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 />
Poster Abstracts 179<br />
P460<br />
Improved sperm tail viability differentiation by coloured<br />
mount<strong>in</strong>g medium<br />
Kovács, A 1-2* , Sarlós, P 1 , Egerszegi, I 1 , Oláh, J 2 , Révay, T 1 , Vass, N 2 and<br />
Jávor, A 2<br />
1Research Institute for Animal Breed<strong>in</strong>g and Nutrition; 2 Debrecen University,<br />
Centre for Agricultural Sciences, Institute for Animal Breed<strong>in</strong>g Sciences<br />
Introduction Head membrane permeability and acrosome status of<br />
spermatozoa of different domestic mammals can be well<br />
differenciated by the comb<strong>in</strong>ed trypan blue – Giemsa sta<strong>in</strong><strong>in</strong>g<br />
(Kovács A., Foote R.H.: Viability and acrosome sta<strong>in</strong><strong>in</strong>g of bull, boar<br />
and rabbit spermatozoa. Biotechnic & Histochemistry 67: 119-124,<br />
1992; Nagy Sz. et al.: Evaluation of sperm tail membrane <strong>in</strong>tegrity by<br />
light microscopy. Theriogenology 52: 1153-1159, 1999), as well as by<br />
the similar Chicago sky blue - Giemsa (Kútvölgyi et al.: Viability and<br />
acrosome sta<strong>in</strong><strong>in</strong>g of stallion spermatozoa by Chicago sky blue and<br />
Giemsa. Biotechnic & Histochemistry 81: 109-117, 2006) sta<strong>in</strong><strong>in</strong>g.<br />
The evaluation of the tail membrane permeability may be often<br />
uncerta<strong>in</strong> due to its narrowness and different backgrounds caused by<br />
sem<strong>in</strong>al plasma and exten<strong>de</strong>r components.<br />
Materials and methods Both comb<strong>in</strong>ed sta<strong>in</strong><strong>in</strong>gs are rout<strong>in</strong>ely<br />
applied <strong>in</strong> our laboratories us<strong>in</strong>g a yellow filter help<strong>in</strong>g the better<br />
differentiation of the acrosome status. Preparations from fresh ram<br />
and boar, and frozen-thawed bull and ram semen samples were<br />
mounted with immersion oil (Merck 4699) control evaluated with a<br />
yellow filter, or with the same immersion oil saturated with Sudan I.<br />
(Pek<strong>in</strong>g Chemical Works, Pek<strong>in</strong>g, Ch<strong>in</strong>a) or with dimethyl yellow<br />
(Sigma D6760). The saturated solutions were the supernatants of the<br />
centrifugated oversaturated ones.<br />
Results and discussion Sudan I. or Dimethyl yellow solved <strong>in</strong><br />
immersion oil resulted <strong>in</strong> not only a good differentiation of the<br />
acrosome status similarly to the control, but also a better<br />
dist<strong>in</strong>guish<strong>in</strong>g of membrane permeable „<strong>de</strong>ad” and impermeable<br />
„live” tail doma<strong>in</strong>s of spermatozoa even <strong>in</strong> the case of background<br />
caused by the exten<strong>de</strong>r. The effect can be expla<strong>in</strong>ed by the relief-like<br />
nature of the smears - the yellow mount<strong>in</strong>g medium is more thick<br />
around the cells than above them. The yellow surround<strong>in</strong>gs of the<br />
cells are lighter than the coloured „<strong>de</strong>ad”, but darker than the<br />
unsta<strong>in</strong>ed „live” sperm tails ensur<strong>in</strong>g their easy and clear<br />
differentiation. The tail membrane of spermatozoa is more sensitive to<br />
the harmful effects of the freez<strong>in</strong>g-thaw<strong>in</strong>g procedure than their head<br />
membrane, therefore its correct evaluation is extremely important for<br />
the quality control.<br />
P461<br />
First foal born <strong>in</strong> Italy us<strong>in</strong>g flow cytometrically sorted<br />
spermatozoa<br />
Mari, G 1 *; Rizzato, G 1 ; Iacono, E 1 ; Merlo, B 1 ; Seren, E 2 ; Taman<strong>in</strong>i, C 2 ; Galeati,<br />
G 2 ; Sp<strong>in</strong>aci, M 2<br />
1 Veter<strong>in</strong>ary Cl<strong>in</strong>ical Department, University of Bologna, Italy; 2 Dep. of<br />
Veter<strong>in</strong>ary Morphophysiology and Animal Production, University of Bologna,<br />
Italy<br />
Introduction Sex-preselection of stallion spermatozoa is possible<br />
us<strong>in</strong>g a modified flow cytometer to separate spermatozoa <strong>in</strong>to X and<br />
Y-chromosome bear<strong>in</strong>g cells on the basis of DNA content. The<br />
objectives of this study were to evaluate vitality, motility<br />
characteristics and fertiliz<strong>in</strong>g ability of equ<strong>in</strong>e sexed semen.<br />
Methods Semen from 4 thoroughbred stallions of proven fertility<br />
(65% pregnancy rate per cycle) was diluted 1:1 with KMT and<br />
analysed by Computer Ai<strong>de</strong>d Sperm Analysis (CASA). Total motility<br />
(TM), progressive motility (PM), velocity average path (VAP) and<br />
rapid spermatozoa (RAP) were recor<strong>de</strong>d. For sort<strong>in</strong>g, samples were<br />
diluted to 200 x10 6 spermatozoa/ml and sta<strong>in</strong>ed with Hoechst 33342<br />
for 1.30 h at 35° C <strong>in</strong> the dark. Just prior to sort<strong>in</strong>g a f<strong>in</strong>al<br />
concentration of 100 x10 6 spermatozoa/ml was reached and red food<br />
dye was ad<strong>de</strong>d. A MoFlo SX® sperm sorter was used. Sorted<br />
spermatozoa were evaluated for motility parameters by CASA and for<br />
viability by SYBR-PI sta<strong>in</strong><strong>in</strong>g. The fertility of sexed spermatozoa was<br />
assessed by <strong>in</strong>sem<strong>in</strong>at<strong>in</strong>g 4 mares (7 estrous cycles). Mares were<br />
histeroscopically <strong>in</strong>sem<strong>in</strong>ated at the utero-tubal junction, ipsilateral to<br />
the preovulatory follicle, with 5 x10 6 spermatozoa <strong>in</strong> 250 μl, 30-32 h<br />
after 2500 IU hCG adm<strong>in</strong>istration for <strong>in</strong>duction of ovulation.<br />
Pregnancy diagnosis was carried out 15 days after ovulation.<br />
Results Sperm motility characteristics were negatively affected by<br />
sort<strong>in</strong>g (pre vs. post-sort<strong>in</strong>g: TM 79.6±5.5 vs. 38.6±14.5; PM<br />
32.3±9.4 vs. 10.6±5.4; VAP 116.4±12.4 vs. 99.9±13.3; RAP<br />
67.0±12.0 vs. 30.2±12.0; p0.05, T<br />
stu<strong>de</strong>nt test). Pregnancy rate was 28.6% (2/7), and a pregnancy was<br />
loss at 30 days while the other was carried to term with the birth of a<br />
healthy filly.<br />
Conclusions Sperm motility parameters are negatively affected by the<br />
sort<strong>in</strong>g process. On the other hand, viability is only slightly and nonsignificantly<br />
affected. Usually, mares are <strong>in</strong>sem<strong>in</strong>ated with 500 x 10 6<br />
motile spermatozoa; be<strong>in</strong>g that the number of sexed spermatozoa/h is<br />
about 15 x 10 6 , and it would take too time to obta<strong>in</strong> the optimal dose,<br />
the histeroscopic <strong>in</strong>sem<strong>in</strong>ation allows to use low numbers of sexed<br />
spermatozoa. Pregnancy rate obta<strong>in</strong>ed is lower than that reported <strong>in</strong><br />
literature (L<strong>in</strong>dsey et al., 2002: 38% with 5 x10 6 motile spermatozoa),<br />
likely due to the lower number of motile spermatozoa <strong>in</strong> the<br />
<strong>in</strong>sem<strong>in</strong>at<strong>in</strong>g dose used <strong>in</strong> the present study. The Authors wish to<br />
thank “Società Italiana Produttori Sementi”.<br />
P462<br />
Viability of equ<strong>in</strong>e spermatozoa recovered from the<br />
epididymis cauda submitted to refrigeration<br />
Mart<strong>in</strong>s, MIM*; Nagao, JF; Gomes, RG<br />
Department of Veter<strong>in</strong>ary Cl<strong>in</strong>ics, University of Londr<strong>in</strong>a State (UEL), Brazil<br />
The recovery of spermatozoa from the epididymis cauda may be an<br />
important tool <strong>in</strong> equ<strong>in</strong>e reproduction because it makes possible the<br />
recovery of viable cells after the <strong>de</strong>ath of valuable stallions. The<br />
purpose of this study was to compare the viability of the spermatozoa<br />
recovered from the epididymis cauda submitted to refrigeration.<br />
Epididymals were obta<strong>in</strong>ed from five horses (English Thoroughbreds<br />
and two mixed –breed) from a slaughterhouse. Dur<strong>in</strong>g the<br />
transportation to the lab, the specimens were ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> a 0,9%<br />
sal<strong>in</strong>e solution <strong>in</strong> an ice box. The recovery of the spermatozoa was<br />
accomplished by compress<strong>in</strong>g the epididymis cauda and part of the<br />
<strong>de</strong>ferent duct with help of an anatomic nipper on a Petri dish<br />
conta<strong>in</strong><strong>in</strong>g the exten<strong>de</strong>r Botu-Sêmen® (Biotech Ltda, Botucatu,<br />
Brazil). The sperm samples were evaluated for motility, vigor, sperm<br />
concentration, percentage of viable cells and morphology. The semen<br />
was diluted (f<strong>in</strong>al concentration of 100x10 6 /mL) and stored <strong>in</strong> 1,5 mL<br />
plastic tubes and packed <strong>in</strong> a transportation system Botu-Ta<strong>in</strong>er®<br />
(Biotech Ltda, Botucatu, Brazil) for 18 hours. After this period, the<br />
tubes were transferred to a conta<strong>in</strong>er with 400mL of water and kept <strong>in</strong><br />
the refrigerator (4ºC) for 24 hours. The semen samples were analysed<br />
<strong>in</strong> three different moments: collection (M1), removal from the<br />
transportation boxes (M2) and after 24 hours of refrigeration at 4ºC<br />
(M3). The average motility (%), vigor (0-5), alive (%) and normal<br />
spermatic cells (%) were 64.0, 3.7, 90.6 e 60.0 (M1); 56.0, 2.9, 91.8<br />
and 57.8 (M2); 56.0, 3.1, 88.4 and 56.0 (M3). The most frequent<br />
spermatic alterations were: abaxial tail <strong>in</strong>sertion (6.3%), proximal<br />
cytoplasmic droplet (7.3%), distal cytoplasmic droplet (3.0%) and<br />
strongly ben<strong>de</strong>d tail (13.4%); probably because the spermatozoas<br />
were from the epididymis cauda. Transportation was consi<strong>de</strong>red<br />
efficient for the ma<strong>in</strong>tenance of spermatic quality after 24 hours at 4<br />
ºC and no significant difference was observed among the evaluations.<br />
The process of spermatozoa recovery and refrigeration obta<strong>in</strong>ed from<br />
the epididymis cauda is promis<strong>in</strong>g, however, new experiments must<br />
be carried out aim<strong>in</strong>g the analyses of <strong>in</strong> vivo and/or <strong>in</strong> vitro fertility of<br />
these cells.