Four experiments determined the kinetics of in vitro maturation and fertilization of cat oocytes and the effects of prolonged cold storage of ovaries before oocyte recovery on in vitro maturation/in vitro fertilization (IVM/IVF) success. Domestic cat ovaries were collected at ovariohysterectomy and stored at 4°C in PBS until oocyte recovery and culture in Eagle's minimal essential medium (EMEM) containing FSH, LH, oestradiol and BSA for maturation. In Expt 1, meiotic maturation was assessed at 0, 12, 24, 38 and 48 h of culture. After 24 h, > 61% of oocytes were in telophase I or metaphase II. In Expt 2, oocytes were recovered from ovaries stored for 24, 48 or 72 h and cultured in EMEM for 24 h. There was no difference among groups (P > 0.05) in the ability to achieve nuclear maturation (mean ± sem, 57.1 ± 5.3%, 60.4 ± 5.4%, 55.4±15.1% for 24, 48 and 72 h, respectively). Fertilization and embryo development after insemination at 16, 24, 32, 40 and 48 h of culture were examined in Expt 3. Of 98 oocytes inseminated at 32 h, 69% cleaved, 59% developed into morulae and 13% into blastocysts, more (P <0.05) than those oocytes inseminated at earlier and later times. Development to blastocysts occurred after insemination at 16 (1.2%), 24 (9.1%) and 32 (13.3%) h of culture, but not after insemination at 40 or 48 h. Expt 4 involved cold storage of ovaries for 24, 48 or 72 h before oocyte recovery and insemination at 32 h of culture (the optimal time measured in Expt 3). Compared with storage for 24 h, fertilization success was lower (P < 0.05) in the 48 and 72 h groups, and, although 9.1% of inseminated oocytes from the 24 h storage group developed to blastocysts, none (P <0.05) achieved this stage after 48 or 72 h of storage. These results indicate that domestic cat oocytes reach nuclear maturity by 24 h in culture and can be fertilized and develop to blastocyts optimally after insemination at 32 h. Oocytes recovered from ovaries stored at 4°C for up to 72 h are capable of reaching telophase I or metaphase II in vitro. However, only oocytes stored within the ovary for 24 h produce blastocysts, indicating that the ability to achieve nuclear maturation is an inadequate indicator of fertilization and developmental competence.
B. A. Wolfe and D. E. Wildt
T. C. Wood and D. E. Wildt
Immature cumulus–oocyte complexes (COCs) were recovered from freshly excised domestic cat ovaries and graded at a magnification of × 40 for the condition of the cumulus oophorus of the oocyte cytoplasm. Grade I and II COCs were those with a uniformly dark cytoplasm and a readily identifiable, eccentrically located germinal vesicle. Grade I COCs had five or more cumulus oophorus cell layers, whereas grade II complements had less than five cell layers. Grade III and IV COCs were those undergoing progressive stages of oocyte cytoplasmic deterioration indicated by transparency or mosaic fragmentation and partial-to-complete loss of cumulus oophorus cells. In Expt 1, 699 oocytes were cultured for maturation and fertilization in vitro. More (P < 0.05) oocytes from grade I COCs matured (59.3%) and fertilized (29.7%) than from all other grades. Maturation and fertilization success did not differ (P > 0.05) for grade II (32.4, 11.6%, respectively) and grade III (21.9, 5.1%) oocytes, but these values were superior (P < 0.05) to those of grade IV (5.1, 1.4%). In Expt 2, 1040 COCs were graded, cultured for maturation and then inseminated. Of grade I oocytes, 24.4% developed into blastocysts compared with only 5.3% of grade II oocytes (P < 0.05). In general, oocytes from grade III and IV COCs were incapable of cleaving or growing in vitro. Of the 1739 COCs collected for both experiments, 12.3% met grade I criteria, the only category that provided consistent maturation, fertilization and development to blastocyst stage in vitro. In summary, a highly heterogeneous population of cumulus–oocyte complexes can be separated in the cat on the basis of grossly apparent morphological characteristics that, in turn, reflect functional differences in the ability of oocytes to mature, fertilize and develop in vitro.
D. E. WILDT, H. D. WOODY and W. R. DUKELOW
A gonadotrophin regimen was developed to induce a high incidence of double and triple ovulations in the beef cow using a single injection of FSH on Day 16 followed by a low dose of HCG on the day of oestrus. Conception rate was not affected by gonadotrophin treatment and 1/9 cows experiencing multiple ovulations gave birth to twin calves.
D. E. WILDT, C. B. MORCOM and W. R. DUKELOW
Endocrine Research Unit, Michigan State University, East Lansing, Michigan 48824, U.S.A.
(Received 17th December 1974)
The technique of laparoscopy has been routinely used for repeated ovarian observation in the gilt (Wildt, Fujimoto, Spencer & Dukelow, 1973) as well as other species (Jewett & Dukelow, 1972; Rawson & Dukelow, 1973; Seeger, 1973; Snyder & Dukelow, 1974). In addition to its advantage for serial observations of ovarian morphology with minimal stress to the animal, laparoscopy has been found to be a practical substitute for laparotomy in other research areas.
There are several reports of the use of pregnancy diagnosis techniques in swine (Walker, 1967; O'Reilly, 1967; Mather, Diehl & Tumbleson, 1970; Lindahl, Martin & Dziuk, 1972; Diehl & Day, 1973), but these procedures are not accurate until 20 days after mating and do not provide an indication of the number of ovulations or of the uterine and ovarian morphology.
Recently, the biochemistry
N Songsasen, T K Woodruff and D E Wildt
The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) μg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E2) and progesterone (P4) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P4 than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 μg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E2 concentration remained unchanged over time (P>0.05) across FSH dosages. However, P4 increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E2 rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.
S. K. Wasser, S. L. Monfort and D. E. Wildt
Summary. A rapid method was developed for extracting and assaying oestradiol and progesterone in faeces (n = 242) of female yellow baboons, free-living in Tanzania. Dose response studies generated slopes of 1·02 (r 2 = 0·99) for oestradiol and 1·09 (r 2 = 0·99) for progesterone, suggesting that this method accurately measured these steroids in faeces. Parallelism was proved by demonstrating that slopes produced from serially diluted samples were not different from those generated from standard curves (mean P value = 0·53 ± 0·17 for oestradiol and 0·44 ± 0·13 for progesterone). Faecal progesterone concentrations measured over several cycles in 2 females increased and decreased in correspondence to visual markers of the luteal phase (i.e. the period between sex-skin detumescence and menses), but the presumed preovulatory oestradiol peak was not observed consistently in all cycles. Progesterone profiles during early to midgestation in 3 females confirmed pregnancy by 25 days (14%) of gestation. Oestradiol profiles were more variable and were not indicative of pregnancy until 40 days (22%) of gestation. Radiolabel-infusion studies revealed that 32% of progesterone (n = 2) but only 11% of oestradiol (n = 2), was cleared through faeces. The latter findings may account for the greater variation observed in temporal oestradiol patterns during the baboon menstrual cycle and pregnancy. Compared with previous techniques, these new methods (i) save considerable time in assaying raw material and (ii) result in high extraction recoveries of faecal steroids ∼88% for oestradiol and 91% for progesterone). This approach may be particularly useful for studying physioloical function and endocrine–environmental interrelationships in free-living primate species.
Keywords: faeces; oestradiol; progesterone; free-ranging; baboon
S. L. Monfort, N. P. Arthur and D. E. Wildt
Summary. Immunoreactive urinary oestrogen conjugates were assessed in daily urine samples (∼ 5 samples/week) collected from 8 Przewalski's mares maintained under semi-free-ranging pasture conditions. The relative percentage contributions of immunoreactive urinary oestrogens during different reproductive states (oestrus, luteal phase, early, mid- and late gestation) were determined using high-pressure liquid chromatography. In general, conjugated forms of oestrone (oestrone sulphate and oestrone glucuronide) were the major excreted immunoreactive oestrogens in non-pregnant and pregnant Przewalski's mares. Variations in urinary oestrogen conjugates indicated that the onset of oestrous cyclicity coincided with increasing daylengths, and the non-conception oestrous cycle was 24·1 ± 0·7 days (n = 17) in duration. Most copulations (29/35, 82·9%) were observed between Day −4 and Day +1 from the preovulatory oestrogen conjugates peak (Day 0). Based on known copulation dates, the mean gestation length was 48·6 ± 0·4 weeks (range 47·3–50·3 weeks). During pregnancy, urinary excretion of oestrogen conjugates increased ∼ 300-fold over levels in non-pregnant mares, reaching peak concentrations by Week +24 (51% of gestation). These results demonstrate that longitudinal reproductive events, including oestrous cyclicity and pregnancy, can be monitored precisely by evaluating urinary oestrogen conjugates in samples from Przewalski's mares maintained under semi-free-ranging conditions.
Keywords: Przewalski's horse; oestrogen; oestrous cycle; pregnancy; urinary steroids
C. C. Platz, D. E. Wildt and S. W. J. Seager
Summary. Methods for collection, freeze preservation and artificial insemination of domestic cat semen were developed. Total sperm count and pre-freeze and post-thaw% motility were significantly greater (P < 0·05) for samples collected by an artificial vagina method than for those collected by electroejaculation. Although conception rate was low (10·6%), 6 pregnancies were achieved, 2 after hormonal induction of oestrus.
K. L. Goodrowe, J. G. Howard and D. E. Wildt
Summary. Domestic cats experiencing a natural or FSH-induced oestrus were studied. Mated cats produced fewer (P < 0·01) unfertilized oocytes and more (P < 0·01) morulato blastocyst-stage embryos of better quality after a natural oestrus than after FSH treatment. Serum oestradiol- 17β concentrations were lower (P < 0·05) and progesterone levels rose earlier (P < 0·05) in the induced oestrus compared to the natural oestrus group. Morula/blastocyst-stage embryos from both groups transferred to 15 FSH/hCG-treated recipients produced 3 pregnancies and 2 live-born litters (1 from a natural oestrus donor and 1 from an FSH-treated donor). These results indicate that fertilization rates and embryo quality in domestic cats appear to be compromised by the FSH treatment, probably because of altered oestradiol-17β and progesterone concentrations.
Keywords: domestic cat; embryo recovery; oestrus; FSH; oestradiol-17β; progesterone.
JoGayle Howard, M. Bush, V. de Vos and D. E. Wildt
Summary. A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anaesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93·3 ml, 2408·6 × 106 spermatozoa/ml, 70%, 3·9 and 7·4, respectively. A high percentage (mean 77·5%) of spermatozoa within each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic droplet defect. When sperm viability was tested in vitro at 37°C, sperm motility rating declined by at least half of the initial assessment within 3·5 h of semen collection. Generally, spermatozoa maintained motility in vitro for < 6 h. Serum testosterone ranged from 1·4 to 8·2 ng/ml in 4 males evaluated in the morning (07:30–08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00–18:00 h), testosterone levels were <0·9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25·6 ng/ml) and a preputial discharge potentially indicative of 'musth'. The present study demonstrates that high quality semen can be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.