Development to the blastocyst stage was assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The proportion of cleaved oocytes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal sheep than for those from adult sheep (7.4% and 24.6% respectively). There were no differences in the metabolism of glucose, glutamine or pyruvate between oocytes matured in vivo and in vitro, or of glucose or pyruvate between oocytes from prepubertal and adult sheep. Glutamine metabolism by mature oocytes from prepubertal sheep was significantly lower than that by oocytes from adult sheep. Ultrastructural studies revealed no differences in the morphology of cytoplasmic organelles of oocytes matured in vitro from prepubertal and adult sheep, but differences in the volume fraction and size of mitochondria and cortical granules were observed. These data suggest that mature oocytes from prepubertal sheep do not possess the developmental potential of their adult-derived counterparts, and this phenomenon may be associated with metabolic and ultrastructural anomalies.

No abstract

Research was conducted to develop an effective method for cryopreserving bottlenose dolphin (Tursiops truncatus) semen processed immediately after collection or after 24-h liquid storage. In each of two experiments, four ejaculates were collected from three males. In experiment 1, three cryopreservation methods (CM1, CM2, and CM3), two straw sizes (0.25 and 0.5 ml), and three thawing rates (slow, medium, and fast) were evaluated. Evaluations were conducted at collection, prefreeze, and 0-, 3-, and 6-h postthaw. A sperm motility index (SMI; total motility [TM] x % progressive motility [PPM] x kinetic rating [KR, scale of 0-5]) was calculated and expressed as a percentage MI of the initial ejaculate. For all ejaculates, initial TM and PPM were greater than 85%, and KR was five. At 0-h postthaw, differences in SMI among cryopreservation methods and thaw rates were observed (P < 0.05), but no effect of straw size was observed. In experiment 2, ejaculates were divided into four aliquots for dilution (1:1) and storage at 4 degrees C with a skim milk- glucose or a N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)-TRIS egg yolk solution and at 21 degrees C with a Hepes-Tyrode balanced salt solution (containing bovine albumin and HEPES) (TALP) medium or no dilution. After 24 h, samples were frozen and thawed (CM3, 0.5-ml straws, fast thawing rate) at 20 x 10(6) spermatozoa ml(-1) (low concentration) or at 100 x 10(6) spermatozoa ml(-1) (standard concentration). The SMI at 0-h postthaw was higher for samples stored at 4 degrees C than for samples stored at 21 degrees C (P < 0.001), and at 6-h postthaw, the SMI was higher for samples frozen at the standard concentration than for samples frozen at the low concentration (P < 0.05). For both experiments, acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol applied to fresh or liquid-stored semen maintained high levels of initial ejaculate sperm characteristics.

No abstract

Theoretical and practical knowledge of sperm function is an essential requirement in almost every aspect of modern reproductive technology, if the overarching objective is the eventual production of live offspring. Artificial insemination (AI) techniques depend on the availability of high quality semen, whether fresh, diluted and stored, or frozen. Assessing such semen for quality and the likelihood of fertility is therefore also important, as much time, resources and effort can easily be wasted by using poor samples. Some semen technologies are aimed not at quality assessment, but at attempting to skew the breeding outcomes. Sex preselection by separating the male- and female-bearing spermatozoa using flow cytometry is now practised routinely in the agricultural industry, but speculatively it may eventually be possible to use other genetic markers besides the sex chromosomes. A moment's reflection shows that although sex-biasing flow cytometry technology is well developed and generally fulfils its purpose if presorting of sperm quality is adequate, other technologies aimed specifically at semen assessment are also sophisticated but provide inadequate data that say little about fertility. This is especially true of instrumentation for objective sperm motility assessment. Here we aim to examine this technological paradox and suggest that although the sperm assessment equipment might be sophisticated, the shortcomings probably lie largely with inappropriate objectives and data interpretation. We also aim to review the potential value and use of sperm sexing technology for non-domestic species, arguing in this case that the limitations also lie less with the technology itself than with the applications envisaged. Finally, the potential application of a sorting method directed at motility rather than sperm DNA content is discussed.

The reproductive physiology of the Pacific white-sided dolphin, Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%, nZ37) and conceptions (83%, nZ18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm 2 ), and sperm concentration (144.3!10 7 sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (nZ22) were 31 d long and were comprised of a 10 d follicular and 21 d luteal phase. Ovulation occurred 31.2 h after the onset of the LH surge and 19.3 h after the LH peak. Follicular diameter and circumference within 12 h of ovulation were 1.52 and 4.66 cm respectively. Estrus synchronization attempts with altrenogest resulted in 17 (22%) ovulatory cycles with ovulation occurring 21 d post-altrenogest. Ten AI attempts using cryopreserved semen resulted in five pregnancies (50%). The mean gestation length was 356 days (range 348-367). These data provide new information on the Pacific whitesided dolphin's reproductive physiology and collectively enabled the first application of AI in this species.

No abstract

The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.