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 />
100 Poster Abstracts<br />
endangered breeds at risk of ext<strong>in</strong>ction. This study reports on the<br />
validation and use of a sperm DNA fragmentation test for domestic<br />
stallion and donkey spermatozoa <strong>in</strong> which the sperm chromat<strong>in</strong><br />
dispersion test (SCD) was applied to both chilled and frozen semen<br />
samples. The SCD test was conducted on spermatozoa that been<br />
processed for rout<strong>in</strong>e chilled and frozen-thawed <strong>in</strong>sem<strong>in</strong>ation. The<br />
SCD test was applied to sperm that were subsequently <strong>in</strong>cubated at<br />
37ºC for 0, 4, 6, 24 and 48h <strong>in</strong> an attempt to try and emulate post<strong>in</strong>sem<strong>in</strong>ation<br />
conditions with<strong>in</strong> the mare's reproductive tract. The<br />
results of this <strong>in</strong>vestigation revealed that there was no significant<br />
difference <strong>in</strong> the sperm DNA fragmentation <strong>in</strong><strong>de</strong>x (sDFI) of sperm<br />
evaluated <strong>in</strong>itially after collection compared to those tested<br />
immediately after chill<strong>in</strong>g or cryopreservation. However, with<strong>in</strong> 1h of<br />
<strong>in</strong>cubation at 37ºC, both chilled and frozen-thawed spermatozoa<br />
showed a significant <strong>in</strong>crease <strong>in</strong> the proportion of sDFI; after 6 h the<br />
sDFI had <strong>in</strong>creased to over 50% and by 48 h almost 100% of the<br />
spermatozoa exhibited DNA damage. While the sDFI of <strong>in</strong>dividual<br />
stallions and donkeys at equivalent times of <strong>in</strong>cubation was variable,<br />
an analysis of the rate of change of sDFI revealed no significant<br />
difference between animals or the way <strong>in</strong> which the semen was<br />
preserved. In terms of sperm DNA fragmentation dynamics, the<br />
highest <strong>in</strong>tensity of sperm DNA damage occurred <strong>in</strong> the first 6 h of<br />
<strong>in</strong>cubation. SDF dynamics varied with respect to <strong>in</strong>dividual animals<br />
and it was possible to separate animals on the basis of the rate of<br />
DNA <strong>de</strong>gradation. In the case of donkeys, the analysis of sperm DNA<br />
fragmentation coupled with other classical parameters of semen<br />
quality provi<strong>de</strong>d a useful measure of the fertility of each animal.<br />
Additionally, the application of the SCD <strong>in</strong> this study leads us to<br />
conclu<strong>de</strong> that sperm chromat<strong>in</strong> organization is analogous <strong>in</strong> stallions<br />
and donkeys, although they do differ with respect to the actual rate of<br />
prote<strong>in</strong> <strong>de</strong>pletion after a standard lys<strong>in</strong>g treatment. We conclu<strong>de</strong> that<br />
the SCD methodology orig<strong>in</strong>ally <strong>de</strong>veloped for domestic stallions can<br />
also be applied for the assessment sperm DNA fragmentation <strong>in</strong> the<br />
donkey or <strong>in</strong> related wild Equid species such about which there is<br />
limited <strong>in</strong>formation about sperm quality.<br />
P218<br />
Is there an effect of dose rate of Cloprostenol given <strong>in</strong><br />
dioestrus on <strong>in</strong>terval from treatment to ovulation <strong>in</strong><br />
mares<br />
Cuervo-Arango, J 1 *; Newcombe, JR 2<br />
1Royal Veter<strong>in</strong>ary College, Department of Veter<strong>in</strong>ary Cl<strong>in</strong>ical Science,<br />
University of London, UK; 2 Equ<strong>in</strong>e Fertility Unit, Warren house farm,<br />
Brownhills, UK<br />
Introduction Although the ovulatory effects of prostagland<strong>in</strong>s are<br />
well documented <strong>in</strong> several domestic species <strong>in</strong>clu<strong>de</strong>d horses, there<br />
has been little attention paid to the use of this drug for cl<strong>in</strong>ical<br />
purposes. Mares often grow large follicles dur<strong>in</strong>g the luteal phase<br />
which may or may not ovulate before progesterone levels <strong>de</strong>cl<strong>in</strong>e.<br />
Cl<strong>in</strong>ical observations of adm<strong>in</strong>ister<strong>in</strong>g prostagland<strong>in</strong>s <strong>in</strong> dioestrus<br />
mares with large follicles suggest that there may be a negative<br />
correlation between follicular diameter and <strong>in</strong>terval from treatment to<br />
ovulation (ITO). The aims of this study were two fold: a) to assess<br />
the effect of different doses of Cloprostenol (a PGF 2 alpha analogue,<br />
Estrumate®) when given to dioestrus mares with a dom<strong>in</strong>ant follicle<br />
larger than 28mm on the ITO and b) to evaluate the effect of the<br />
diameter of the dom<strong>in</strong>ant follicle at the time of treatment on ITO.<br />
Materials and methods Data from 529 TB mares from several stud<br />
farms and breed<strong>in</strong>g seasons were analysed. Mares with a dom<strong>in</strong>ant<br />
follicle > 28mm were given either 12.5µg (n=99), 75µg (n=203),<br />
250µg (n=108) or 625µg (n=119) of Estrumate® (250µg<br />
Cloprostenol/ml) while <strong>in</strong> dioestrus as i<strong>de</strong>ntified by ultrasonographic<br />
exam<strong>in</strong>ation of a visible CL and absence of uter<strong>in</strong>e oe<strong>de</strong>ma. For data<br />
analysis mares were classified as hav<strong>in</strong>g a dom<strong>in</strong>ant follicle of either<br />
28-31mm (n=190), 32-35mm (n=163) or >36mm (n=176). Mares<br />
were scanned every other day until ovulation was <strong>de</strong>tected. A general<br />
l<strong>in</strong>ear mo<strong>de</strong>l of variance was used to test the effect of dose rate and<br />
follicular diameter on ITO.<br />
Results There was a significant effect of dose rate (P=.003) and<br />
follicular diameter (P=.000) on ITO. Higher doses of Cloprostenol<br />
<strong>in</strong>duced ovulation faster than lower doses (4.5, 4.4, 3.8 and 3.2 days<br />
for 12.5, 75, 250 and 625µg respectively) regardless of follicular<br />
diameter. In the same way, mares with larger follicles at the time of<br />
prostagland<strong>in</strong> <strong>in</strong>duction ovulated faster than those with smaller<br />
follicles (4.5, 3.9 and 3.4 days for follicles of 28-31, 32-35 and<br />
>36mm respectively) regardless of dose. The fastest ITO was <strong>in</strong>duced<br />
by 625µg of Cloprostenol <strong>in</strong> mares with a dom<strong>in</strong>ant follicle >36mm<br />
(mean ITO 2.4 days).<br />
Conclusion Prostagland<strong>in</strong> dose and follicular diameter at the time of<br />
<strong>in</strong>duction have a significant effect on <strong>in</strong>terval to ovulation and<br />
therefore can be useful tools for the prediction of ovulation. Doses as<br />
low as 12.5µg of Cloprostenol (0.05ml Estrumate®) are sufficient to<br />
<strong>in</strong>duce luteolysis, oestrus and ovulation when the CL is mature.<br />
P219<br />
Histological characterisation of mucus secret<strong>in</strong>g cells <strong>in</strong><br />
the lower equ<strong>in</strong>e reproductive tract<br />
Cumm<strong>in</strong>s, C*, Duggan, V, Fitzpatrick, E, Reid, C, Carr<strong>in</strong>gton, S<br />
UCD Veter<strong>in</strong>ary Sciences Centre, UCD, Belfield, Dubl<strong>in</strong> 4, Ireland<br />
Introduction Surface epithelial cells of the equ<strong>in</strong>e cervical and<br />
vag<strong>in</strong>al mucosa secrete a mucus gel which fulfils a <strong>de</strong>fensive function<br />
by prevent<strong>in</strong>g colonisation of the epithelium by pathogens. The<br />
physical characteristics of this gel vary at different stages of the<br />
reproductive cycle <strong>de</strong>pend<strong>in</strong>g on the secretion of steroid hormones.<br />
Around the time of ovulation, the low viscosity of the mucus gel<br />
allows transport of sperm. Dur<strong>in</strong>g dioestrus, the mucus becomes more<br />
viscous prevent<strong>in</strong>g migration of pathogens <strong>in</strong>to the uterus and dur<strong>in</strong>g<br />
pregnancy a thick mucus plug forms. Recent studies on normal<br />
cervical muc<strong>in</strong>s <strong>in</strong> women have i<strong>de</strong>ntified neutral, sialic acid- and<br />
sulphate-conta<strong>in</strong><strong>in</strong>g oligosacchari<strong>de</strong>s. We have un<strong>de</strong>rtaken an <strong>in</strong>itial<br />
histological characterisation of the mucus of the equ<strong>in</strong>e cervix and<br />
vag<strong>in</strong>a. This knowledge improves our un<strong>de</strong>rstand<strong>in</strong>g of the normal<br />
equ<strong>in</strong>e reproductive tract and its <strong>de</strong>fence mechanisms and will be<br />
useful <strong>in</strong> <strong>de</strong>tect<strong>in</strong>g pathologies such as ascend<strong>in</strong>g placentitis.<br />
Materials and Methods Samples of tissue were taken from 19 postmortem<br />
mares, of these 6 mares were <strong>in</strong> oestrus, 12 were <strong>in</strong> dioestrus<br />
and 1 mare was pregnant. Serum progesterone levels were measured<br />
to <strong>de</strong>term<strong>in</strong>e the stage of the reproductive cycle. No vag<strong>in</strong>al sample<br />
was available from the pregnant mare. Samples were fixed <strong>in</strong> 4%<br />
paraformal<strong>de</strong>hy<strong>de</strong>. Muc<strong>in</strong>s were <strong>de</strong>monstrated <strong>in</strong> paraff<strong>in</strong> sections<br />
us<strong>in</strong>g the periodic acid Schiff (PAS) and alcian blue sta<strong>in</strong><strong>in</strong>g methods.<br />
Lect<strong>in</strong> b<strong>in</strong>d<strong>in</strong>g was also <strong>in</strong>vestigated to <strong>de</strong>tect specific sugars.<br />
Results Cervix: Positive sta<strong>in</strong><strong>in</strong>g for muc<strong>in</strong>s dur<strong>in</strong>g oestrus was<br />
conf<strong>in</strong>ed to the apical cytoplasm of surface epithelium. Dur<strong>in</strong>g<br />
dioestrus and pregnancy, sta<strong>in</strong><strong>in</strong>g exten<strong>de</strong>d throughout the<br />
supranuclear cytoplasm. Dur<strong>in</strong>g pregnancy, the cervical mucus plug<br />
can be i<strong>de</strong>ntified as positively-sta<strong>in</strong>ed secreted material. Epithelial<br />
cells sta<strong>in</strong>ed positively for both acidic and neutral muc<strong>in</strong>s. Neutral<br />
sta<strong>in</strong><strong>in</strong>g appeared to predom<strong>in</strong>ate. With lect<strong>in</strong> b<strong>in</strong>d<strong>in</strong>g epithelial cells<br />
sta<strong>in</strong>ed positive for (α-2,6)-l<strong>in</strong>ked sialic acid <strong>in</strong> the cervices of both<br />
dioestrus and oestrus mares. Sta<strong>in</strong><strong>in</strong>g was positive only <strong>in</strong> low levels<br />
<strong>in</strong> the pregnant mare’s sample. Vag<strong>in</strong>a: The normal vag<strong>in</strong>al<br />
epithelium is non-kerat<strong>in</strong>ised stratified squamous epithelium. The<br />
epithelial cells are covered by a th<strong>in</strong> layer of mucus. The author has<br />
found no reference to mucus secret<strong>in</strong>g cells <strong>in</strong> the equ<strong>in</strong>e vag<strong>in</strong>a.<br />
However, columnar secretory epithelial cells were found on squamous<br />
epithelium <strong>in</strong> the cranial part of the equ<strong>in</strong>e vag<strong>in</strong>a. This <strong>de</strong>scription is<br />
similar to that of the bov<strong>in</strong>e vag<strong>in</strong>a. The columnar secretory cells<br />
produced both acidic and neutral muc<strong>in</strong>s. Acidic sta<strong>in</strong><strong>in</strong>g appeared to<br />
predom<strong>in</strong>ate. The cells sta<strong>in</strong>ed positively for (α-2,6)-l<strong>in</strong>ked sialic acid.<br />
Conclusions Previous studies suggest that the cervix is solely<br />
responsible for the secretion of mucus <strong>in</strong> the lower equ<strong>in</strong>e<br />
reproductive tract. Our histological study suggests that the vag<strong>in</strong>a may<br />
play an important role <strong>in</strong> mucus production such as the formation of<br />
the mucus plug of pregnancy. This may be important <strong>in</strong> ascend<strong>in</strong>g<br />
placentitis where failure of the mucus plug is thought to be an<br />
important factor.