Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Poster Abstracts 179
P460
Improved sperm tail viability differentiation by coloured
mounting medium
Kovács, A 1-2* , Sarlós, P 1 , Egerszegi, I 1 , Oláh, J 2 , Révay, T 1 , Vass, N 2 and
Jávor, A 2
1Research Institute for Animal Breeding and Nutrition; 2 Debrecen University,
Centre for Agricultural Sciences, Institute for Animal Breeding Sciences
Introduction Head membrane permeability and acrosome status of
spermatozoa of different domestic mammals can be well
differenciated by the combined trypan blue – Giemsa staining
(Kovács A., Foote R.H.: Viability and acrosome staining of bull, boar
and rabbit spermatozoa. Biotechnic & Histochemistry 67: 119-124,
1992; Nagy Sz. et al.: Evaluation of sperm tail membrane integrity by
light microscopy. Theriogenology 52: 1153-1159, 1999), as well as by
the similar Chicago sky blue - Giemsa (Kútvölgyi et al.: Viability and
acrosome staining of stallion spermatozoa by Chicago sky blue and
Giemsa. Biotechnic & Histochemistry 81: 109-117, 2006) staining.
The evaluation of the tail membrane permeability may be often
uncertain due to its narrowness and different backgrounds caused by
seminal plasma and extender components.
Materials and methods Both combined stainings are routinely
applied in our laboratories using a yellow filter helping the better
differentiation of the acrosome status. Preparations from fresh ram
and boar, and frozen-thawed bull and ram semen samples were
mounted with immersion oil (Merck 4699) control evaluated with a
yellow filter, or with the same immersion oil saturated with Sudan I.
(Peking Chemical Works, Peking, China) or with dimethyl yellow
(Sigma D6760). The saturated solutions were the supernatants of the
centrifugated oversaturated ones.
Results and discussion Sudan I. or Dimethyl yellow solved in
immersion oil resulted in not only a good differentiation of the
acrosome status similarly to the control, but also a better
distinguishing of membrane permeable „dead” and impermeable
„live” tail domains of spermatozoa even in the case of background
caused by the extender. The effect can be explained by the relief-like
nature of the smears - the yellow mounting medium is more thick
around the cells than above them. The yellow surroundings of the
cells are lighter than the coloured „dead”, but darker than the
unstained „live” sperm tails ensuring their easy and clear
differentiation. The tail membrane of spermatozoa is more sensitive to
the harmful effects of the freezing-thawing procedure than their head
membrane, therefore its correct evaluation is extremely important for
the quality control.
P461
First foal born in Italy using flow cytometrically sorted
spermatozoa
Mari, G 1 *; Rizzato, G 1 ; Iacono, E 1 ; Merlo, B 1 ; Seren, E 2 ; Tamanini, C 2 ; Galeati,
G 2 ; Spinaci, M 2
1 Veterinary Clinical Department, University of Bologna, Italy; 2 Dep. of
Veterinary Morphophysiology and Animal Production, University of Bologna,
Italy
Introduction Sex-preselection of stallion spermatozoa is possible
using a modified flow cytometer to separate spermatozoa into X and
Y-chromosome bearing cells on the basis of DNA content. The
objectives of this study were to evaluate vitality, motility
characteristics and fertilizing ability of equine sexed semen.
Methods Semen from 4 thoroughbred stallions of proven fertility
(65% pregnancy rate per cycle) was diluted 1:1 with KMT and
analysed by Computer Aided Sperm Analysis (CASA). Total motility
(TM), progressive motility (PM), velocity average path (VAP) and
rapid spermatozoa (RAP) were recorded. For sorting, samples were
diluted to 200 x10 6 spermatozoa/ml and stained with Hoechst 33342
for 1.30 h at 35° C in the dark. Just prior to sorting a final
concentration of 100 x10 6 spermatozoa/ml was reached and red food
dye was added. A MoFlo SX® sperm sorter was used. Sorted
spermatozoa were evaluated for motility parameters by CASA and for
viability by SYBR-PI staining. The fertility of sexed spermatozoa was
assessed by inseminating 4 mares (7 estrous cycles). Mares were
histeroscopically inseminated at the utero-tubal junction, ipsilateral to
the preovulatory follicle, with 5 x10 6 spermatozoa in 250 μl, 30-32 h
after 2500 IU hCG administration for induction of ovulation.
Pregnancy diagnosis was carried out 15 days after ovulation.
Results Sperm motility characteristics were negatively affected by
sorting (pre vs. post-sorting: TM 79.6±5.5 vs. 38.6±14.5; PM
32.3±9.4 vs. 10.6±5.4; VAP 116.4±12.4 vs. 99.9±13.3; RAP
67.0±12.0 vs. 30.2±12.0; p0.05, T
student test). Pregnancy rate was 28.6% (2/7), and a pregnancy was
loss at 30 days while the other was carried to term with the birth of a
healthy filly.
Conclusions Sperm motility parameters are negatively affected by the
sorting process. On the other hand, viability is only slightly and nonsignificantly
affected. Usually, mares are inseminated with 500 x 10 6
motile spermatozoa; being that the number of sexed spermatozoa/h is
about 15 x 10 6 , and it would take too time to obtain the optimal dose,
the histeroscopic insemination allows to use low numbers of sexed
spermatozoa. Pregnancy rate obtained is lower than that reported in
literature (Lindsey et al., 2002: 38% with 5 x10 6 motile spermatozoa),
likely due to the lower number of motile spermatozoa in the
inseminating dose used in the present study. The Authors wish to
thank “Società Italiana Produttori Sementi”.
P462
Viability of equine spermatozoa recovered from the
epididymis cauda submitted to refrigeration
Martins, MIM*; Nagao, JF; Gomes, RG
Department of Veterinary Clinics, University of Londrina State (UEL), Brazil
The recovery of spermatozoa from the epididymis cauda may be an
important tool in equine reproduction because it makes possible the
recovery of viable cells after the death of valuable stallions. The
purpose of this study was to compare the viability of the spermatozoa
recovered from the epididymis cauda submitted to refrigeration.
Epididymals were obtained from five horses (English Thoroughbreds
and two mixed –breed) from a slaughterhouse. During the
transportation to the lab, the specimens were maintained in a 0,9%
saline solution in an ice box. The recovery of the spermatozoa was
accomplished by compressing the epididymis cauda and part of the
deferent duct with help of an anatomic nipper on a Petri dish
containing the extender Botu-Sêmen® (Biotech Ltda, Botucatu,
Brazil). The sperm samples were evaluated for motility, vigor, sperm
concentration, percentage of viable cells and morphology. The semen
was diluted (final concentration of 100x10 6 /mL) and stored in 1,5 mL
plastic tubes and packed in a transportation system Botu-Tainer®
(Biotech Ltda, Botucatu, Brazil) for 18 hours. After this period, the
tubes were transferred to a container with 400mL of water and kept in
the refrigerator (4ºC) for 24 hours. The semen samples were analysed
in three different moments: collection (M1), removal from the
transportation boxes (M2) and after 24 hours of refrigeration at 4ºC
(M3). The average motility (%), vigor (0-5), alive (%) and normal
spermatic cells (%) were 64.0, 3.7, 90.6 e 60.0 (M1); 56.0, 2.9, 91.8
and 57.8 (M2); 56.0, 3.1, 88.4 and 56.0 (M3). The most frequent
spermatic alterations were: abaxial tail insertion (6.3%), proximal
cytoplasmic droplet (7.3%), distal cytoplasmic droplet (3.0%) and
strongly bended tail (13.4%); probably because the spermatozoas
were from the epididymis cauda. Transportation was considered
efficient for the maintenance of spermatic quality after 24 hours at 4
ºC and no significant difference was observed among the evaluations.
The process of spermatozoa recovery and refrigeration obtained from
the epididymis cauda is promising, however, new experiments must
be carried out aiming the analyses of in vivo and/or in vitro fertility of
these cells.