In Vitro Fertilization and Embryo Development in Vitro and in Vivo in the Tiger (Panthera Tigris)1

Abstract

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domestic cat. In Trial I (July 1989), 10 female tigers were treated with either 2,500 (n = 5) or 5,000 (n = 5) IU eCG i.m. and with 2,000 IU hCG i.m. 84 h later. In Trial II (January 1990), 6 females (5 of which were treated in Trial I) were given 2,500 IU eCG i.m. and 2,000 IU hCG i.m. 84 h later. Twenty-four to twenty-six hours after hCG treatment, all tigers were subjected to laparoscopy, and oocytes were aspirated transabdominally. On the basis of follicular development (follicles greater than or equal to 2 mm in diameter), all females responded to exogenous gonadotropins (range, 6-52 follicles/female). Follicle number and oocyte recovery rate were unaffected (p greater than 0.05) by eCG dose or time of year. A total of 456 oocytes were collected from 468 follicles (97.4% recovery; mean, 28.5 +/- 3.4 oocytes/female). Of these, 378 (82.9%) qualified as mature, 48 (10.5%) as immature, and 30 (6.6%) as degenerate. During Trial I, 8 electroejaculates were collected from 7 male tigers, and in Trial II, 3 semen samples were collected from 3 males. Motile sperm were recovered on each occasion; the overall mean (+/- SEM) ejaculate volume was 7.5 +/- 0.7 ml, the number of motile sperm/ejaculate was 105.9 +/- 20.6 x 10(6), and the percentage of structurally normal sperm/ejaculate was 81.4 +/- 2.0%. After swim-up processing, 0.05 x 10(6) motile sperm were co-cultured with 10 or fewer tiger oocytes in a humidified atmosphere (38 degrees C) of 5% CO2 in air. Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized. Oocytes from 2 females became contaminated in culture and, therefore, were excluded from embryo cleavage calculations. Of the remaining 195 fertilized oocytes, 187 (95.9%) cleaved to the two-cell stage. No parthenogenetic cleavage was observed in noninseminated control oocytes (n = 20). Eighty-six good-to-excellent-quality two- to four-cell embryos were transferred surgically into the oviducts of 4 of the original oocyte donors in Trial I and 2 females in Trial II. A pregnancy occurred in 1 female in Trial II, and 3 live-born cubs were delivered by Caesarean section 107 days after embryo transfer. Of the 56 cleaved embryos cultured in vitro in Ham's F10 for 72 h, 14 (25.0%) were at the sixteen-cell stage, and 15 (26.8%) were morulae.(ABSTRACT TRUNCATED AT 400 WORDS)

Ability of thawed tiger (Panthera tigris) spermatozoa to fertilize conspecific eggs and bind and penetrate domestic cat eggs in vitro

“…Electroejaculate and ovarian egg characteristics and subsequent IVF rates using fresh spermatozoa were consistent with earlier results (Wildt et al, 1987Byers et al, 1989;Donoghue et al, 1990) indicating that the animals and gametes used here were typical for the tiger species. The results demonstrated that a conventional egg yolk-lactose-glycerol cryodiluent combined with a semen pelleting technique could be used to recover sperm motility after thawing and permitted comparatively high rates of fertilization and embryo development in vitro.…”

“…The finding that IVF and subsequent embryo culture rates did not differ between the fresh and thawed sperm treatments suggested that tiger spermatozoa were relatively unaffected by this cryopreservation approach. We have speculated (Donoghue et al, 1990) that one reason that tiger spermatozoa interact so readily with eggs in vitro is related to their robustness and the lack of structural abnormalities commonly observed in other felid species (Wildt et ai, 1983(Wildt et ai, , 1988Howard et al, 1984;Miller étal., 1989;Donoghue et al, 1992). In nonfelid taxa, cryopreservation can cause extensive gross and ultrastructural sperm damage that can markedly compromise fertility (Pursel et ai, 1972;Berndston et al, 1981).…”

“…All males were of proven fertility (on the basis of live cubs sired) or had produced consistently high quality electroejaculates in a previous study (Donoghue et al, 1990). Each tiger was housed separately with free access to an out-of-doors area (5-5 6-8 m2) during daylight; at night, each tiger was confined indoors in a 2-6 2-6 m2 enclosure.…”

“…Collection, evaluation and cryopreservation of semen Ejaculates were collected using a standardized anaesthesia and electroejaculation technique reported previously (Wildt et al, 1983(Wildt et al, , 1987Howard et al, 1986b;Donoghue et al, 1990). In brief, after inducing anaesthesia with ketamine hydrochloride (5-0 mg kg" ', i.m.…”

“…Tigers are particularly attractive for such studies because an IVF system has already been developed that routinely results in >63% of inseminated eggs forming cleaved embryos in vitro (Donoghue et ai, 1990). Tiger embryos generated by IVF have also resulted in live young (Donoghue et al, 1990). The high efficiency of IVF in tigers, compared with that in other wild felids (Goodrowe et ai, 1989;Miller et al, 1989;Donoghue et al, 1992), appears to be related to the ability of tigers to ejaculate semen containing high proportions of structurally normal spermatozoa of high motility (Donoghue et al, 1990;Wildt et ai, 1992).…”