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 />
178 Poster Abstracts<br />
P457<br />
Microfluidic sort<strong>in</strong>g of boar spermatozoa<br />
Holt, W 1 *, Satake, N 1 , Smith, GD 2 , Alhai<strong>de</strong>r, A 3 , Takayama, S 4 , Watson, PF 3<br />
1Institute of Zoology, Zoological Society of London, United K<strong>in</strong>gdom;<br />
2Department of Obstetrics and Gynecology, University of Michigan, United<br />
States; 3 Veter<strong>in</strong>ary Basic Sciences, Royal Veter<strong>in</strong>ary College, United<br />
K<strong>in</strong>gdom; 4 Department of Biomedical Eng<strong>in</strong>eer<strong>in</strong>g, University of Michigan,<br />
United States<br />
Microfluidic sperm sorter <strong>de</strong>vices, <strong>de</strong>veloped at the University of<br />
Michigan, are able to sort a highly motile subpopulation of human<br />
spermatozoa, where motile spermatozoa traverse the boundary<br />
between two lam<strong>in</strong>ar flow<strong>in</strong>g streams (one conta<strong>in</strong><strong>in</strong>g spermatozoa<br />
and the other compris<strong>in</strong>g culture media alone) and are collected at an<br />
outlet reservoir (1). The aim of the present study was to <strong>in</strong>vestigate<br />
the application of this technology to the physical separation of<br />
putative boar sperm subpopulations. We have previously shown<br />
statistically that bicarbonate exposure significantly stimulates the<br />
motility of a (boar-<strong>de</strong>pen<strong>de</strong>nt) sperm subpopulation (2), while others<br />
rema<strong>in</strong> unaffected; physical separation of these populations has never<br />
been achieved. Percoll washed spermatozoa from 3 boars were<br />
<strong>in</strong>cubated (10 m<strong>in</strong> at 38ºC) <strong>in</strong> Tyro<strong>de</strong>’s based medium without<br />
bicarbonate, then 15mM bicarbonate/CO 2 was ad<strong>de</strong>d to the medium<br />
before load<strong>in</strong>g <strong>in</strong>to sperm-sorters (provi<strong>de</strong>d by Strex Inc., Japan); the<br />
sorters were then left for 30 m<strong>in</strong>utes at 38°C. Two outlet populations<br />
were collected and 60μl samples of each were subsequently analysed<br />
us<strong>in</strong>g a CASA system (Hobson Sperm Tracker, Hobson Track<strong>in</strong>g<br />
Systems, Sheffield, UK). In a prelim<strong>in</strong>ary study, spermatozoa were<br />
sta<strong>in</strong>ed with SYBR14 and Propidium Iodi<strong>de</strong> before load<strong>in</strong>g <strong>in</strong>to sperm<br />
sorters; vi<strong>de</strong>omicrography of the sort<strong>in</strong>g channel showed that >95% of<br />
spermatozoa <strong>in</strong> the ‘sorted’ channel were live (PI−ve), although they<br />
were highly diluted. The proportion of immotile spermatozoa was<br />
reduced from 45% <strong>in</strong> the unsorted channel to 26% <strong>in</strong> the sorted<br />
channel. The proportion of progressively motile spermatozoa also<br />
<strong>de</strong>creased upon sort<strong>in</strong>g (from 45% to 22%), but there was a surpris<strong>in</strong>g<br />
<strong>in</strong>crease <strong>in</strong> the proportion of hyperactivated spermatozoa (from 10%<br />
to 52%). These results <strong>de</strong>monstrate the feasibility of us<strong>in</strong>g the sperm<br />
sorters for the isolation of a boar sperm subpopulation. Given that<br />
progressive motility is required for the sort<strong>in</strong>g process to occur, the<br />
prevalence of hyperactivated spermatozoa after sort<strong>in</strong>g suggests that<br />
the sort<strong>in</strong>g may <strong>in</strong>duce the f<strong>in</strong>al stages of capacitation.<br />
P458<br />
Time oriented A.I. <strong>in</strong> cattle with reduced number of sperm<br />
cells<br />
Kanitz, W. 1 *, Becker, F. 1 , Bhojwani, S. 1 , Alm, H. 1 , Nehr<strong>in</strong>g, H. 2 , Nürnberg, G. 1<br />
1Research Institute for the Biology of Farm <strong>Animals</strong>, Dummerstorf, Germany,<br />
2Institute for <strong>Reproduction</strong> of Farm <strong>Animals</strong>, Schönow, Germany<br />
Among others number of spermatozoa per A.I. and time of<br />
<strong>in</strong>sem<strong>in</strong>ation with regard to oestrus symptoms or ovulation are<br />
important <strong>de</strong>term<strong>in</strong>ants for pregnancy rate <strong>in</strong> cattle. In addition, the<br />
necessary number of spermatozoa per A.I. is un<strong>de</strong>r <strong>in</strong>vestigation with<br />
an aim of better utilisation of A.I. sires and the <strong>in</strong>sem<strong>in</strong>ation of sorted<br />
spermatozoa. Because, data on fertilisation rate after A.I. with reduced<br />
sperm number are unavailable, we performed the follow<strong>in</strong>g<br />
experiment. Altogether 116 heifers (German Holste<strong>in</strong>) received<br />
PGF 2 (0.5 mg Cloprostenol ® , Jenapharm, Germany; i.m.) between<br />
day 8 to 14 of oestrous cycle. 60 hours later 50 µg GnRH agonist<br />
(Depherel<strong>in</strong> ® ; Veyx, Germany, i.m.) was <strong>in</strong>jected. Subsequently, 13<br />
hours later the animals were <strong>in</strong>sem<strong>in</strong>ated once with frozen/thawed<br />
semen of 4 proven sires. The number of spermatozoa was 15x10 6 ,<br />
5x10 6 or 1x10 6 per A.I. (G1/G2/G3). After surgical recovery of the<br />
oviduct and the tip of the uter<strong>in</strong>e horn ipsilateral to the C.l. embryos<br />
and oocytes were flushed from the oviduct on day 4 after<br />
<strong>in</strong>sem<strong>in</strong>ation. The recovered oocytes and embryos were classified<br />
accord<strong>in</strong>g their morphology and fixed us<strong>in</strong>g BFS medium (buffered<br />
formol solution, Merck, Germany). All oocytes and embryos were<br />
sta<strong>in</strong>ed with HOECHST 33258 (Sigma, Germany) to i<strong>de</strong>ntify<br />
accessory sperm cells. For statistical analyses fertilisation rates and<br />
portions of <strong>in</strong>tact embryos were compared by means of Chi-squaretest.<br />
The <strong>in</strong>fluence of the factors sire, and sperm number on<br />
fertilisation rate was tested by GLM-procedure of SAS ® (Release 8.2,<br />
SAS Institute, Inc., Cary, NC 1999). Of the 116 heifers treated 106<br />
animals ovulated (ovulation rate 91.5 %). The mean recovery rate for<br />
oocytes and embryos was 80.2 %. Mean fertilisation rates and the<br />
portions of <strong>in</strong>tact embryos were 92.3/84.6 % (G1), 96.2/80.7% (G2)<br />
and 78.8/75.8 % (G3). Mean number of accessory sperm cells per<br />
embryo was significantly higher <strong>in</strong> G1 and G2 than <strong>in</strong> G3 (G1:<br />
26.6±8.4, G2: 45.3±8.6, G3: 6.5±7.2). From the data obta<strong>in</strong>ed it can<br />
be conclu<strong>de</strong>d that time oriented A.I. can result <strong>in</strong> high fertilisation<br />
rate. Moreover, the portion of <strong>in</strong>tact embryos does not <strong>de</strong>pend on<br />
sperm number per <strong>in</strong>sem<strong>in</strong>ation after time oriented A.I. F<strong>in</strong>ally, the<br />
number of accessory sperm cells is not correlated with the portion of<br />
<strong>in</strong>tact embryos after time oriented A.I.<br />
P459<br />
A simple technique for m<strong>in</strong>imal dose DIUI <strong>in</strong> PMSG<br />
multiple-ovulat<strong>in</strong>g dairy heifers<br />
Kornmatitsuk, B 1 *; Charoenyongyoo, P 1 ; Pattharanukulkit, K 1 ;<br />
Akkhawattanangkul, Y 1 ; Chaiprasat, S 2 ; Kornmatitsuk, S 1<br />
1Faculty of Veter<strong>in</strong>ary Science, Mahidol University, Phutthamonthon, Nakhon<br />
Pathom, 73170; 2 Livestock Semen Production Center-Inthanont Royal<br />
Project, Department of Livestock Development, M<strong>in</strong>istry of Agriculture and<br />
Cooperatives, Maung, Chiang Mai, 50300<br />
The aim of the present study was to <strong>in</strong>vestigate an accomplishment of<br />
m<strong>in</strong>imal dose <strong>de</strong>ep <strong>in</strong>tra-uter<strong>in</strong>e <strong>in</strong>sem<strong>in</strong>ation (DIUI) us<strong>in</strong>g a simple<br />
embryo transfer (ET) pistolet, <strong>in</strong> connection to relative fertilisation<br />
rates and early embryo qualities on day 7 post-service, study<strong>in</strong>g <strong>in</strong><br />
pregnant mare serum gonadotrop<strong>in</strong> (PMSG) multiple-ovulat<strong>in</strong>g dairy<br />
heifers. Ten heifers of crossbred Holste<strong>in</strong> Frisian (≥75%) were<br />
<strong>in</strong>clu<strong>de</strong>d. They were characterised for their oestrous cycles,<br />
afterwards subjected to the superovulatory programme us<strong>in</strong>g PMSG/<br />
Prostagland<strong>in</strong> F2alpha (PGF2alpha) protocol, and fixed-time artificial<br />
<strong>in</strong>sem<strong>in</strong>ation: Group 1, 3 heifers <strong>in</strong>sem<strong>in</strong>ated with 20×10 6 sperm cells<br />
at body of uterus; Group 2, 4 heifers <strong>in</strong>sem<strong>in</strong>ated with 10×10 6 sperm<br />
cells at tip of the uter<strong>in</strong>e horns and Group 3, 3 heifers <strong>in</strong>sem<strong>in</strong>ated<br />
with 10×10 6 sperm cells at body of the uterus.. Embryos were nonsurgically<br />
recovered and categorised on 7 days subsequent to the<br />
<strong>in</strong>sem<strong>in</strong>ation. On the day of each treatment, the ovaries of the heifers<br />
were ultrasonographically exam<strong>in</strong>ed. Prior to the treatment, majorities<br />
of the follicles observed <strong>in</strong> all heifers were of small sizes (