
reproductive cycle depending on the secretion of steroid hormones.
Around the time of ovulation, the low viscosity of the mucus gel
allows transport of sperm. During dioestrus, the mucus becomes more
viscous preventing migration of pathogens into the uterus and during
pregnancy a thick mucus plug forms. Recent studies on normal
cervical mucins in women have identified neutral, sialic acid- and
sulphate-containing oligosaccharides. We have undertaken an initial
histological characterisation of the mucus of the equine cervix and
vagina. This knowledge improves our understanding of the normal
equine reproductive tract and its defence mechanisms and will be
useful in detecting pathologies such as ascending placentitis.
Materials and Methods Samples of tissue were taken from 19 postmortem
mares, of these 6 mares were in oestrus, 12 were in dioestrus
and 1 mare was pregnant. Serum progesterone levels were measured
to determine the stage of the reproductive cycle. No vaginal sample
was available from the pregnant mare. Samples were fixed in 4%
paraformaldehyde. Mucins were demonstrated in paraffin sections
using the periodic acid Schiff (PAS) and alcian blue staining methods.
Lectin binding was also investigated to detect specific sugars.
Results Cervix: Positive staining for mucins during oestrus was
confined to the apical cytoplasm of surface epithelium. During
dioestrus and pregnancy, staining extended throughout the
supranuclear cytoplasm. During pregnancy, the cervical mucus plug
can be identified as positively-stained secreted material. Epithelial
cells stained positively for both acidic and neutral mucins. Neutral
staining appeared to predominate. With lectin binding epithelial cells
stained positive for (α-2,6)-linked sialic acid in the cervices of both
dioestrus and oestrus mares. Staining was positive only in low levels
in the pregnant mare’s sample. Vagina: The normal vaginal
epithelium is non-keratinised stratified squamous epithelium. The
epithelial cells are covered by a thin layer of mucus. The author has
found no reference to mucus secreting cells in the equine vagina.
However, columnar secretory epithelial cells were found on squamous
epithelium in the cranial part of the equine vagina. This description is
similar to that of the bovine vagina. The columnar secretory cells
produced both acidic and neutral mucins. Acidic staining appeared to
predominate. The cells stained positively for (α-2,6)-linked sialic acid.
Conclusions Previous studies suggest that the cervix is solely
responsible for the secretion of mucus in the lower equine
reproductive tract. Our histological study suggests that the vagina may
play an important role in mucus production such as the formation of
the mucus plug of pregnancy. This may be important in ascending
placentitis where failure of the mucus plug is thought to be an
important factor.