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 />
164 Poster Abstracts<br />
morphological changes approximately 200 tubule-cross-sections were<br />
evaluated and grouped (A-D) accord<strong>in</strong>g to the most <strong>de</strong>veloped germ<br />
cell observed. Testosterone (T) and estradiol-17ß (E) were estimated<br />
<strong>in</strong> blood collected dur<strong>in</strong>g downregulation and at castration. Tim<strong>in</strong>g of<br />
recru<strong>de</strong>scence showed dist<strong>in</strong>ct <strong>in</strong>dividual differences yield<strong>in</strong>g<br />
different numbers of dogs per group: Gr. A, spermatocytes (n=4); Gr.<br />
B, round spermatids (n=3); Gr. C, elongat<strong>in</strong>g spermatids (n=6) and<br />
Gr. D, elongated spermatids (n=17). T and E concentrations <strong>in</strong>creased<br />
from Gr. A to B (T: 0.14 ± 0.10 to 2.54 ±1.57ng/ml; E: 6.40 ± 2.19 to<br />
9.73 ± 4.16pg/ml) and were constant thereafter. These first results<br />
imply that onset of spermatogenesis occurs at low steroid hormone<br />
levels, accompanied by expression of the androgen receptor, and is<br />
rapidly stimulated by a further <strong>in</strong>crease. To our knowledge, this is the<br />
first study giv<strong>in</strong>g <strong>de</strong>tailed <strong>in</strong>formation about recru<strong>de</strong>scence of<br />
spermatogenesis of the dog follow<strong>in</strong>g downregulation.<br />
P414<br />
Early Detection of Membrane Asymmetry <strong>in</strong> Frozenthawed<br />
Buffalo Spermatozoa<br />
Gov<strong>in</strong>dasamy, K 1 *; Kumar, S 2 and Sharma, B 3<br />
1PhD Scholar, 2 Pr<strong>in</strong>ciple Scientist, Division of Animal <strong>Reproduction</strong>; 2 Indian<br />
Veter<strong>in</strong>ary Research Institute, Izantnagar, Bareilly (UP), India; 3 Pr<strong>in</strong>ciple<br />
Scientist, Division of Animal Biochemistry, Indian Veter<strong>in</strong>ary Research<br />
Institute, Izantnagar, Bareilly (UP), India<br />
Introduction Mammalian spermatozoa un<strong>de</strong>rgo tremendous chemical<br />
and physical stresses dur<strong>in</strong>g cryopreservation. The sperm plasma<br />
membrane is one of the key structures affected by cryopreservation.<br />
When the cell membrane is disturbed, phospholipid<br />
phosphatidylser<strong>in</strong>e (PS) is translocated from the <strong>in</strong>ner to the outer<br />
leaflet of the plasma membrane, which is one of the earliest signs of<br />
membrane disruption. Annex<strong>in</strong> V conjugated to Fluoresce<strong>in</strong><br />
isothiocyanate (FITC) fluorochrome reta<strong>in</strong>s its high aff<strong>in</strong>ity for PS<br />
and therefore, serves as a sensitive probe that can be used for flow<br />
cytometric <strong>de</strong>tection characterized by the loss of membrane<br />
asymmetry.<br />
Methods Fifty six semen ejaculates, eight ejaculates from seven<br />
healthy Murrah buffalo bulls (4-6 years age) ma<strong>in</strong>ta<strong>in</strong>ed at standard<br />
management conditions were used for the study. The standard<br />
conventional cryopreservation protocol was adopted. Comb<strong>in</strong>ation of<br />
two fluorescent dyes, Annex<strong>in</strong> V and propidium iodi<strong>de</strong> (PI) was used<br />
to evaluate the membrane asymmetry <strong>in</strong> fresh and frozen thawed<br />
spermatozoa <strong>in</strong> conjugation fluorescent microscope and flow<br />
cytometry.<br />
Results Four groups of spermatozoa were i<strong>de</strong>ntified: (i) viable<br />
spermatozoa (Annex<strong>in</strong> V-negative and PI-negative); (ii) viable<br />
spermatozoa with early membrane disruption but <strong>in</strong>teger plasma<br />
membrane (Annex<strong>in</strong> V-positive and PI-negative); and two categories<br />
of PI positive, <strong>de</strong>ad spermatozoa (iii) early necrotic (Annex<strong>in</strong> V-<br />
positive and PI-positive); and (iv) Late necrotic cells (Annex<strong>in</strong> V-<br />
negative and PI-positive). The four sperm population varied<br />
significantly between fresh and frozen thawed spermatozoa. In fresh<br />
semen, the early sperm membrane change was observed only <strong>in</strong> 17%<br />
of the total number of sperm. However, after freez<strong>in</strong>g and thaw<strong>in</strong>g,<br />
these sperm accounted for more than 31%. After freez<strong>in</strong>g-thaw<strong>in</strong>g,<br />
there was significant <strong>in</strong>crease <strong>in</strong> the percentage of live cells with<br />
phosphatidylser<strong>in</strong>e externalization (early membrane changes) and<br />
early necrotic cells, while reduc<strong>in</strong>g the percentage of live normal<br />
cells.<br />
Conclusions The Annex<strong>in</strong> V-b<strong>in</strong>d<strong>in</strong>g assay is an effective tool to<br />
provi<strong>de</strong> early <strong>de</strong>tection of membrane asymmetry <strong>in</strong> the viable<br />
spermatozoa. Further, cryopreservation of buffalo spermatozoa<br />
<strong>in</strong>duces translocation of phosphatidylser<strong>in</strong>e as <strong>in</strong> the early apoptosis<br />
of somatic cells.<br />
P415<br />
I<strong>de</strong>ntification, Isolation and <strong>in</strong> vitro long term culture of<br />
Male Germl<strong>in</strong>e Stem Cell <strong>in</strong> Porc<strong>in</strong>e<br />
Su Young, H*; Gupta, MK; Uhm, SJ; Lee, HT<br />
Dept of Bioscience & Biotechnology, ARRC, Konkuk University, Seoul 143-<br />
701, Korea<br />
Male germl<strong>in</strong>e stem cells are the basis of spermatogenesis and resi<strong>de</strong><br />
on basement membrane of sem<strong>in</strong>iferous tubule <strong>in</strong> testis. Due to the<br />
lack of specific markers, i<strong>de</strong>ntification of male germl<strong>in</strong>e stem cells is<br />
difficult to study <strong>in</strong> pig. In this study, we <strong>in</strong>vestigated to <strong>de</strong>term<strong>in</strong>e<br />
isolation and long-term culture system of male germl<strong>in</strong>e stem cell <strong>in</strong><br />
neonatal pig testis. We used farm piglets 5~10days old, testis were<br />
digested by sequential enzymatic system, <strong>in</strong>clud<strong>in</strong>g 1mg/ml<br />
collagenase, 1mg/ml hyaluronidase and 0.25% tryps<strong>in</strong>/EDTA. Cells<br />
were separated by discont<strong>in</strong>uous <strong>de</strong>nsity gradient and differential<br />
plat<strong>in</strong>g to <strong>in</strong>crease of purity. Isolated cells were cultured <strong>in</strong> DMEM<br />
medium supplemented with 15% FBS, and specific growth factors as<br />
1000 IU/ml leukemia <strong>in</strong>hibitory factor (LIF) and 10ng/ml glial cell<br />
l<strong>in</strong>e-<strong>de</strong>rived neurotrophic factor (GDNF) at 37℃ <strong>in</strong>cubator with 5%<br />
CO2. We have used STO cell l<strong>in</strong>e for fee<strong>de</strong>r layer treated by<br />
mitomyc<strong>in</strong> C for mitotically <strong>in</strong>activation. After 7-8 days culture, three<br />
dimensional colonies appeared as orig<strong>in</strong>al generation. Alkal<strong>in</strong>e<br />
phosphatase (AP) sta<strong>in</strong><strong>in</strong>g expressed positively. Also these germ cells<br />
expressed stem cell markers OCT-4, SSEA-1, and spermatogonial<br />
stem cell markers PGP9.5, Dolichos Biflourus Agglut<strong>in</strong><strong>in</strong> (DBA) <strong>in</strong><br />
immunocytochemistry. These cells have been <strong>in</strong>vestigated by RT-<br />
PCR us<strong>in</strong>g specific primers, pgp 9.5 and pigvasa, which is expressed<br />
specifically <strong>in</strong> the porc<strong>in</strong>e undifferentiated spermatogonia. We<br />
established porc<strong>in</strong>e male germl<strong>in</strong>e stem cell l<strong>in</strong>es from neonatal testis<br />
and <strong>in</strong> vitro long-term culture system. These results could be used for<br />
i<strong>de</strong>ntify<strong>in</strong>g the mechanism of spermatogenesis and apply<strong>in</strong>g for<br />
transgenesis.<br />
P416<br />
In vitro effect of sodium nitroprussi<strong>de</strong> (SNP) on sheep<br />
sperm motility<br />
Hassanpour, H*<br />
Department of Basic Sciences, College of Veter<strong>in</strong>ary Medic<strong>in</strong>e, Sharekord<br />
University, Iran<br />
Nitric oxi<strong>de</strong> (NO) has been recently shown to regulate many functions<br />
of sperm such as Acrosome reaction, sperm chemotaxis and motility.<br />
The aim of this study is to <strong>in</strong>vestigate the effects of sodium<br />
nitroprussi<strong>de</strong> (SNP) as nitric oxi<strong>de</strong> donor on sperm motility of sheep.<br />
After collect<strong>in</strong>g of normozoospermic samples by artificial vag<strong>in</strong>a<br />
from twenty Bakhtiari rams, motile spermatozoa were harvested by<br />
the swim-up technique us<strong>in</strong>g SOF-HEPES medium then <strong>in</strong>cubated for<br />
120 m<strong>in</strong>utes <strong>in</strong> the presence of SNP (0.1, 0.5, 0.7 μM). sperm motility<br />
assessed <strong>in</strong> four gra<strong>de</strong>s (rapid progressive motility, gra<strong>de</strong> A; slow or<br />
sluggish progressive motility, gra<strong>de</strong> B; non-progressive motility,<br />
gra<strong>de</strong> C; or immotility, gra<strong>de</strong> D.).In this study, SNP <strong>in</strong> the<br />
concentration of 0.1µM non-significantly <strong>in</strong>creased sperm motility at<br />
gra<strong>de</strong>s A & C, and <strong>de</strong>creased at gra<strong>de</strong>s B & D. Concentration of 0.5<br />
µM <strong>in</strong>creased gra<strong>de</strong> B & D, and <strong>de</strong>creased gra<strong>de</strong> C. SNP at the<br />
concentration of 0.7 µM significantly <strong>de</strong>creased sperm motility at<br />
gra<strong>de</strong> A (15.1%) and <strong>in</strong>creased at gra<strong>de</strong> D (16.7%), <strong>in</strong> comparison<br />
with their control groups (p