Superovulation and embryo transfer in wood bison (Bison bison athabascae)

Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.     

Ovarian superstimulation and oocyte collection in wood bison (Bison bison athabascae) during the ovulatory season

The objective of the study was to establish an effective ovarian superstimulatory protocol and subsequently obtain oocytes from bison by transvaginal ultrasound-guided follicular aspiration. Two experiments involving 22 wood bison were done during the breeding season (September to December). In experiment 1, the bison were given a luteolytic dose of prostaglandin (Day 0) and underwent follicular ablation (Day 8) to induce ovarian synchrony. Synchronized bison were then assigned randomly to two groups (n = 11 per group) and given either 200 mg FSH diluted in saline sc, or 200 mg FSH diluted in a proprietary slow-release formulation (SRF) im on Days 9 and 11. Prostaglandin was given to both groups on Day 11 followed by 25 mg LH on Day 13. Oocytes were collected by transvaginal ultrasound-guided aspiration of follicles ≥5 mm on Day 14. In experiment 2, bison were synchronized as in experiment 1 and assigned randomly to one of two groups (n = 11 per group) and given either a single dose of 2500 IU eCG im on Day 9, or 200 mg FSH sc on Days 9 and 11. Prostaglandin was given to both groups on Day 11, and LH (25 mg) was given on Day 13. Oocyte collection was done as described in experiment 1. Cumulus-oocyte-complexes (COC) were classified according to morphologic characteristics. In experiment 1, more follicles ≥5 mm were detected on Day 14 in bison treated with FSH versus eCG (12.2 ± 1.73 vs. 5.8 ± 0.52; P < 0.05), and more COC were collected from FSH-treated animals (7.2 ± 1.41 vs. 3.4 ± 0.62; P < 0.05). In experiment 2, the FSH-saline and FSH-SRF groups had a similar number (mean value ± standard error of the mean) of follicles ≥5 mm on Day 14 (12.4 ± 1.49 vs. 13.8 ± 1.24, respectively) and a similar number of COC were collected (6.5 ± 1.13 vs. 6.3 ± 0.96, respectively). The proportion of COC collected per follicle aspirated and the percentage of compact, expanded, and denuded oocytes did not differ between groups in either experiment 1 or 2. In summary, a two-dose regimen of FSH diluted in saline and given sc or in a SRF and given im induced a similar ovarian response in wood bison, whereas a single dose of eCG resulted in a significantly lower ovarian response. Overall, COC were collected from 55% of follicles after transvaginal, ultrasound-guided needle aspiration in wood bison.     

Sperm characteristics in plains (Bison bison bison) versus wood (Bison bison athabascae) bison

The objective was to compare sperm characteristics between the two subspecies of North American bison, plains bison (Bison bison bison) and wood bison (Bison bison athabascae). Frozen-thawed ejaculated sperm from age-matched plains (n = 3) and wood (n = 2) bison were evaluated for morphometry, motility, viability, protein profile, and in vitro fertilization characteristics. Sperm morphometry and motility were assessed with computer-based systems, viability was assessed with SYBR-14 and propidium iodide, and fertilizing ability was determined using a heterologous in vitro fertilization system (using bovine oocytes). For plains versus wood bison, there were significant differences for head width (4.76 ± 0.22 vs 4.71 ± 0.19 μm; mean ± SD), head area (35.64 ± 1.91 vs 34.72 ± 2.64 μm²), head perimeter (23.61 ± 0.68 vs 23.31 ± 0.98 μm), midpiece length (14.58 ± 0.4 vs 14.36 ± 0.51 μm), midpiece width (0.81 ± 0.06 vs 0.79 ± 0.07 μm), and tail length (46.61 ± 2.15 vs 45.98 ± 2.08 μm). However, there was no significant difference in head length (overall, 9.04 ± 0.37 μm), progressive motility (41.16 ± 8.39%), or viability (41.58 ± 5.58%). Based on two-dimensional gel electrophoresis, 93 out of 113 protein spots were similar in their expression patterns. Furthermore, we inferred that differences in sperm biometry between these subspecies did not affect in vitro fertilization percentage (overall, 82.62 ± 12.13%). Based on these findings, we concluded that plains bison were an appropriate research model for developing reproductive technologies for wood bison.     

Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison)

The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.     

Exercise affects both ovarian follicular dynamics and hormone concentrations in mares

The objectives were to evaluate the effects of exercise on ovarian folliculogenesis and related hormones in mares. Mares (n = 11) were randomly assigned into a control (non-exercised) or treatment (exercised) group. Treatment mares (n = 5) were moderately exercised for 30 min, 6 d/wk. All mares underwent daily transrectal ultrasonographic examinations and ovarian follicles > 6 mm were measured. Blood samples were collected during the first (Cycle 1) and last (Cycle 4) cycle, and serum concentrations of cortisol, LH, and FSH were determined. Mean cortisol concentrations were elevated (P < 0.05) in exercised mares, 6.29 ± 0.22 compared with 5.62 ± 0.16 ng/dL (mean ± SEM), 30 min post exercise. There were no significant differences between groups in mean FSH concentrations; however, exercised mares had lower (17.3 ± 6.4 vs 41.1 ± 5.5 ng/mL; P < 0.05) peak LH concentrations. Furthermore, exercised mares experienced a longer (24.7 ± 0.8 vs 22.2 ± 0.8 d; P < 0.05) mean interovulatory interval for all cycles combined, fewer (P < 0.05) follicles 6 to 20 mm in diameter, and an increased (P < 0.05) number of follicles >20 mm following deviation. The dominant and largest subordinate follicle in exercised mares had a greater (P < 0.05) mean diameter on the day of deviation, suggesting delayed deviation. Exercised mares also tended (P = 0.06) to have an increased number of cycles with at least two dominant follicles compared to control (62 vs 36%, respectively), indicating a decreased ability of the largest follicle to assert dominance. Under the conditions of this study, moderately exercising mares induced higher cortisol concentrations, lowered peak LH concentrations, and altered ovarian follicular dynamics.     

Ovarian follicle development in wapiti (Cervus elaphus) during the anovulatory season

The ovaries of 12 mature wapiti hinds were studied by transrectal ultrasonography during the anovulatory season to characterize follicular dynamics and to test the hypothesis that follicle development occurs in a wave-like fashion. The hinds were examined daily, standing without sedation. Follicle size and numbers were recorded, and individual follicles were identified serially. Follicle development was considered wave-like if periodic changes in follicle numbers could be associated temporally with the development of a dominant follicle. There were non-random changes (P<0.01) in the number of follicles > or =4 mm in diameter detected per day. Each peak in follicle numbers was associated with the development of a single dominant follicle. The dominant follicle of the cohort was larger (P<0.05) than the other follicles 1 day after its emergence. Intervals between successive peaks (6.8 +/- 0.4 day) and troughs (6.8 +/- 0.4 day) in follicle numbers, and emergence of sequential dominant follicles (7.1 +/- 0.5 day) were not different (P=0.86). Results confirmed the hypothesis that ovarian follicles develop in a wave-like fashion in wapiti during the anovulatory season.     

Animal and temporal effects on ovarian follicular dynamics in Brahman heifers

The aims of the current study were to determine if the pattern of ovarian follicular growth and development in Bos indicus heifers is different to that reported in Bos taurus breeds, and to examine the factors that determine which dominant follicle will ovulate. In addition, the extent to which variation in follicular dynamics is attributable to variation between animals and over time was evaluated. The ovaries of 17 Brahman heifers were examined daily by transrectal ultrasonography using a 7.5 MHz transducer for a total of 117 interovulatory intervals over a period of 10 months. Size and position of individual follicles ⪖5 mm in diameter, and size of corpora lutea (CL) were recorded. Circulating progesterone concentrations were determined from plasma samples obtained twice weekly. Although size of dominant follicles and CL within the ovaries of Bos indicus heifers were smaller than reported for Bos taurus breeds, the overall patterns of dominant follicle growth were similar. There were significant correlations between number of dominant follicles occurring prior to ovulation and time of appearance of the second dominant follicle, duration of detection of CL and size of the ovulatory follicle in the preceding oestrous cycle (P < 0.05). There were significant animal effects on a number of ovarian characteristics including number of dominant follicles per oestrous cycle (P < 0.001), with one heifer having four dominant follicles in more than a third of oestrous cycles observed. In addition, changes in daylength over the 10 month period were related to changes in duration of the interovulatory interval, persistence and maximum diameter of CL and size of ovulatory follicles. Liveweight change over the same period was related to changes in maximum diameter of the first dominant follicle.     

Systemic and intraovarian effects of dominant follicles on ovine follicular growth

The objective was to study the endocrine activity in sheep with large ovarian follicles and the effects of dominant follicles on other follicles, looking for possible intraovarian differences. Induction of dominant follicles was achieved using controlled exogenous LH pulses every 90 min over 14 days in eight Scottish Blackface ewes. During this period, follicular development was assessed by daily transrectal ultrasonography and jugular venous blood samples were collected every 12 h for FSH, LH inhibin and oestradiol assay. The exogenous LH pulses caused the appearance of large follicles in all the ewes, which reached a maximum mean diameter of 7.2 +/- 0.5 mm on Day 5.5 +/- 2.6 after first detection. In the presence of a dominant follicle, no other follicle grew to a diameter larger than 4 mm and there was a decrease in the number of new growing follicles (P < 0.05) and in the number of smaller follicles (P < 0.01). This effect of dominance was mediated by changes in FSH concentration, since FSH level decreased (P < 0.05) as dominant follicles grew and the decrease in FSH levels was related to a decline in the number of remaining follicles (P < 0.05). However, the greatest decrease in the number of small follicles growing to larger sizes was observed in the ovary ipsilateral to the dominant follicle (P < 0.05). These data confirm that the presence of a large follicle depresses the recruitment and growth of other follicles by systemic factors and provide some evidence of local inhibitors blocking the final development of other putative large follicles.     

Characterization of ovarian follicular dynamics in dromedary camels (Camelus dromedarius)

Ovarian follicular dynamics was monitored by transrectal ultrasonography, for a period of 60 to 90 days, and its correlation with plasma estradiol-17β (E2) and progesterone (P4) were studied in seventeen, multiparous, non-lactating, 12 to 20-year-old dromedary camels. The average number of follicles recruited (12.77 ± 0.93) in each wave between animals varied (P < 0.001). The number of follicles recruited during different follicular waves was highly repeatable (0.95) within individual animals. The growth and mature phase periods of the dominant follicle (DF) were 6.10 ± 0.15 and 10.20 ± 0.47 days, respectively with a linear growth rate of 1.17 ± 0.02 mm/day between Day 0 and 10 of the follicular wave. There was an inverse relationship between the diameter of the largest DF and number of follicles (r = −0.95, P < 0.001). The DF development did not regularly alternate between the ovaries and the incidence of codominance was 45%. The mean maximum diameter of DF during its mature phase was 27.30 ± 0.78 mm and oversized follicle was 38.43 ± 1.41 mm. In 73.3% waves, the DF continued its growth for a period of 10.64 ± 1.53 days even after losing its dominance and developed into oversized follicle. The duration of the regression phase of DF and oversized follicle were 24.71 ± 3.79 and 18.50 ± 2.23 days. The mean duration of a complete follicular wave was 47.11 ± 2.94 days with an interwave interval (IWI) of 16.36 ± 0.37 days. The IWI within an individual was repeatable (0.88) and between the animals was variable (P < 0.001). Plasma E2 concentration profiles showed a wave like pattern. The peak plasma E2 concentrations were attained approximately 12 days after beginning of the growth phase, when the largest DF grew to a diameter of 18.7 mm. Plasma concentration of P4 was below 1.0 ng/mL in 85% of waves and above 1.0 ng/mL in 15% of the waves for a period of 3 to 6 days in the absence of spontaneous ovulation. It is concluded that ovarian follicular development and plasma E2 concentrations occurs in a wave like pattern in dromedary camels and the IWI and follicle numbers recruited per wave are variable between the animals and repeatable within an individual animal.     

Association of uterine edema with follicle waves around the onset of the breeding season in pony mares

During spring transition, when estrus may be exhibited for prolonged periods, it is important for veterinarians and stud farm personnel to be able to predict whether a large follicle will ovulate or regress. It is thought that the presence of ultrasonically detectable uterine edema indicates that a follicle will ovulate, however, there is little evidence to support this. In the present study, 16 mares were regularly examined by transrectal ultrasonography to follow growth and regression of follicles from seasonal anestrus in February until second ovulation. Blood samples were collected daily for measurement of estradiol concentrations when a large ovarian follicle was present. Estrous-like uterine edema was detected during 7 of 11 (64%) anovulatory follicle waves, in 12 of 14 (86%) mares before their first ovulation, and in 100% of mares before their second ovulation. Uterine edema was first detected 43+/-6.7 days before first ovulation. Large anovulatory follicles tended to be present for longer periods of time than ovulatory follicles. Uterine edema was present for a significantly greater proportion of time in the presence of a large follicle at second ovulation than at first ovulation (P<0.05) or for anovulatory follicles (P<0.01). Peak plasma estradiol concentrations and mean plasma estradiol concentrations were significantly higher (P<0.001) when a dominant preovulatory follicle was present compared with a dominant anovulatory follicle, but there was no difference in estradiol concentrations between first and second ovulations. It was apparent, therefore, that uterine edema was not a reliable indicator of follicular steroidogenic competence, or of whether the follicle would ovulate.     

Hemorrhagic ovarian follicles in llamas

The ovaries of 74 llamas were examined daily by transrectal ultrasonography for at least 30 d. Hemorrhagic follicles were observed in 13 (18%) llamas (incidence per anovulatory dominant follicle, 16%). The proportion of llamas in which a hemorrhagic follicle was detected was different among groups (nonmated, 8 25 ; mated to a vasectomized male, 4 21 ; mated to an intact male, nonpregnant, 1 10 ; mated to an intact male, pregnant, 0 18 ; P<0.05). A hemorrhagic follicle, observed grossly after ovariectomy, was large (13 mm) and fluctuant, with a thin translucent wall and dark red contents. No ovulatory stigma was detected, and after incising the wall, bloody fluid escaped and the follicle collapsed leaving only a small blood clot within the antrum. Ultrasonically, the formation of a hemorrhagic follicle was indicated by scattered free-floating echogenic spots within the follicular antrum which swirled upon ballottement of the ovary. The antral contents appeared to become organized (did not swirl when ballotted) after follicle growth ceased. Ultrasonic indications of antral hemorrhage were not observed in any follicles in which ovulation was later detected (0 45 ovulatory follicles). All of the hemorrhagic follicles (13 13 ) involved the dominant follicle of a wave during which no copulatory stimulus was applied. Hemorrhagic follicles were apparently anovulatory and were repeatable (P<0.05) within individuals. The interval from first detection to the first day of maximum diameter was longer (P<0.05) and maximum diameter was greater (P<0.0001) for hemorrhagic follicles than nonhemorrhagic follicles (16.4 versus 13.1 d and 22.1 versus 12.8 mm, respectively); however, the interwave interval was not affected by the presence of a hemorrhagic follicle. Luteinization of the hemorrhagic follicle was indicated (thickened wall) in two llamas by an elevated plasma progesterone concentration and/or by ultrasound. By their large size, hemorrhagic follicles may be interpreted as hemorrhagic follicular cysts; however, they were not associated with other ovarian irregularities or with infertility.     

Hormonal control of estrous cyclicity and attempted superovulation in wood bison (Bison bison athabascae)

The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05).     

Preliminary assessment of reproductive technologies in wood bison (Bison bison athabascae): implications for preserving genetic diversity

Since the high prevalence of bovine tuberculosis and brucellosis in free-ranging wood bison in the Canadian north poses a threat to nearby healthy bison populations, commercial bison and cattle ranches, and potentially to humans, there is considerable impetus to salvage the genetics of infected bison and maintain a disease-free herd. In that regard, there is a great need to develop appropriate reproductive technologies. Therefore, the objective of this study was to develop protocols to produce and cryopreserve wood bison embryos (based on protocols used for cattle). Cumulus oocyte complexes (COC) aspirated from ovaries recovered after slaughter were matured in vitro, and fertilized with either frozen-thawed semen or chilled epididymal spermatozoa. Although both sources of spermatozoa resulted in acceptable rates of fertilization (64.4%, n=45; 89.2%, n=28, respectively) and cleavage (75.0%, n=40; 92.5%, n=40), production of morulae (7.5%, n=40; 25.0%, n=40) and blastocysts (7.5%, n=40; 10.0%, n=40) was low. Morulae- and blastocyst-stage embryos were frozen-stored by vitrification. To our knowledge, this is the first report regarding the in vitro production and cryopreservation of bison embryos for genetic recovery of diseased wood bison. These techniques have substantial potential for conserving and managing the genetic diversity of wild bison, and may also have important management implications for genetic salvage of diseased bison populations in North America.     

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up

The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n = 10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9 mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2 ± 0.3 mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P < 0.0001). There was a tendency (P = 0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P < 0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P < 0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present.     

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.     

Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.     

Follicular activity and hormonal secretory profile in vicuna (Vicugna vicugna)

The objective of the present study was to characterize ovarian activity in non-mated vicunas, relating ovarian structures (evaluated by transrectal ultrasonography, daily for 30 days) to changes in plasma concentrations of estradiol-17beta and progesterone. Ovarian follicular activity occurred in waves, characterized by the follicle emergence, growth and regression. The mean duration of follicular waves was 7.2+/-0.5 days (mean+/-S.E.M.), with a range of 4-11 days. The follicular growth phase averaged 3.0+/-0.2 days, the static phase 1.4+/-0.1, the regression phase 2.9+/-0.3 days, and the inter-wave interval was 4.2+/-0.3 days. The mean growth rate during the growing phase was 1.8+/-0.1mm/day, while the duration of the interval from 6mm to maximum diameter was 1.4+/-0.1 days. The mean maximum diameter of the dominant follicle was 8.4+/-0.3mm (range: 6.2-11.2) and mean diameter of the largest subordinate follicle was 5.4+/-0.1mm. There was an inverse relationship between the size of the largest follicle and the total number of follicles (r=-0.21, P=0.002). Follicle activity alternated between ovaries in 77% of the waves, with 40% of dominant follicles present in the left ovary and 60% in the right ovary. Plasma estradiol-17beta concentrations also had a wave-like pattern, varying between 12.0 and 62.8 pmol/l. Plasma progesterone concentrations remained below 5.0 nmol/l and there was no ultrasonographic evidence of ovulation during the study.     

Quantitative echotexture analysis of bovine ovarian follicles

Computer-assisted image analysis was used to evaluate ultrasound images of bovine ovarian follicles. The ovaries of 8 sexually mature heifers were examined daily by transrectal ultrasonography for 2 estrous cycles. Ultrasonographic examinations of the ovaries were then videotaped, and the dominant and subordinate follicles of successive waves were individually identified and monitored. Recorded images of the dominant anovulatory follicle of the first wave (n = 15) and the ovulatory follicle of the last wave (n = 15) of the estrous cycle were subsequently digitized for computer analysis of echotexture (mean pixel value and pixel heterogeneity). Regions of the image spanning the breadth of the follicle wall were selected, and image analysis revealed that mean pixel value of the dominant anovulatory follicle changed over time (P = 0.0005). Mean pixel value decreased (P = 0.0005) dramatically during the early static phase (Days 6 to 8, Day 0 = day of ovulation), increased (P = 0.0005) at the onset of the regressing phase (Day 12), and reached maximal levels (P = 0.0005) on Day 14. Similarly, image echotexture of the ovulatory follicle revealed a time-dependent effect (P = 0.0001) due to a rapid decrease in mean pixel values between 7 and 4 d before ovulation, followed by an increase until the day before ovulation. The echotexture of images of the follicular antrum were also evaluated and with regard to the dominant anovulatory follicle, a time-dependent effect was not detected for mean pixel value (P = 0.62) but was observed for pixel heterogeneity (P = 0.02). In addition, there was a positive correlation between mean pixel value and heterogeneity (r = 0.61, P = 0.0001). Heterogeneity initially decreased (P = 0.02) and remained low until the emergence of the second follicular wave (mean Day 9). Values subsequently increased and became variable during the late static and regressing phases (> Day 9). Mean pixel value of the antrum of the dominant ovulatory follicle increased (P = 0.0001) as the day of ovulation approached. Heterogeneity did not change (P = 0.14), nor was there any correlation between mean pixel value and heterogeneity for the antrum of the ovulatory follicle (r = 0.06, P = 0.49). We concluded that changes in echotexture (mean pixel value and heterogeneity) of bovine ovarian follicles assessed by computer analysis of ultrasound images were temporally related to functional status (i.e., anovulatory versus ovulatory; growing, static or regressing). The results were strongly supportive of the concept that ultrasonographically detected image attributes are a reflection of physiologic status.     

Development of dominant follicles and length of ovarian cycles in post-partum dairy cows

The resumption of ovarian activity after normal calvings was studied in 18 lactating Friesian cows. Since, in 17 cows, first post-partum ovulation occurred without overt oestrous behaviour being detected, the resultant cycles were called 'ovarian cycles'. The mean (+/- s.d.) length of the ovarian cycles was 21.0 +/- 8.7 days. The duration of cycles tended to be normal (18-24 days) or long (greater than or equal to 25 days) when the ovulatory dominant follicles were identified before Day 10 post partum; they were consistently short (9-13 days) when dominant follicles identified after Day 20 post partum ovulated. When such follicles were detected between Days 10 and 20 post partum, long, normal and short ovarian cycles were detected. The number of waves of follicular growth with associated dominant follicles observed during the ovarian cycles tended to be related to cycle length; short cycles had 1 dominant follicle, normal cycles predominantly 2, and long cycles mostly 3 dominant follicles. The mean (+/- s.d.) duration of 13 oestrous cycles studied was 23.1 +/- 2.1 days. Of these cycles, 7 had 3 and 6 had 2 dominant follicles. The oestrous cycles with 3 dominant follicles had a mean (+/- s.d.) duration of 24.0 +/- 1.2 days and the respective dominant non-ovulatory follicles reached maximum sizes on Days 8 and 18, respectively; oestrous cycles with 2 dominant follicles were 22.2 +/- 2.6 days in duration, and the dominant non-ovulatory follicle reached maximum size by Day 8. Ovarian follicular development during the first 45 days of pregnancy was characterized by the growth and regression of successive dominant follicles, each lasting 10-12 days. These results show that the first ovarian cycle was predominantly short when the ovulatory dominant follicle was first detected after Day 20 post partum.     

Follicular growth monitoring in the female cat during estrus

Follicular growth in the feline ovary is usually detected indirectly, through behavior observation, vaginal smears, or more invasively, by estradiol assay in blood. This study was designed to describe follicular dynamics by transabdominal ultrasonography. Secondly, the stage of follicular growth was associated to behavioral and vaginal changes. Ovarian ultrasonography was performed during nine anovulatory and 12 ovulatory cycles. Forty-eight follicles were followed during anovulatory cycles: on the first day of estrus behavior, 4.8 ± 0.2 follicles (2 to 7 per female) of 2.3 ± 0.01 mm mean diameter were present. Follicular growth continued at a rate of 0.2 ± 0.04 mm per day. At least one follicle in the cohort reached a diameter greater than 3.0 mm. Maximal follicular growth (when one follicle of the cohort reached the maximal diameter observed for the whole estrus) was reached 3.8 ± 0.3 days after the onset of estrus with the largest follicle reaching a diameter of 3.5 ± 0.04 mm. Growth of the various follicles within a cohort was not exactly synchronous. When no ovulation took place, the follicular diameter decreased by 0.1 ± 0.01 mm per day until the end of estrus. The first day after the end of behavioral estrus, the diameter of the largest follicle in each cohort was 2.7 ± 0.05 mm. No correlation was found between follicular development and either vaginal smear characteristics, or time elapsed since the onset of estrus. When ovulations were mechanically induced after one follicle had reached 3.0 mm in diameter, artificial insemination produced normal pregnancy rate and litter size: four pregnant females out of nine, and 2 to 4 kittens per litter. Ultrasonography proved thus to allow the monitoring of follicular growth in the female cat, with low correlation with behavior and vaginal smear modifications. Further studies are needed to evaluate the interest of an ultrasonographic ovarian follow-up to determine the optimal moment for ovulation induction prior to artificial insemination.