possum natural mating behaviour has led to the development of a laparoscopic artificial Ž
. Ž
. insemination AI procedure Molinia et al., 1998 . As possums are monovular, it has
been necessary to concurrently develop superovulation and AI protocols to maximise potential numbers of ovulated eggs available for fertilization in vivo. Further, hormone
manipulation permits control of the timing of insemination relative to ovulation, and fertilization. Using both AI and superovulation with the exogenous hormones pregnant
Ž .
Ž .
mare serum gonadotrophin PMSG rLH, fertilized eggs or embryos 1–2 per possum Ž
. are consistently recovered Molinia et al., 1998 . Accordingly, the combination of
superovulation and AI provides a model system to investigate sperm transport and pre-fertilization events in the possum.
The main aim of the current study was to investigate sperm transport in vivo following intrauterine AI of PMSGrLH superovulated possums. Specific objectives
were to determine the distribution of spermatozoa through the female reproductive tract in relation to ovulation, to characterise the epithelial lining of the oviduct with particular
emphasis on the presence of any sperm storage regions within the lower isthmus, and to examine the morphology and motility of spermatozoa within each of the isthmus, middle
and ampulla oviduct segments.
2. Materials and methods
2.1. Animals Ž
. Brushtail possums Trichosurus Õulpecula were captured in box traps and transferred
to the Landcare Research Animal Facility at Lincoln, New Zealand. The animals were weighed and separate sexes were housed in individual cages. The animals were
maintained on a diet of cereal pellets supplemented with fruit and vegetables and their food and water requirements were attended to daily. Experiments were approved by the
Animal Ethics Committee of Manaaki Whenua Landcare Research and the University of Newcastle Animal Care and Ethics Committee.
2.2. SuperoÕulation A PMSGrLH hormone protocol was used to superovulate all female possums prior
Ž .
to laparoscopic AI. PMSG Folligon; Intervet, Oss, The Netherlands was administered as a single 15 IU i.m. injection followed by a single i.m. injection of 4 mg LH
Ž .
Ž Lutropin-V; Vetrepharm, Essendon, Victoria, Australia 78 h later Glazier and Molinia,
. 1998 . LH was provided as a generous gift by Vetrepharm, Australia.
2.3. Semen collection and processing Ž
y1
. Ž .
Male possums were anaesthetised by CO rO 2 lr1 l min
Jolly et al., 1995
2 2
Ž and then killed with a single intra-cardiac injection of sodium pentobarbitone Pento-
.
y1
barb; Chemstock Animal Health, Christchurch, NZ at a dose rate of 125 mg kg . The
caudal epididymis was separated from the testis, and the tissue surface lacerated with a
Ž .
sharp blade and resuspended in phosphate-buffered saline PBS to release spermatozoa. Spermatozoa were assessed for motility by phase contrast microscopy and for concentra-
Ž .
tion using a haemocytometer Rodger et al., 1991 . Semen samples were diluted with PBS to a concentration of 1 = 10
7
motile spermatozoa and stored at 48C for up to 2 h prior to insemination.
2.4. Laparoscopic intrauterine AI Females were inseminated at 27 h after LH directly into both uteri using a
Ž .
laparoscopic procedure as previously described Molinia et al., 1998 . Each uterus Ž
received a single dose of spermatozoa 0.4 ml containing approximately 4 million motile .
spermatozoa . The animals were allowed to recover in a quiet corner before examination of the reproductive tracts.
2.5. Examination and processing of reproductiÕe tracts Ž
. At 1.5, 3, 6, 9 and 12 h after AI n s 5 per group , females were euthanised by
barbiturate overdose as detailed above for male possums. Reproductive tracts were removed and the ovaries separated for assessment of numbers of recent ovulation sites
Ž .
and remaining un-ovulated follicles 2 mm . Vaginal smears were taken and the slides examined for the presence of spermatozoa using Hoffman modulation contrast mi-
Ž .
croscopy Zeiss Axiovert 35 microscope . Each ovaryroviductruterus complex was separated from the vagina at the cervices.
Ž .
Each oviduct was clamped Vascu-Statt; Scanlan, Victoria, Australia by visual assess- Ž
. ment into three approximately equal segments; the isthmus nearest the uterus , middle
Ž .
segment and ampulla nearest the ovary . As detailed below, the left side of the tract was flushed and the right side processed for either histological staining or electron mi-
croscopy.
2.6. Flushing and estimation of sperm numbers The left uterus and oviduct regions were excised from the remainder of the tract and
Ž .
Ž .
Ž flushed with either 1.0 ml uterus or 0.6 ml oviduct segments of heparinised 12.5 IU
y1
. ml
PBS. After flushing, tissue was fixed in Bouin’s fluid, processed for routine light Ž
. microscopy as detailed below and examined for the presence of spermatozoa to ensure
that the procedure had been successful. The flushes were first examined under a phase-contrast microscope to assess motility
and headrtail orientation of spermatozoa. To estimate sperm numbers, a constant Ž
. Ž
volume of the flush 0.5 ml was centrifuged at 150 = g for 5 min. The supernatant 0.4 .
Ž .
ml was discarded and the pellet resuspended with 0.5 triton 0.1 ml to remove red blood cells. Sperm were counted using an Improved Neubauer Bright-Line haemocy-
tometer. Replicate counts were carried out on both chambers of the haemocytometer for all samples examined.
2.7. Histological processing For half of the possums examined, the right side of the tract was fixed whole in
Bouin’s solution. Segments were immersed in Bouin’s fixative for 48 h, then washed in several changes of 70 ethanol and stored in 70 ethanol. Wax blocks were prepared
Ž .
and 7-mm sections were cut and stained with Harris’ haematoxylin and eosin HNE , or with one of the following mucopolysaccharide histochemical methods: 1 alcian blue,
Ž . Ž
. pH 2.5 or periodic acid-Schiff PAS
Pearse, 1968 . 2.8. Electron microscopy
For the remaining possums, the right side of the tract was fixed whole in Karnovsky’s Ž
. solution
Karnovsky, 1965 at pH 7.6 overnight at 48C before storage in 0.1 M
Ž .
phosphate buffer, pH 7.4 at room temperature Mate et al., 1992 . Post-fixation was in Ž
. 1 wrv OsO
in 0.1 M phosphate buffer for 1 h at 48C. This was followed by
4
washing in distilled water, dehydration and embedding in Spurr’s resin. Ultrathin sections were placed on nickel grids, stained with uranyl acetate and lead citrate and
examined on a Joel CX-100 electron microscope.
2.9. Statistical analysis Ovarian assessments are presented as mean values with range intervals. The numbers
of spermatozoa recovered from flushes of the tract are presented as means standard Ž
. error of the means S.E.M. . Comparison of treatment means was examined non-para-
metrically using a Kruskal–Wallis one way ANOVA on ranks. Dunns’ method was used for pairwise comparison to determine which treatment groups were different.
3. Results
