Percentage Of Viable Embryos Research Articles

P-449 Storage of vitrified oocytes up to 8 years does not affect pregnancy and live birth rates: analysis of 5,362 cycles

Abstract Study question Does long-term storage of vitrified oocytes affect laboratory and reproductive outcomes after ICSI? Summary answer The time spent by vitrified oocytes in liquid nitrogen banks does not affect oocyte survival, fertilization rate nor reproductive outcomes up to live birth. What is known already Vitrification is the most efficient approach to oocyte cryopreservation and is commonly applied in oocyte donation programs. The survival rate can be >90% and vitrified-warmed oocytes provide comparable reproductive results to fresh oocytes. However, even with modern cryobanking technology, there are potential temperature fluctuations caused by access to cryopreserved material, transportation, stocktaking events, and regular maintenance. While some studies indicate that a multiyear storage of vitrified oocytes does not affect pregnancy rates, others found decreased reproductive outcomes after as little as 6 months, and further investigations in large cohorts are needed to confirm the safety of long-term oocyte storage. Study design, size, duration Retrospective cohort study of 5,362 cycles with vitrified oocytes from oocyte donors carried out between 2013 and 2021. All cycles included fresh embryo transfer (ET). Oocyte vitrification and warming were carried using the Cryotop® protocol (Kitazato). We analyzed the effect of storage time of vitrified oocytes in liquid nitrogen vapors on laboratory outcomes (oocyte survival, fertilization rate (FR), percentage of viable embryos) and reproductive outcomes (pregnancy -biochemical and clinical- and live birth (LB) rates). Participants/materials, setting, methods Eight categories of storage time were established: 0-0.25 years (reference group, ≤3 months), 0.25-0.5, 0.5-1, 1-1.5, 1.5-2, 2-3, 3-4, and >4 years. The effect of oocyte storage time on laboratory and reproductive outcomes was analyzed by linear and logistic regression, respectively. These multivariate analyses were adjusted by multiple factors: sperm origin (partner vs donor), sperm parameters, number of oocytes inseminated, number of embryos transferred and day of ET (2-3 vs 5), among others. Main results and the role of chance Mean recipient age was 42.0±4.6, while oocyte donors age was 26.1±4.6. The mean number of thawed oocytes was 8.0±2.5, all surviving oocytes were inseminated by ICSI with partner (84.9%) or donor sperm (15.1%). The oocyte storage time ranged from 3 days to 8.2 years (mean: 0.7 ± 0.9 years). We did not find significant differences in oocyte storage time between positive and negative pregnancy and LB, nor between different survival rates (<100 vs = 100%), FR (<70 vs ≥ 70%) and percentages of viable embryos (<50 vs ≥ 50%) at univariate analysis (p > 0.05 in all cases). The mean oocyte survival rate was 90.2%±14.7% and, after adjusting for confounders, did not decrease with longer storage time (i.e. 88.9% for time >4 years, p = 0.963). Similarly, the linear regression model did not show a significant effect of storage time on FR, which was close to 70% in all categories (range: 67.6-70.6, p > 0.05), nor a decrease in the percentage of viable embryos. Finally, reproductive outcomes were similar across storage times (p > 0.05 for all categories when compared to the reference 0-0.25); specifically, long-term oocyte storage (>4 years) did not affect the chances of clinical pregnancy (OR: 0.657 [0.395-1.092], p = 0.194) and LB (OR: 0.666 [0.393-1.128], p = 0.231). Limitations, reasons for caution These results cannot be extended to cycles using the patient’s own oocytes or involving a severe male factor (testicular spermatozoa or criptozoospermia), which were excluded. Due to the retrospective nature of the study, some uncontrolled variables could affect the results. Wider implications of the findings This is the first study evaluating the effect of long-term oocyte storage on a large cohort of patients undergoing oocyte donation, thus partially discarding the effect of the female factor. Reassuringly, these results indicate that long-term storage of oocytes is a safe option for young patients and oocyte banks. Trial registration number Not applicable

AUTOMATIC EMBRYO CLASSIFICATION ALGORITHM APPLIED ON CLEAVAGE STAGE FOR BLASTULATION, ANEUPLOIDY AND IMPLANTATION PREDICTION

To analyze the relationship of the automatic classification test provided on Day 3 by the EevaXtend algorithm (Early embryo viability assessment) system with percentage of viable embryos, blastocyst and implantation rate, blastocysts available for biopsy and aneuploidy rates.

Follicular dynamics and in vivo embryo production in Santa Inês ewes treated with smaller doses of pFSH

To evaluate follicular dynamics, there was assessment of superovulatory response and in vivo embryo production in ewes treated with relatively smaller doses of exogenous pFSH than typically used in combination with a dose of eCG at the beginning of the gonadotropin treatment period. Santa Inês ewes (n = 24) were randomly divided into three groups, based on mg dose of pFSH administered: G200 (n = 8), G133 (n = 8) and G100 (n = 8) in eight decreasing doses at 12 -h intervals. All ewes were treated with 300 IU of eCG concomitantly starting with first pFSH administration. Ovulatory follicular dynamics and follicular wall vascularization (FWV) were evaluated using a B-mode and color Doppler ultrasonic machine, respectively. Superovulatory response and embryo production were evaluated 6 days after estrous detection. In the G200 group, the preovulatory follicle size (PFS) were less (P < 0.05), ovulation time later (P < 0.05), and PFS rate greater (P < 0.05); while in the G100 group ovulation rate, and number and percentage of unfertilized eggs were greater (P < 0.05) than in the G133 group (P < 0.05). Number and percentage of viable embryos were greater in the G200 and G100 compared to G133 group (P < 0.05). The dose of 100 mg of FSH was as efficacious as the traditional dose of 200 mg, in combination with a dose of eCG, for superovulatory response and viable embryo production but there was a greater percentage of unfertilized eggs with this treatment.

El uso de la corifolitropina alfa en donantes de óvulos obtiene la misma eficiencia reproductiva y efectividad que la FSH recombinante, con una mayor comodidad para la donante

IntroductionConventional ovarian stimulation with daily injections of FSH for oocyte donors, besides the discomfort of the medication, involves a great demand of time. Treatments need to be simplified without losing effectiveness and safety. Corifollitropin alpha (Elonva) enables the first 7injections of FSH to be replaced with one single injection. ObjectiveTo compare the effectiveness and convenience of corifollitropin alpha (Elonva) compared to daily recombinant FSH (rFSH) in the ovarian stimulation of oocyte donors. Materials and methodsAn analysis was performed on 90 cycles of Elonva and 96 cycles of rFSH. The statistical analysis included the Student t-test for means and the chi-squared test for percentages. Effectiveness has been evaluated through reproductive effectiveness defined as the total number of live births in relation to the total number of mature eggs. An analysis was also performed on variables, such as number of mature oocytes, percentage of viable embryos, implantation rate, and clinical pregnancy rate per embryo transfer. Comfort has been evaluated by the number of visits to the clinic for medication administration. ResultsSignificant differences were only found in the number of injections administered to donors (3.20 Elonva vs. 8.55 rFSH, P<.005). Reproductive effectiveness was 5.61 vs. 5.49, with implantation rate 44.44 vs. 44.34, and clinical pregnancy 56.25 vs. 58.73. There no significant differences in any of variables analysed, including the mean cost. No cases of OHSS or adverse drug reactions were observed. ConclusionsThe use of corifollitropin alpha in oocyte donors offers the same reproductive effectiveness and is more convenient for the donor compared to stimulation with daily rFSH.

Inbreeding effects on in vitro embryo production traits in Guzerá cattle

Inbreeding has been associated with the impairment of reproductive performance in many cattle breeds. Although the usage of reproductive biotechnologies has been increasing in bovine populations, not much attention has been given to the impact of inbreeding over cow’s performance on artificial reproduction. The objective of this study was to estimate the impact of inbreeding on in vitro embryo production in a Guzerá breed population. The inbreeding coefficient (F), calculated as half of the co-ancestry of the individual’s parents, was used as an estimate of inbreeding. The inbreeding coefficients of the donor, sire (used on in vitro fertilization) and of the embryos were included, separately, in the proposed models either as classificatory or continuous variables (linear and quadratic effects). The percentage of non-inbred individuals (or embryos) and mean F of donors, embryos and sires were 29.38%; 35.76%; 42.86% and 1.98±2.68; 1.32±3.13; 2.08±2.79, respectively. Two different models were considered, one for oocyte production traits and other for embryo production traits. The increase of F of the donor significantly (P<0.05) impaired the number of viable oocytes (NOV), number of grade I oocytes (NGI) and number of cleaved embryos (NCLV). Moreover, the donor’s F influenced the percentage of grade I oocytes (PGI), percentage of viable embryos (PEMB) and percentage of cleaved embryos that turned into embryos (PCXE). No significant (P>0.05) effects were observed for the sire (father of the embryos) inbreeding coefficient over the traits analysed. Embryo’s F influenced (P<0.05) the number of viable embryos (NEMB), percentage of viable embryos (PEMB) and percentage of cleaved embryos that turn into embryos (PCXE). Results suggested that an increase in the inbreeding coefficient might impair the embryos ability to survive through challenges imposed by the in vitro environment. Submitting highly inbred Guzerá female donors to in vitro embryo production may, in the long-term, have negative implications on the number of embryos obtained per cow and increase the relative costs of the improvement programmes based on this technology. High levels of inbreeding should be avoided when selecting Guzerá female donors and planning in vitro fertilization mating.

Effects of preincubation of eggs and activation medium on the percentage of eyed embryos in ide (Leuciscus idus), an externally fertilizing fish

Standardization of fertilization protocols is crucial for improving reproductive techniques for externally fertilizing fish in captive breeding. Therefore, the objectives of this study were to determine the effects of preincubation of eggs and activation medium on the percentage of eyed embryos for ide (Leuciscus idus). Pooled eggs from five females were preincubated in three different activating media for 0, 30, 60, 90, and 120 seconds and then fertilized by pooled sperm from five males. At the eyed-egg stage, the percentage of viable embryos was later calculated. Results showed that preincubation time was significant for the freshwater activation medium (P < 0.001), such that the percentage of eyed embryos declined across the preincubation time gradient. Additionally, there was an effect on the percentage of eyed embryos when eggs were incubated with Woynarovich solution (P < 0.001), such that a decline was detected at 90 seconds, whereas no effect was detected for the saline water medium. Activating medium had a significant effect on the percentage of eyed embryos for each preincubation time (P < 0.05). More precisely, freshwater produced the lowest percentage of eyed embryos at all preincubation times (ranged from 1.9% at 120 seconds to 43.6% at 0 seconds), whereas saline water and Woynarovich solution produced the highest percentage of eyed embryos at 0 seconds and 30 seconds before incubation. Woynarovich solution produced the highest percentage of eyed embryos at 60 seconds (65.26%), whereas saline water produced the highest percentage at 90 seconds (68.37%). No difference was detected between saline water and Woynarovich solution at 120 seconds. Examination of sperm traits showed no impact of activating medium on computer assisted sperm analysis parameters. Together, these results suggest that saline water or Woynarovich solution improve fertilization rate in ide during IVF; thus, these media are useful for standardizing fertilization protocols and controlled reproduction for this species.

Successful use of artificial insemination in the production of red-legged partridges (Alectoris rufa)

This paper reports the first successful artificial insemination (AI) of red-legged partridges (Alectoris rufa), the fertility rate achieved, and the length of time sperm cells can survive inside the oviduct (i.e., the post-AI fertile period). Semen from 20 mature males was collected by massage and pooled for use in single intravaginal inseminations (20 μL of fresh, undiluted semen) of eight females (15 × 106 sperm/female). The latter’s eggs were then collected for 4 weeks, and the fertilizing capacity of the sperm used in the preceding AIs was determined by observing the development of the blastoderm. The duration of the post-AI fertile period was determined by subjecting fertilized eggs to the SP-holes assay. A second experiment was then performed to measure the percentage of viable embryos at 20 days of incubation (30 %) and hatchability (40 %). The mean fertility rate was 34.5 ± 11.7 % and the SP-holes value 17.3 ± 4.3. The mean duration of the post-AI fertile period was 3 weeks. In conclusion, the present work reports the first ever birth of partridge chicks following AI and shows that this procedure may be of use in the conservation of this species.

The influence of female body weight class on the effectiveness of controlled reproduction of carp Cyprinus carpio Linnaeus, 1758

The effects of Ovopel treatment and hypophysation on five different size classes (age = 4–16 years) of female carp, Cyprinus carpio L., from 10 breeding lines were assessed (class I: >3≤5 kg; class II: >5≤7 kg; class III: >7≤9 kg; class IV: >9≤11 kg; class V: >11 kg). Ovulation stimulation was done with carp pituitary homogenate i.e. CPH (0.3+2.7 mg kg−1 BW) or [(D-Ala6, Pro9-NEt)-mGnRH+metoclopramide] i.e. Ovopel (1/5+1 pellet kg−1 BW). Analysis of variance included the results obtained in controlled spawnings carried out during 15 breeding seasons. The classification according to the body weight classes significantly (P ≤ 0.01) differentiated the weight of eggs (g) obtained both from CPH and Ovopel treated fish, the percentage of viable embryos (36 h) after CPH (P ≤ 0.05), number of eggs after CPH and Ovopel treatment (P ≤ 0.01) and number of living embryos (36 h) calculated per kg−1 CPH treated fish. The lowest weight of eggs was obtained after both the CPH and Ovopel application in class I and the highest in class V. The highest mean for the total number of eggs from CPH treated fish was noted for size classes IV and V and the lowest for class I. From Ovopel treated females the highest mean was noted in classes V and IV and the lowest in class I. The highest mean for the number of live embryos (36 h) calculated per kilogram CPH and Ovopel treated fish was recorded in female weight classes I and II. The lowest mean for this trait was found for size class V after both CPH and Ovopel treatment. The group of hypophysed females provided the best results of reproduction when the body weight was over 7 kg but not exceeding 9 kg (class III). Females treated with Ovopel reached such results when body weight was higher than 9 kg but did not exceed 11 kg (class IV). After both CPH and Ovopel treatment the poorest results were noted in females exceeding 11 kg body weight (class V). The predicted regressions for the number of viable embryos (36 h incubation) obtained per 1 kg female BW allows to determine in which weight class which stimulator offered the best predictability for this trait.

150 USING A LUTEINIZING HORMONE SURGE DETECTION TEST, PREDI&amp;apos;BOV&amp;reg;, TO OPTIMIZE THE TIME OF ARTIFICIAL INSEMINATION DURING A SUPEROVULATION PROTOCOL IN A HOLSTEIN HEIFERS HERD

Insemination of superovulated bovine donors in due time is of central importance for fertilization and embryo viability. A preliminary test focusing on LH surge detection during superovulation (unpublished datas) indicated that one quarter of the donors present LH surges 12 to 24 h before heat observation (which could correspond, in the case of AI after heat observation, to post ovulation AI). Therefore, it was hypothesised that the average number of embryos per flush could be improved by inseminating donors with early LH surge 12 h after the beginning of the LH peak whenever the heat occurs.In a donor herd station, a trial was performed with 54 Holstein heifers, equipped with Heatime® tags (system detecting the peak activity linked to the heat) collected twice or 3 times after the following superovulation protocol: D-6 to D-11 = reference heat; D0 = input of an implant of norgestomet (Crestar®); D2 8:00 = first FSH (Stimufol®) injection (FSH1); D4 8:00 = cloprostenol (Estrumate®) injection; D4 16:00 = implant removal; D5 8:00 = FSH7 and first LH surge detection test (Predi'Bov®); D5 16:00 = FSH8 and 2nd Predi'Bov® test. Two AI's at interval of 8 to 16h were done (AI's were performed either at 9:00, or between 17:00 and 19:00). For the standard protocol (= STA), thefirst AI occurred after heat observation or activity peak detection by Heatime (whatever the Predi'Bov® test results were). For the adjusted protocol (= ADJ), the first AI occurred from 11 to 16 h after the first positive Predi'Bov® test result or like STA protocol if both results were negative. Heifers followed alternately the 2 protocols, 27 beginning with the ADJ protocol, 27 others with the STA one. LH surge precocity was not repeatable among donors. In the case of an early LH surge detection (one positive Predi'Bov® test), the heat activity peak occurred from 2 to 8 h after the FSH8 for 44% of the flushes, from 8 to 24 h after FSH8 for 54% of the flushes and never for 2% of the flushes. When no early LH surge was observed, the heat activity peak occurred more than 8 h after FSH8 for 78% of the flushes. Interval of heat activity peak-FSH8, IA1-heat activity peak and IA1-early LH surge were highly variable but did not effect the mean number of viable embryos. However, we observed a significant effect (P = 0.04) of the precocity of the heat on the average number of total embryos: 13.8 ± 8.4 v. 11.1 ± 8.1 when the interval heat activity peak-FSH8 had been respectively &lt;8 h or ≥8 h, respecively. Among the 148 collections, 74 were done after the STA protocol, 74 after the ADJ protocol and 70 followed an early LH surge. The adjustment of the AI depending on the detection of an early LH surge (ADJ protocol) had a significant positive (P = 0.04) effect on the mean percentage of viable embryos per flush (52% ± 28 in STA group and 62% ± 31 in ADJ group). Nevertheless, regarding the mean number of viable embryos, this effect failed to reach significance (P = 0.23) (respectively 5.7 ± 5.1 in STA group and 6.7 ± 6.7 in ADJ group). A larger study on more animals is necessary to obtain a significant difference in the number of viable embryos.

229 INFLUENCE OF HIGH OR LOW INTAKE OF DRY MATTER AND ENERGY ON IN VIVO PRODUCTION OF BOVINE EMBRYOS

The aim of this study was to evaluate the influence of high or low dry matter (DM) intake and/or energy on embryo quality and production in vivo. Nonlactating Nelore cows (n = 32, 4 to 10 years old) weighing 489.5 ± 11.3 kg and with a body condition score of 3.25 (1 to 5) were used. After 15 days on the adaptation diet, cows were blocked by initial body weight (BW) and randomly divided in 4 experimental groups. The maintenance group (M) received a diet to provide 1.2% of DM/kg of BW. The restriction group (0.7M) received the equivalent of 70% of the group M diet (0.84% of DM per kg of BW). The high intake group (1.5M) received the equivalent of 150% of the M group (1.8% of DM/kg of BW). The energy group (E) received a diet with a DM similar to the M group but with an energy level equivalent to the 1.5 M group. All cows were submitted to aspiration of follicles &gt;2 mm for ovum pick-up (OPU). Recovered oocytes were used in another experiment. The embryo donors received an intravaginal device (IVD) of progesterone release (Sincrogest®, Ouro Fino, Ribeirão Preto, Brazil), soon after OPU. Two days after OPU, the cows received 8 decreasing doses of FSH (100 mg, IM, Folltropin-V®, Bioniche Animal Health, Belleville, Ontario, Canada) and concomitant with the fifth and sixth treatments of FSH, PGF2α (500 μg each, IM, cloprostenol, Sincrocio®, Ouro Fino) was administered. The IVD was removed at the time of the last FSH injection. Twelve hours after IVD removal ovulation was induced with GnRH (0.01 mg, IM, Buserelin acetate, Sincroforte®, Ouro Fino). Cows were inseminated 12 and 24 h later. Embryo collection was performed 7 or 8 days after GnRH injection. The cows were offered all diets in a crossover design study. There were 4 sessions of embryos flushing, each 42 days apart. Data were analysed by PROC GLIMMIX of SAS (SAS Institute Inc., Cary, NC, USA) and the results are presented as least-squares means ± SE, always following the order of treatments 0.7 M, M, 1.5 M and E. There were differences in the superstimulatory response (12.6 ± 1.4b; 14.6 ± 1.6a; 13.6 ± 1.5ab; and 11.0 ± 1.2c follicles &gt;6 mm; P &lt; 0.01) and superovulatory response (9.8 ± 1.3ab; 11.0 ± 1.4a; 10.2 ± 1.3a; and 8.6 ± 1.3b CL; P &lt; 0.01) among groups. Despite the lower responses observed especially in the high-energy group, no difference among groups was observed for total embryos/ova (4.5 ± 0.7; 5.0 ± 0.8; 5.0 ± 0.8; and 4.7 ± 0.7; P = 0.60), viable embryos (2.0 ± 0.4; 2.3 ± 0.5; 2.6 ± 0.6; and 2.2 ± 0.5; P = 0.40), or freezable embryos (1.7 ± 0.4; 2.0 ± 0.4; 2.1 ± 0.5; and 1.9 ± 0.4; P = 0.60). There was also no difference among groups for fertilization rate (75.8 ± 9.6; 82.3 ± 8.0; 87.8 ± 6.6; and 81.1 ± 8.4%; P = 0.71) and percentage of viable embryos (54.5 ± 10.8; 50.8 ± 10.6; 50.5 ± 10.9; and 54.4 ± 10.7%; P = 0.98). In conclusion, in contrast to our initial hypothesis, a period of 42 days under high feed/energy intake in nonlactating zebu cows apparently did not compromise in vivo embryo production. This may be because cows had a moderate body condition score or because the feeding period was not long enough to compromise oocyte/embryo quality. Financial support from FAPESP and CNPq is acknowledged.

Effect of dietary organic zinc, manganese, copper, and cobalt supplementation on milk production, follicular growth, embryo quality, and tissue mineral concentrations in dairy cows

This study evaluated potential effects of organic trace mineral supplementation on reproductive measures in lactating dairy cows. Cows were blocked by breed and randomly assigned at dry-off to receive inorganic trace mineral supplementation (control; n = 32) or to have a portion of supplemental inorganic Zn, Cu, Mn, and Co replaced with an equivalent amount of the organic forms of these minerals (treatment; n = 31). Trace minerals were provided through control or treatment premixes fed at 100 g·cow(-1)·d(-1). Premixes were fed to dry cows (range = 40 to 72 d before calving) in 1.8 kg·cow(-1)·d(-1) concentrate pellets through a computer feeder to provide 40, 26, 70, and 100% of supplemented Zn, Mn, Cu, and Co, respectively, and to lactating cows (range = 69 to 116 d after calving) in a total mixed ration to provide 22, 14, 40, and 100% of supplemented Zn, Mn, Cu, and Co, respectively. Treatment increased milk production at wk 14 (P = 0.047) postcalving, milk urea N content (P = 0.039), and BW loss from calving to 1 mo postcalving (P = 0.040), and decreased milk fat percentage (P = 0.045) and BCS (P = 0.048). Treatment tended to increase milk production at wk 13 (P = 0.089) postcalving and endometrial tissue concentrations of Fe (P = 0.070), BW at mo 1 (P = 0.056), and milk protein percentage (P = 0.064). Treatment did not affect (P > 0.1) DMI, health events, first-wave follicular dynamics, first cycle luteal measures, embryo quality, liver trace mineral concentrations, or luteal trace mineral concentrations. Cows with a rectal temperature ≥39°C at the time of AI had a smaller percentage of fertilized entities (P < 0.001). However, of the entities that were fertilized, the percentage of viable embryos, embryo quality, accessory sperm number, and embryo cell number were not affected (P > 0.1) by treatment. We conclude that replacing a portion of inorganic supplemental trace minerals with an equivalent amount of these organic trace minerals (Zn, Mn, Cu, and Co) increased milk production in mid-lactation, but did not affect postpartum follicular dynamics, embryo quality, or liver and luteal trace mineral concentrations.

111 EFFECT OF ETHYLENE GLYCOL IN VITRIFICATION MEDIUM ON BOVINE OOCYTE ACTIVATION AND IN VITRO EMBRYO PRODUCTION

In this study the effect of increasing ethylene glycol (EG) concentrations in the vitrification media (VM) and its relation with oocyte activation and in vitro embryo production were evaluated. Cumulus-oocytes complexes (COC) were matured in vitro for 22 h and then partially denuded by gently pipetting with hyaluronidase, and randomly assigned to 4 treatments: T1 = control group; T2 = COC exposed to 10% EG + 10% DMSO (VM1), 20% EG + 20% DMSO (VM2); T3 = 15% EG + 5% DMSO (VM1), 30% EG + 10% DMSO (VM2); T4 = 20% EG + 0% DMSO (VM1), 40% EG + 0% DMSO (VM2). Exposition to VM1 and VM2 lasted 3 min and 30 s, respectively. After treatment COC were incubated in maturation medium up to 24 h. In Experiment 1 COC were cultured for 24 h in fertilization TALP supplemented with 50 μg mL-1 of heparin, then completely denuded and cultured 24 h in CR1-aa. After this, oocytes were stained with bisbenzimide (Hoechst 33342) and the number of nuclei and cells were recorded. Structures presenting 2 cells or 2 nuclei were considered as activated oocytes. In Experiment 2 matured COC exposed to cryoprotectants as in Experiment 1 were fertilized and cultured for 6 days as previously described (Mucci et al. 2006). Variables analyzed included oocyte activation, cleavage rate at 48 h, and percentage of viable embryos at Day 7. Data were analyzed by PROC GENMOD (SAS Institute Inc., Cary, NC, USA). Activated oocytes percentage did not differ between EG concentrations in VM and were higher (T2, 24.7%, n = 101; T3, 25.0%, n = 96; T4, 30.2%, n = 119) compared with controls (T1, 9.8%, n = 61). In Experiment 2 no differences were found in cleavage rates (T1, 81.9%, n = 68; T2, 87%, n = 67; T3, 85.9%, n = 61; T4, 84.2%, n = 64) and Day 7 percentage of viable embryos (T1, 34.9%, n = 29; T2, 28.6%, n = 22; T3, 26.8%, n = 19; T4, 27.6%, n = 21) in treated COC. The exposition of COC to cryoprotectants per se could trigger oocyte activation in the range of 10 to 40%. Thanks toAdriana Cano (Instituto Nacional de Tecnología Agropecuaria) for contributions in statistical analysis.

409 SUPERSTIMULATED BOVINE DONORS INSEMINATED WITH SEMEN WITH DIFFERENT LINEARITY EVALUATED BY COMPUTER-ASSISTED SPERM ANALYSIS

There is a highly significant relationship between semen quality and the percentage of fertilized ova and transferable embryos in superovulated donors (Stroud B and Hasler JF 2006 Theriogenology 65, 65-76). Computer-assisted sperm analysis (CASA) provides an objective and highly repeatable system for sperm analysis. It has been observed that linearity (LIN) measured by CASA has a high correlation with fertility (Foote RH et al. 1998 Theriogenology 49, 871-879). In the present report, retrospective analysis was done to determine the effect of sperm linearity as assessed by CASA on the number of ova and viable embryos recovered from superstimulated cows. This research was carried out using different breeds of donors (n = 150, 80% Angus) during the last 5 years in an embryo transfer center in Buenos Aires province, Argentina. Donors with a CL received an intravaginal progesterone device (DIB®, Syntex, Buenos Aires, Argentina), 2 mg of 17? estradiol, and 50 mg of progesterone (Rio de Janeiro, Brazil), i.m. on the same day. On Day 4 after DIB® insertion, superstimulatory treatment was initiated as a decreasing dose regimen of FSH i.m. (Pluset®, Callier, Spain, or Folltropin®, Bioniche Animal Health, Inc., Belleville, Ontario, Canada) every 12 h during 4 days. On Day 6, DIB® devices were removed and cows were administered cloprostenol (2 mL) twice at 12-h intervals. When estrus was detected, donors received GnRH (2 mL i.m. Dalmarelin®, Von Franken, Buenos Aires, Argentina) and were inseminated 12 and 24 h thereafter. Semen was thawed in a water bath at 37°C for 1 min. Before insemination, every semen dose was evaluated for motility characteristics using the IVOS Sperm Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA). Two chambers of 20-(im depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). According to CASA results, 3 groups were defined based on LIN: Group 1 = &lt;50%; Group 2 =≥50% and &lt;60%; and Group 3 =≥60%. Seven days later, embryo collection was performed and ovarian response was evaluated by transrectal ultrasonography to assess number of CL + anovulatory follicles. Ova/embryos were evaluated and classified according to the IETS manual. Kruskal-Wallis test was used to compare variables among groups, and results are shown in Table 1. There was a significant difference between Groups 1 and 3 for total number and percentage of viable embryos. More embryos were recovered from donors inseminated with higher LIN semen. Also, there was a significant difference between Group 1 and both Groups 2 and 3 in the number of total fertilized ova, percentage of fertilized ova, and percentage of unfertilized ova. The present results suggest that insemination of donors with semen of high LIN results in higher fertilization rates and number of viable embryos. Table 1.Number of fertilized ova and viable embryos of donor cows inseminated with semen with different linearity

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

324 ADJUSTING SPERM CONCENTRATION USED TO INSEMINATE SUPERSTIMULATED BEEF COWS, IN ORDER TO AVOID DECLINE IN EMBRYO PRODUCTION

The quantity and quality of semen may affect conception rate after artificial insemination (AI). The concentration of motile spermatozoa, after thawing, varies according to the sire and lot of frozen semen used. The minimum amount of spermatozoa required in a semen straw was determined to obtain pregnancy after a single AI of a non-superovulated animal. However, superstimulation of embryo donors results in the availability of many more oocytes (an average 10–20) for fertilization than in a non-superovulated female (1 oocyte). The purpose of the present work was to verify if by adjusting the concentration of motile spermatozoa, in straws with low sperm concentration, the percentage of viable embryos is comparable to those obtained using straws with high sperm concentration after thawing. Nelore cows (Bos taurus indicus) were superstimulated with a protocol termed P36 (Barros CM et al. 2003 Theriogenology 59, 524 abst), in which the ovulation is induced by exogenous LH (12.5 mg, Lutropin®, Vetrepharm, London, Ontario, Canada), administered 36 h after PGF2α. One sample of each lot of semen was analyzed by CASA (computer-assisted semen analysis), and motile sperm concentration, after thawing, was adjusted to a minimum of 25–30 × 106 spermatozoa, which is approximately 3 to 4 times higher than the sperm concentration used for a regular AI. Fixed-time AI (FTAI) was performed 12, 24, and sometimes 36 h after exogenous LH. The number of semen straws necessary to obtain at least 25 × 106 spermatozoa varied from 2 to 6 (Groups 2 to 6, respectively). Since at least two semen straws were used per animal, there is no Group 1. The number of FTAI was adjusted according to the number of straws used, i.e., 2 straws (FTAI 12 and 24 h after LH), and 3 or more straws (12, 24, and 36 h after LH). Mean total structures (oocytes, viable embryos and degenerate embryos), mean viable embryos per flushing, and viability rate (percentage of viable embryos/total structures) were, respectively: 12.2, 8.9, and 73.6% (Group 2, n = 19 flushings); 13.5, 9.6, and 70.9% (Group 3, n = 101); 13.3, 9.4, and 70.9% (Group 4, n = 22); 5.5, 4.0, and 72.7% (Group 5, n = 4); and 24.0, 13.0, and 54.2% (Group 6, n = 1). When the results from Groups 4, 5, and 6 were pooled, total structures, viable embryos, and viability rate were: 12.5, 8.7, and 69.8% (n = 27). The statistical analysis was performed using the ratio of viable embryos/total structures for each flushing, transformed in square root followed by arc sin. Data from groups 4, 5 and 6 were pooled before comparing to the other groups by ANOVA. In order to facilitate the comprehension of the results, data were presented as viability rate instead of means of arc sin. There was no difference when comparing pooled data from Groups 4, 5, and 6 with the other groups (2 or 3; P = 0.87; ANOVA). It is concluded that by adjusting the concentration of motile spermatozoa in straws with low sperm concentration (Groups 4, 5, and 6), the viability rates are comparable to those obtained using semen with high sperm concentration (Group 2 or 3). Nogueira has a fellowship from FAPESP (Sã Paulo).

335EFFECT OF PATERNAL LINE OF DONOR ON SUPEROVULATORY RESPONSE AND EMBRYO QUALITY IN JAPANESE BLACK HEIFERS

The objective of this study was to investigate the effect of paternal line of donor on superovulatory response and embryo quality in Japanese Black cattle. Japanese Black cattle have paternal lines that can be classified into the following, according to meat quality and the growth rate: ‘high marbling meat type’, ‘high growth rate type’ and ‘high marbling meat and high growth rate (intermediate type)’. We hypothesized that paternal line of donor may affect superovulatory response. One hundred and forty superstimulations were performed on 56 Japanese Black heifers, and data on superovulatory response and embryo collection were analyzed. Japanese Black donors used in this study were from the following 4 paternal lines: Kedaka (large-framed with high growth rate;; n=9, 30 times), Fujiyoshi (intermediate type;; n=22, 47 times), Shigekane (high marbling meat type;; n=13, 40 times), and Tajiri (high marbling meat type and small-frame with slow growth rate;; n=12, 23 times). Donors were synchronized using a CIDR-B (EAZI-BREED, InterAg, NZ) for 8 to 12 days, and 500μg PGF analog (Resipron-C; Teikoku Zouki, Japan or Clopromate-C; Sumitomo, Japan) administered at CIDR removal. Superstimulation was initiated on Day 10 (Day 0=day of estrus) of the synchronized cycle;; FSH (Antrin R10;; Denka, Japan) was administered twice daily for 4 days with decreasing doses for a total of 28 Armour units FSH. PGF analog was administered in the morning on the last day of FSH treatment. Donors were given 50 or 100μg GnRH analog (Sporunen, Denka, or Conceral, Takeda Schering prau, Japan) at the time of estrus detection, and were inseminated 12h after the onset of estrus. Embryos were recovered on Day 7 after AI. The numbers of CL and follicles were recorded with transrectal ultrasonography immediately after embryo recovery. Embryos were classified according to the IETS Manual. Data were analyzed by Kruskal-Wallis test combined with Scheffe’s multiple comparison test. There were no significant differences in CL numbers among paternal lines: Kedaka (9.5±4.2), Fujiyoshi (8.5±5.1), Shigekane (7.8±5.0) and Tajiri (8.6±4.1). Mean number of recovered ova/embryos per donor was significantly (P&amp;lt;0.05) higher in the Kedaka line (8.4±5.1) than in the Fujiyoshi (4.9±4.1) and Shigekane (5.5±7.0) lines. The percentages of viable embryos was greater (P&amp;lt;0.05) in the Fujiyoshi (73.7%) and Shigekane lines (62.8%) than in the Tajiri line (50.9%), whereas the percentage of unfertilized oocytes was significantly (P&amp;lt;0.05) higher in the Tajiri line (41.5%) than in the Fujiyoshi (17.5%) or Shigekane (26.1%) lines. The percentages of freezeable and transferable embryos were not significantly different among paternal lines. Data suggest that the paternal line had an effect on the superstimulatory response in Japanese Black heifers, and especially the number and percentage of viable embryos per donor.

Social environment and reproductive interference affect reproductive success in the frog Rana latastei

Variation in the social environment produces selection on morphological and behavioral traits. It is less clear how the social environment generates variation in demography through behavioral mechanisms. Theory suggests that one aspect of social environment, relative abundance of heterospecifics, influences the intensity of reproductive interference and its demographic effects. These effects are countered by species recognition and female preferences. We studied the effects of social environment on reproductive success in replicated, mixed breeding populations of two ranid frogs, Rana latastei and Rana dalmatina, the former being of international conservation concern. We manipulated the social environment of female R. latastei experimentally by varying the relative abundance of potential conspecific and heterospecific sexual partners. We measured amplexus frequency and recorded the reproductive success of R. latastei females. When conspecific males were relatively uncommon, (1) the absolute and relative frequencies of conspecific amplexus decreased, indicating a breakdown of sexual isolation, (2) oviposition was less frequent, and (3) the percentage of viable embryos in deposited clutches decreased. R. latastei females in an environment of low relative conspecific abundance (1:5, R. Latastei:R. dalmatina) demonstrated 6.8% the reproductive success of females in an environment exclusively with conspecifics. We present a model for the dependence of conspecific amplexus on the social environment. We discuss several mechanisms that may influence reproduction by R. latastei, and we support conservation of the species' preferred habitat to reduce opportunities for reproductive interference that occur at shared breeding sites.

Effect of immunization on reproducvive performance, embryo quality and progesterone in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone

A total of 217 Rasa Aragonesa ewes were used to test two immunization treatments: 1.Active immunization against androstenedione: ewes immunized in previous matings (androstenedione, reimmunized; AR groups, n=58) or not (first immunization; AF groups n=64) were boosted either 2 or 4 wk before mating. 2.Passive immunization against testosterone: antisera were injected either at sponge withdrawal (zero time; T0 group, n=21) or 1 wk previously (Tl group, n=22). We used 52 ewes as controls (C group). Half of each group was used either to record reproductive performance or to embryo viability assessment. Prolificacy was significantly increased in ewes which reached a moderate antibody level, independently of the treatment. Fertility was lower in AR ewes that attained a high antibody titre (P<0.01). The percentage of viable embryos recovered was lower in AF ewes (P<0.01), and in ewes whose testosterone antibody titre was high (P<0.05), compared to C group. It was proven that similar or lower antibody levels were more harmful for ewes from AF and Tl than for ewes from AR or T0 groups. The proportion of nonfertilized recovered ova was not significant. Progesterone levels were notably increased in AR ewes (P<0.001) independently of ovulation rate and were positively correlated to antibody titre at mating (P<0.01) but these events were not observed in T ewes. These findings indicate that after androgen immunoneutralization, only those ewes having antibody titres within a limited range at mating had improved reproductive performance. Further research is needed in order to understand the role that progesterone plays in immunized ewes.

Frequency of pregnancy and ova implantation in triethyl lead-treated mice

Female mice were treated with triethyl lead chloride orally on Days 3–5 after mating. Decreased frequencies of pregnancy were observed at doses of 3.0 ( p < 0.0005) and 2.2 mg/kg body wt ( p < 0.025). Animals given 1.5 mg/kg showed a decreased frequency of implanted ova ( p < 0.0005). No effects upon the percentage of viable embryos were observed at doses of 0.5 and 1.5 mg/kg.