CONTRACEPTIVE INTERVENTION BY VAS OCCLUSION USING COPOLYMER STYRENE MALEIC ANHYDRIDE (SMA) » IN MALE RATS AND IN VITRO STUDY IN DOGS
Loading...
Date
2021
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
The non-hormonal, copolymeric, contraceptive agent, Styrene maleic
anhydride (SMA) is expected to provide a valuable addition to the current options of
canine male contraception. The present study was conducted to determine the in
vitro spermicidal action of copolymer SMA on canine ejaculatory spermatozoa and
to study the in vivo vas occlusion contraceptive effect of copolymer SMA, followed
by its reversal using dimethyl sulphoxide (DMSO) and sodium bicarbonate
(NaHCOs) in male Wistar rats.
The copolymer SMA was developed by Radical addition-fragmentation chain
transfer (RAFT) polymerization in the ratio of 2:1 of styrene and maleic anhydride
and characterized by Zeta potential analysis. Matrix assisted laser desorption
(MALDI-ToF), Nuclear magnetic resonance spectroscopy (NMR) and Fourier-
Transfoim Infrared (FTIR) spectroscopy. The sperm rich second fraction of semen
collected from twelve adult dogs was subjected to varying dose levels of copolymer
SMA (1.0 and 2.0 mg / 100 pl of DMSO ) in vitro and were microscopically
analysed for its motility, viability and acrosomal integrity. A significant decline in
motility and viability percentage and increase in acrosomal damage of ejaculatory
sperm cells were noticed in both 1.0 and 2.0 mg of copolymer SMA treated canine
spermatozoa at different time intervals viz. 0, 15, 30, 45 and 60 min when compared
to the DMSO treated and untreated control. Both 1.0 and 2.0 mg of copolymer SMA
showed 100 per cent spermicidal effect on spermatozoa at 60 min. Hence, all further
in vitro studies were carried out using the 1.0 mg dose of SMA.
The morphological, topological and ultrastructural changes of canine
spermatozoa induced by copolymer SMA (Img/lOOpl of DMSO) were examined
using Atomic force microscopy (AFM), Scanning electron microscopy (SEM) and
Transmission electron microscopy (TEM). As a result, deleterious changes were
observed in the acrosomal and plasma membrane of canine spermatozoa treated with
copolymer SMA and no such changes were detected in DMSO and untreated control
group of canine spermatozoa.
In vivo vas occlusion trials were conducted in experimental groups (G II to G
V) of Wistar rats using 5mg of copolymer SMA dissolved in lOpl of DMSO injected
intravasally while, only lOpl of DMSO was injected in sham control rats (G I). After
15-19 days post mating, 75 per cent of female rats mated with sham control males
and 17, 8, 0 and 0 per cent of the female rats mated with vas occluded group II, HI,
rv and V male rats, respectively were found pregnant on ultrasound examination.
However, no significant differences were noticed in the sperm count, motility,
viability and abnormality percentage of epididymal spermatozoa between sham
operated group and SMA treated groups of rat. AFM, SEM and TEM studies also
did not show any morphological changes in the head, mid piece and tail regions of
epididymal spermatozoa.
In vivo vas occlusion reversal experiment was also carried out in twelve
Wistar rats (G VI) using solvents viz., DMSO and 5 per cent NaHCOj. At 60 days of
post-reversal period, fertility trials showed 67 and 83 per cent restoration of fertility
in DMSO and NaHCOs reversed group of rats, respectively. SEM studies showed
that the vas lumen was occluded with white, amorphous substances which anchored
into the vas mucosal wall by invagination and blocked the patency. Following vas
occlusion reversal, vas lumen regained its patency with loss of SMA copolymer plug
in the vas lumen and complete regeneration in the vas epithelium was seen. All other
parameters viz. haematology, serum biochemistry and testosterone remained
unaltered in vas occluded and reversal rats. Thus, use of copolymer SMA as vas
occlusive agent was found to be safe, effective and could provide hope as a
reversible male contraceptive in canine.