Time Of Embryo Collection Research Articles

Relationship among circulating anti-Müllerian hormone, insulin like growth factor 1, cadmium and superovulatory response in dairy cows

The objectives of this study were 1. to determine the associations among circulating anti-Mullerian hormone (AMH), insulin like growth factor 1 (IGF1) and cadmium (Cd) concentrations of lactating Holstein cows at the time of superovulation and 2. to determine the effect of circulating AMH, IGF1 and Cd concentrations on the superovulatory response in Holstein dairy cows. Holstein cows (n = 30) were milked thrice daily and housed and fed in free stall barn as a separate group. All animals were synchronized for superovulation and flushed. Three blood samples for AMH, IGF1 and Cd analysis were collected prior to superovulation, at estrus and at the time of embryo collection. The concentrations of blood makers prior to superovulation were highly correlated to superovulatory response. Circulating concentrations of AMH, IGF1 prior to superovulation were negatively correlated to Cd concentrations (P < 0.05). There was no correlation between circulating concentrations of AMH and IGF1. The number of corpus luteum (r = 0.71), total embryo (r = 0.67), total transferable embryo (r = 0.51) and total grade 1 embryo (r = 0.5) were positively correlated to AMH concentrations (P < 0.05). There was a trend for negative correlation found between circulating cadmium concentrations and total grade 1 embryo yield (P < 0.1). When cows were classified into quartiles (Q) of circulating AMH concentration, number of corpus luteum, and total embryos, total transferable embryos and total grade 1 embryos yield was significantly different for AMH quartiles. The superovulatory response parameters evaluated were increased with increased AMH concentrations; particularly we observed a >2-fold difference between first and fourth AMH quartiles in total transferable embryo yield and total grade 1 embryo yield. In conclusion, circulating AMH concentration was strongly associated with superovulatory response. Measuring AMH before enrolling cows in superovulation programs will likely allow practitioners to improve numbers of embryos produced and, thereby, reduce costs per embryo produced.

Effects of eCG and progesterone on superovulation and embryo production in wood bison (Bison bison athabascae)

Experiments were done to determine if inclusion of eCG and progesterone in the superstimulation protocol will increase the ovarian response and embryo production in wood bison, and to provide preliminary information regarding the effect of season. In Experiment 1 (anovulatory season), bison (n=26) were synchronized by follicular ablation (Day −1) and given FSH on Days 0 and 2, and assigned to 3 groups: Progesterone (Days 0–4), eCG (Day 3), or progesterone+eCG. On Day 5, bison were given hCG and inseminated 12 and 24h later. Ova/embryos were collected 8days after hCG. In Experiment 2 (ovulatory season), bison (n=24) were synchronized and assigned randomly to two groups in which superstimulation was induced with FSH, either with or without eCG, as in Experiment 1. No differences among groups were found in ovarian response or embryo production in either experiment. The follicular count at wave emergence was positively correlated with the number of large follicles at the end of superstimulation in all groups. A significantly greater number of follicles present at wave emergence in the anovulatory vs. ovulatory season was associated with a greater number of CL at the time of embryo collection, but only half the number of freezable embryos. In conclusion, the number of transferable embryos collected (1–2/bison) was higher than in any previous report, but was not attributable to the inclusion of eCG or progesterone in the superovulatory protocol. The apparent effect of season on oocyte competence, and not superovulatory response, is worthy of further investigation.

Embryo production in heifers with low or high dry matter intake submitted to superovulation

This study investigated the influence of feed intake on superovulatory response and embryo production of Nelore heifers. Pubertal heifers were kept in a feedlot and were submitted to the same diets, but with different levels of feed consumption: High (1.7 M; n = 20) or Low (0.7 M; n = 19) feed intake. Heifers in the 1.7 M treatment consumed 170% (2.6% of body weight [BW] in dry matter) and the 0.7 M heifers ate 70% (1.1% of BW in dry matter) of a maintenance diet. After 7 wk on these diets, heifers were treated with eight decreasing doses of follicle-stimulating hormone (FSH) given every 12 h, totaling 133 mg Folltropin (Folltropin-V; Bioniche Animal Health, Canada) per heifer. Seven d after AI, heifers had their uteri flushed and embryos were recovered and graded according to the International Embryo Technology Society standards. Data were analyzed using the GLIMMIX procedure of SAS and results are presented as least-squares means ± SEM (P < 0.05). At the onset of the FSH treatment (Day 0 of the protocol), 1.7 M heifers had greater body condition score (BCS), BW and serum insulin concentrations than 0.7 M heifers (4.1 ± 0.1 vs. 3.0 ± 0.1; 462.5 ± 10.1 vs. 382.7 ± 10.4 kg; and 14.3 ± 1.7 vs. 3.5 ± 0.8 μIU/mL, respectively). The 0.7 M heifers had more follicles ≥6 mm at the time of the last FSH (Day 7; 47.9 ± 6.4 vs. 23.5 ± 4.3 follicles), related to a better follicle superstimulatory response to FSH. Similarly, 0.7 M heifers had more corpora lutea at the time of embryo collection (33.6 ± 1.4 vs. 15.7 ± 0.9) than the 1.7 M heifers, which resulted in greater number of recovered embryos and ova (9.9 ± 0.7 vs. 6.7 ± 0.6) and viable embryos (5.3 ± 0.5 vs. 3.8 ± 0.4), despite having similar proportions of viable embryos (∼62%). A negative correlation between circulating insulin and follicle superstimulatory response to FSH was observed (r = −0.68). Therefore, we conclude that high feed intake, for a long period of time, compromised the superovulatory response and embryo production potential of Bos indicus heifers possibly related to the elevation in circulating insulin.

Neurotrophins and their specific receptors in the oviduct of Japanese quail (Coturnix coturnix japonica)

The objectives of this study were 1. to determine the associations among circulating anti-Mullerian hormone (AMH), insulin like growth factor 1 (IGF1) and cadmium (Cd) concentrations of lactating Holstein cows at the time of superovulation and 2. to determine the effect of circulating AMH, IGF1 and Cd concentrations on the superovulatory response in Holstein dairy cows. Holstein cows (n = 30) were milked thrice daily and housed and fed in free stall barn as a separate group. All animals were synchronized for superovulation and flushed. Three blood samples for AMH, IGF1 and Cd analysis were collected prior to superovulation, at estrus and at the time of embryo collection. The concentrations of blood makers prior to superovulation were highly correlated to superovulatory response. Circulating concentrations of AMH, IGF1 prior to superovulation were negatively correlated to Cd concentrations (P < 0.05). There was no correlation between circulating concentrations of AMH and IGF1. The number of corpus luteum (r = 0.71), total embryo (r = 0.67), total transferable embryo (r = 0.51) and total grade 1 embryo (r = 0.5) were positively correlated to AMH concentrations (P < 0.05). There was a trend for negative correlation found between circulating cadmium concentrations and total grade 1 embryo yield (P < 0.1). When cows were classified into quartiles (Q) of circulating AMH concentration, number of corpus luteum, and total embryos, total transferable embryos and total grade 1 embryos yield was significantly different for AMH quartiles. The superovulatory response parameters evaluated were increased with increased AMH concentrations; particularly we observed a >2-fold difference between first and fourth AMH quartiles in total transferable embryo yield and total grade 1 embryo yield. In conclusion, circulating AMH concentration was strongly associated with superovulatory response. Measuring AMH before enrolling cows in superovulation programs will likely allow practitioners to improve numbers of embryos produced and, thereby, reduce costs per embryo produced.

233 ANTI-MÜLLERIAN HORMONE AS A PREDICTIVE ENDOCRINE MARKER FOR SUPEROVULATORY RESPONSE AND EMBRYO PRODUCTION IN BEEF CATTLE

A study investigated the use of anti-Müllerian hormone (AMH) and/or follicle counts as a predictor of subsequent superovulatory response and embryo production in 79 beef cows. Before initiation of superovulation, ultrasonography was used to scan the ovaries of each donor cow to record the number of 3 to 5 mm follicles present, and a blood sample was collected for measure of serum AMH. At the time of embryo collection, the ovaries of donor cows were palpated to estimate the number of corpora lutea (CL) present on each ovary. Recovered embryos were evaluated for stage of development and morphological quality. Across cows, serum AMH ranged from 0.013 to 0.898 ng mL–1, with a mean of 0.293 ng mL–1. The distribution of AMH concentrations was divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest, ng mL–1) for analysis (ANOVA). Number of donors/collections in AMH Q1 through Q4 was 26, 22, 24, and 24, respectively. Donor cows in AMH Q4 had a greater (P &lt; 0.001) number of 3- to 5-mm follicles at the start of superovulation than did donors in either Q1 or Q2. At embryo collection, cows in AMH Q3 and 4 had more (P &lt; 0.001) palpable CL than cows in AMH Q1. The mean number of embryos recovered from donor cows in AMH Q4 (20.1 ± 1.8) was greater (P &lt; 0.001) than those recovered from cows in either AMH Q1 (9.8 ± 1.8) or 2 (9.4 ± 1.9), but similar to that of AMH Q3 (15.5 ± 1.8). Percentages of recovered embryos classified as transferrable, degenerate, or unfertilized were similar (P ≥ 0.275) across AMH quartiles. Analysis indicated that AMH was positively correlated (P &lt; 0.001) with mean follicles (r = 0.458), CL (r = 0.452), and embryos recovered (r = 0.430). To determine if follicle counts at the start of superovulation are predictive of superovulatory response, the distribution of follicle counts was divided into quartiles (F Q1 through Q4, with Q1 the lowest and Q4 the highest) for analysis. Donor cows with higher follicle counts (F Q3 and 4) at the start of superovulation had more (P &lt; 0.001) palpable CL at embryo collection than donor cows in F Q1 or 2. More (P &lt; 0.001) embryos (20.0 ± 2.2 v. 9.6 ± 1.8 and 11.6 ± 1.6) were recovered from cows with the highest follicle counts (F Q4) compared with cows having lower (F Q1 and 2) follicle counts, respectively. The percentage of transferable embryos and unfertilized ova were similar (P ≥ 0.688) across follicle count quartiles. As was noted for AMH, mean number of follicles at the start of superovulation was positively correlated (P &lt; 0.001) with mean CL (r = 0.556) and mean embryos (r = 0.423) but not percentages of viable or degenerate embryos or unfertilized oocytes (P ≥ 0.153). Results confirm that relative AMH concentration was positively correlated with number of small antral follicles in the ovaries of cows and might be used to either predict superovulatory response or possibly adjust superovulatory regimen to improve superovulatory response. Further study is needed to determine the effectiveness of using either AMH concentration or follicle counts to adjust superovulatory regimens.

108 COMPARATIVE EFFICIENCY OF GONADOTROPIN-RELEASING HORMONE AND LUTEINIZING HORMONE IN THE INDUCTION OF OVULATION IN SUPEROVULATED ALPACAS

Alpacas are induced ovulators, responding to copulation and (or) exogenous application of ovulation-inducing hormones. The objective of this study was to determine the efficiency of the injection of a gonadotropin-releasing hormone (GnRH) agonist versus LH in the induction of ovulation and the presence and size of non-ovulated follicles at the time of embryo collection and its relationship to the yield of transferable embryos in superovulated alpacas. Twenty-one adult (3 to 7 years old) female alpacas under extensive grazing at 4300 m elevation in the Peruvian Andes that had been synchronized and treated for superovulation were induced to ovulate 6 days after the application of the superovulatory hormone (1000 IU of eCG, Folligon®, Intervet International BV, Boxmeer, the Netherlands) by mating with fertile males and injection immediately after copulation of either an IM dose of 0.0084 mg of buserelin acetate (Buserelina Zoovet®, Laboratorio Zoovet, Santa Fe, Argentina) to 10 alpacas (T1) or an IM dose of 5-mg Armour standard of LH (Lutropin®, Bioniche Animal Health, Belleville, ON, Canada) to 11 alpacas (T2). All alpacas had a second mating 12 h after the first mating and were subjected to ovarian inspection by ultrasonography and previous embryo collection by nonsurgical transcervical embryo flushing 6.5 days after the first mating. On average, the embryo recovery rate for T1 was 34.6% and there were 7.8 ± 3.99 corpora lutea (CL), 2.7 ± 4.08 collected embryos, and 3.6 ± 2.95 total, 0.5 ± 0.85 small (&lt;6 mm), 1.8 ± 1.99 medium (≥6 but ≤14 mm), and 1.3 ± 2.11 large (≥15 mm) non-ovulated follicles. For T2, the embryo recovery rate was 59.4% and there were 6.73 ± 1.49 CL, 4.0 ± 2.57 collected embryos, and 0.64 ± 0.81 total, 0.0 ± 0.0 small, 0.36 ± 0.67 medium, and 0.27 ± 0.47 large non-ovulated follicles. The differences between treatments were nonsignificant (P &gt; 0.05) for all the parameters; however, there was a clear tendency for a better recovery rate, more embryos collected, and lower number of non-ovulated follicles in T2. The Pearson correlation coefficient between the presence of large follicles in the ovaries at the time of embryo collection and the total number of embryos collected was negative (r = –0.26) and highly significant (P ≤ 0.001). The use of LH for ovulation induction of superovulated alpacas seems to be more advisable than the use of GnRH agonist; further comparisons with larger number of observations per treatment are recommended. This study was financed by the Peruvian Fund for Innovation, Science and Technology (FINCYT).

312 INFLUENCE OF LARGE NONOVULATED FOLLICLES ON ESTROUS INDUCTION AFTER EMBRYO COLLECTION IN SUPEROVULATED JAPANESE BLACK COWS

Rapid oestrous induction after embryo collection shortens the calving interval of donor cows and improves the efficiency of embryo production. This study aimed to evaluate whether large nonovulated follicles present at the time of embryo collection affect oestrous induction after embryo collection in Japanese Black donor cows. Superovulation treatments were initiated during mid-cycle (9–12 days after oestrus) with 20 AU of FSH (Antrin; Kyoritsu Seiyaku Corp., Tokyo, Japan) administered intramuscularly through 6 injections of decreasing doses at 12-h intervals. On the third day, the cows received 0.5 mg of prostaglandin F2α (PGF; Resipron-C, Asuka Seiyaku Corp., Tokyo, Japan) to induce regression of the corpus luteum and were artificially inseminated 60 to 72 h later. Embryos were collected 7 days after AI. All cows received 0.5 mg of PGF immediately after embryo collection and were allocated to 1 of 2 groups: cows (n = 6) with one or more large nonovulated follicles &gt;18 mm in diameter (Group 1) and cows (n = 5) with no large follicles (Group 2). Oestrus detection was performed by visual observation conducted twice daily after embryo collection. Ovarian ultrasonographic examinations and blood collections (for serum progesterone [P4] and oestradiol [E2] measurements) were performed once daily until the next ovulation. Statistical significance was determined using Student’s t-test. There were no differences in the numbers of corpora lutea, ova/embryos, and transferable embryos or the percentages of transferable embryos between the 2 groups. The interval (mean ± SEM) from embryo collection to the appearance of the dominant follicle and to ovulation was longer (P &lt; 0.05) in Group 1 (5.3 ± 0.7 and 11.0 ± 1.0 days, respectively) than in Group 2 (2.6 ± 0.7 and 8.2 ± 0.7 days, respectively). However, the interval from embryo collection to luteolysis (P4 &lt;1 ng mL–1) did not differ between the 2 groups (4.0 ± 0.7 v. 3.6 ± 0.7 days). In conclusion, the presence of large nonovulated follicles at the time of embryo collection may delay oestrous induction after embryo collection.

315 RETURN TO ESTRUS AND OVULATION FOLLOWING ADMINISTRATION OF PROSTAGLANDIN IN SUPEROVULATED BEEF COWS

Two experiments were designed to evaluate the effect of administering 1 or 2 doses of prostaglandin F2α (PGF) at the time of ova/embryo collection on luteal regression and return to oestrus and ovulation in donor cows. In Experiment 1, 14 Angus cows were blocked at the time of embryo collection by the number of corpora lutea (CL) determined by rectal palpation [6 to 10 CL (n = 6), 11 to 15 CL (n = 4), &gt;15 CL (n = 4)] and were randomly assigned to 2 groups to receive a single injection of 500 µg of sodium cloprostenol (PGF, Ciclase, Syntex S.A., Buenos Aires, Argentina) on the day of embryo collection (Day 0) or a single injection of PGF on Day 0 and a second injection of PGF 24 h later. All cows were bled daily to determine progesterone concentrations by RIA and ultrasonically scanned daily (Falco 100, Pie Medical, Maastricht, the Netherlands) for 19 days after embryo collection, to determine ovulation of a newly recruited dominant follicle. In Experiment 2, 47 superovulated Angus cows were blocked by CL numbers [1 to 4 CL (n = 5), 5 to 10 (n = 11), 11 to 15 (n = 13), &gt;15 CL (n = 18)] and treated as in Experiment 1. In this experiment, cows were bled and scanned on Days 0 (i.e. embryo collection), 4, 7, 10, 13, 16, and 19. The interval from embryo collection to luteal regression (i.e. plasma progesterone &lt;1 ng mL–1) and to ovulation was analysed using ANOVA. Proportions were compared by Chi-square test. In Experiment 1, the mean (± SEM) number of CL in donor cows was 11.9 ± 2.0, and the effect of CL numbers on luteal regression, oestrus, and ovulation was not significant (P &gt; 0.5). The interval from treatment to luteal regression was not affected by PGF treatment (1 PGF: 2.0 ± 1.2 days; 2 PGF: 2.4 ± 1.0 days). Furthermore, 6/7 animals (85.7%) were observed in oestrus in both groups, and 6/7 (85.7%) and 4/7 (57.1%) ovulated within 19 days in the 1 PGF and 2 PGF groups, respectively (P &gt; 0.2). The interval to oestrus and ovulation did not differ among groups (7.0 ± 0.7 days and 8.5 ± 0.6 days v. 9.7 ± 0.5 days and 10.0 ± 0.7 days, for 1 PGF and 2 PGF, respectively; P &gt; 0.1). In Experiment 2, the mean number of CL in donor cows did not differ from that in Experiment 1 (13.9 ± 1.1; P &gt; 0.37), and the effect of CL numbers on luteal regression and ovulation was not significant (P &gt; 0.7). Although luteal regression occurred earlier (P = 0.03) in the 2 PGF group (4.1 ± 0.6 days) compared with the 1 PGF group (5.3 ± 2.2 days), no differences were detected between PGF groups in any of the other parameters. The proportion of cows in oestrus and the interval to oestrus was 19/24 (79.2%) and 7.2 ± 0.4 days v. 18/23 (78.3%) and 7.2 ± 0.3 days for cows in the 1 PGF and 2 PGF groups, respectively (P &gt; 0.7). Finally, the proportion of cows ovulating within 19 days after collection and the interval to ovulation were 20/24 (83.3%) and 10.3 ± 0.4 days v. 21/23 (91.3%) and 10.1 ± 0.4 days for cows in the 1 PGF and 2 PGF groups, respectively (P &gt; 0.4). In summary, treatment with PGF at the time on embryo collection results in luteal regression in 4 to 5 days and ovulation of a new dominant follicle in 80 to 90% of the donors within 19 days. The addition of a second PGF treatment did not shorten the interval to oestrus and ovulation.

141 EFFECT OF ENVIRONMENTAL TEMPERATURE ON EMBRYO PRODUCTION AND CONCEPTION RATES IN BEEF AND DAIRY CATTLE

The aim of this study was to evaluate the effect of environmental temperature at the time of embryo collection and transfer on embryo quality and conception rates after transfer of fresh and frozen embryos. Purebred dairy (Holstein, Czech spotted cattle, n = 174) and beef (Charolais, Blonde d’Aquitaine, Piedmontese, Hereford, Limousin and Simmental; n = 72) donors were collected during 2005 to 2007. Donor cows received one application of PGF2α and superstimulation was initiated 9 to 11 days following estrus with pFSH (Folltropin-V; Bioniche Animal Health, Belleville, ON, Canada) given twice daily for 4 days. Prostaglandin2α was given on Day 3 of the superstimulation treatment. Donors were inseminated 3 times, with 1 unit of semen on Days 5 and 6 of treatment. Embryo recovery was carried out on Day 7 after insemination. The recipients were synchronized with PGF2α and embryos were transferred on Day 7 after estrus. The effect of temperature at the time of 246 embryo recoveries and 1338 transfers (fresh and frozen embryos) was analyzed using the GLM, assuming quasi-poisson and quasi-binomial error distribution by R software (www.r-project.org). We evaluated the total number of ova/embryos collected, embryo quality, and conception rates after transfer at the environmental temperatures shown below. Temperatures were divided into the following ranges: A) from –5 to 5°C; B) from 6 to 15°C; C) from 16 to 20°C; and D) over 20°C. The average temperature values were obtained from the Czech hydrometeorological institute and corresponded to the following seasons: winter (range A and B); spring (range B and C); summer (range C and D); fall (range C and B). Dairy cows were managed intensively in barns whereas beef cows were managed in barns during the winter and on pasture from April until October. In beef donors the mean number of total ova/embryos collected and transferable embryos were 9.1 ± 7.9 and 4.2 ± 4.5, respectively and no significant effect of temperature was detected (P &gt; 0.05). In dairy donors the mean number of total ova/embryos collected and transferable embryos were 7.9 ± 6.0 and 4.9 ± 4.5, respectively and no significant effect of temperature was detected (P = 0.27). Conception rate was greatest (P &lt; 0.001) in temperature range D (63.64%) compared with the other temperature ranges (A = 27.66%; B = 43.65%; C = 43.21%;) in beef cows and in the range C (65.31%) than in the other temperature ranges (A = 34.7%; B = 52.67%; D = 56.25%) in dairy cows. It was concluded that temperature at the time of embryo collection did not affect embryo production and quality. However, environmental temperature did have a significant effect on conception rates in both beef and dairy recipients. Nutritional status may also have contributed to the increased conception in beef cattle during warmer temperatures. However, the optimal period for conception in dairy cows was in temperature range from 16 to 20°C, because of the potential adverse effect of heat stress in high-yielding cows. Supported by MEYS CR MSM 2678846201, LA 330; NAZV 1B44034.

297 LACK OF IMPROVEMENT ON EMBRYO PRODUCTION BY THE REPLACEMENT OF THE LAST TWO DOSES OF pFSH BY eCG IN SUPERSTIMULATED NELORE HEIFERS

Results from a previous experiment have shown that the replacement of pFSH by eCG on the last day of the superstimulatory treatment in Nelore (Bos indicus) cows resulted in a greater superovulatory (SOV) response as compared to the treatment exclusively with pFSH (Barros CM et al. 2008 Repr. Fertil. Dev. 20, 152 abst). The aim of this study was to investigate if a similar approach would be beneficial for embryo production in nulliparous Nelore heifers. Forty heifers were randomly divided into two SOV groups: FSH Group: eight pFSH injections or FSH-eCG Group: six pFSH injections followed by two eCG injections. Each female received both treatments 65 days apart in a cross-over design. The SOV protocols consisted of an IM injection of 2 mg estradiol benzoate (Estrogin, Farmavet, São Paulo, Brazil) and insertion of an intravaginal progesterone releasing device (1.9 g progesterone, CIDR, Pfizer, Hamilton, New Zealand) on Day 0. On Day 4.5, the superstimulatory treatments (70 mg pFSH; Folltropin-V, Bioniche Animal Health; Belleville, ON, Canada) were initiated and given in decreasing doses of 28, 21, 14, and 7 mg twice a day, over a 4-day period. The FSH-eCG Group had the last two doses of pFSH replaced by two doses of 150 IU eCG (Folligon, Bioniche). At the time of the fifth and sixth injections of FSH, 25 mg dinoprost tromethamine (Lutalyse, Pfizer, Paulinia, Brazil) was injected IM. The CIDR was removed at the time of the seventh superstimulatory injection. Ovulation was induced with an IM injection of 0.05 mg GnRH (gonadorelin acetate; Gestran Plus; ARSA S.R.L., Buenos Aires, Argentina) 12 h after the last superstimulatory injection. All heifers were artificially inseminated with frozen/thawed semen from the same bull 12 and 24 h after GnRH. Seven days after the first AI, embryos/ova were recovered using a nonsurgical uterine flushing technique and classified according to IETS standards. To determine the superstimulatory (number of follicles ≥6 mm 12 h prior to GnRH) and SOV (number of ovulated follicles 48 h after GnRH, confirmed by CL number at the time of embryo collection) responses, transrectal ultrasonography was performed. Data were analyzed by paired t test and are presented as mean ± SEM. There was no difference between FSH and FSH-eCG groups regarding superstimulatory (23.2 ± 1.9 v. 22.3 ± 1.6 follicles ≥6 mm, P = 0.56) or SOV (15.2 ± 1.1 v. 17.5 ± 1.4 CL, P = 0.21) responses. Treatments were also similar for number of total embryos/ova (9.6 ± 0.9 v. 9.5 ± 1.0, P = 0.91), viable embryos (4.9 ± 0.7 v. 3.7 ± 0.5, P = 0.17), or degenerate embryos (3.0 ± 0.6 v. 4.3 ± 0.7, P = 0.10) recovered. Contrasting with the results using Nelore cows, the present study did not observe improvement on embryo production by replacing of the last two doses of pFSH by eCG in superstimulated heifers. Financial support from CNPq, FAPESP, EMBRAPA and Pfizer of Brazil.

The dominant follicle exerts an interovarian inhibition on FSH-induced follicular development

An experiment was conducted to evaluate the role of the dominant follicle (DF) of the first wave in regulating follicular and ovulatory responses and embryonic yield to a superovulation regime with FSH-P. Twenty normally cycling Holstein-Freisian heifers (n = 20) were synchronized with GnRH and pgf 2α and randomly assigned to a control or a treated group (n = 10 each). Treated heifers had the first wave dominant follicle removed via transvaginal, ultrasound-guided aspiration on Day 6 after a synchronized estrus. All heifers received a total of 32 mg FSH-P given in decreasing doses at 12 h intervals from Day 8 to Day 11 plus two injections of pgf 2α (35 mg and 20 mg, respectively) on Day 10. Heifers were inseminated at 6 h and 16 h after onset of estrus. Follicular dynamics were examined daily by transrectal ultrasonography from Day 4 to estrus, once following ovulation, and at the time of embryo collection on Day 7. Blood samples were collected daily during the superovulatory treatment and at embryo collection. Follicles were classified as: small, ≤ 5 mm; medium, 6–9 mm; or large, ≥ 10 mm. Aspiration of the dominant follicle was associated with an immediate decrease in large follicles, and a linear rate increase in small follicles from Day 4 to Day 8 just prior to the FSH-P injections, (treatment > control: +0.33 vs. −0.22, number of small follicles per day; P < 0.10). During FSH-P injections, the increase in number of medium follicles was greater (P < 0.01) for treatment on Day 9–11 (treatment > control: Day 9, 3.2 > 1.8; Day 10, 9.2 > 4.7; Day 11, 13.1 > 8.3; ± 0.56). Number of large follicles was greater in treatment at Day 11 (5.12 > 1.4 ±0.21; P < 0.01). Mean number of induced ovulatory follicles (difference between number of follicles at estrus and Day 2 after estrus) was greater in treatment (13.4 > 6.3 ± 1.82; P < 0.01). Plasma estradiol at Day 11 during FSH-P treatment was greater in treatment (32.5 > 15.8 ± 2.6; P < 0.01). Plasma progesterone at embryo flushing (Day 7 after ovulation) was greater in treatment (7.4 > 4.9; P < 0.02); technical difficulties at embryo recovery reduced sensitivity of embryonic measurements. No changes in the distribution of unfertilized oocytes and embryo developmental stages were detected between control and treatment groups. Presence of dominant follicle of the first wave inhibited intraovarian follicular responses to exogenous FSH.

Serum anti-eCG improves luteal function and increases ova/embryos recovery in eCG-superovulated ewes

Superovulatory response to an eCG treatment (1200 IU) alone (Group eCG, n= 10) or with anti-eCG serum (Group AS, In = 10) given 12 h after the onset of estrus was studied during the breeding season in ewes. Blood samples were taken for LH and progesterone determination and on Day 5 post-estrus all ewes were flushed and ova/embryos recovered. Mean overall interval to first signs of standing estrus for all ewes was 23.6 ± 1.8 h (mean ± SEM) and peak of LH surge ranged between −11 h to 9 h after onset of estrus (2.0 ± 1.6 h). Mean number of CL (8.8 ± 1.5 vs. 10.6 ± 0.9 for eCG and AS groups, respectively; P = NS) and large anovulatory follicles (2.8 ± 2.0 vs. 0.9 ± 0.4, P = NS) were not different. A higher ova/embryo recovery was obtained in the AS group (4.1 ± 1.0 vs. 0.9 ± 0.5, P < 0.05) and this was related to a lower proportion of ewes that had small (4.0 ± 0.1 mm in diameter, n = 75), pale and non-protruding corpora lutea at time of embryo collection ( 2 10 vs. 5 10 , P = 0.14). Pooled data of all ewes that displayed this CL ( n = 7) show that this condition: (1) is related to the endocrine status of the ewes at time of eCG treatment (low endogenous levels of progesterone); (2) is characterised by a premature luteinisation (increase of progesterone at the onset of oestrus) and a subsequent premature luteolysis (decrease of progesterone levels at Day 3 to 5 post-estrus) and (3) is associated with a lower number of horns that yielded some ovum/embryo ( 4 14 vs. 16 24 , P ≤ 0.01). We conclude that the administration of anti-eCG serum at 12 h after the onset of estrus increases the ova/embryos recovery rate because of lower number of ewes that displayed prematurely regressing corpora lutea in the eCG-superovulated ewes.

Influence of day-0 and day-7 superovulated cow serum during development of bovine oocytes in vitro.

Oocytes were matured in medium supplemented with 5% serum collected from superovulated cows at oestrus (Day-0 SCS) or at the time of embryo collection (Day-7 SCS), or in medium supplemented with fetal calf serum (FCS). After insemination using frozen-thawed sperm, oocytes were cultured in vitro with medium supplemented with 5% Day-0 SCS or 5% Day-7 SCS or 5% FCS. The proportions of embryos that cleaved were not significantly different among treatments, whereas development of the embryo to a blastocyst was significantly higher in the presence of SCS than FCS. When the four possible combinations of Day-0 SCS and Day-7 SCS were used in the maturation and culture media, there were no differences among treatments, except that the cleavage rate was significantly higher (P < 0.05) with Day-0 SCS in the maturation medium and Day-7 SCS in the culture medium than with Day-7 SCS in the maturation medium and Day-0 SCS in the culture medium. The proportions of embryos that cleaved and developed to blastocysts were not related with the level of progesterone and luteinizing hormone in the serum added to the maturation and culture media. However, the use of serum with low concentrations of glucose, fatty acids and cholesterol in the maturation medium and the culture medium tended to be associated with a higher rate of cleavage and blastocyst development.

胚移植法を用いたセンダイウイルス感染マウスの清浄化

Mouse embryos (8-16 cells) were collected from Sendai virus (SV)-infected mice at 5 or 7 weeks after inoculation. All donors having embryo(s) were positive when tested by CF test or ELISA for SV antibody at the time of embryo collection. Most of the morphologically normal embryos developed (90.6%, 259/286) to morulae or blastocysts after culture for 26-28 hr. A total of 76 embryos cultured were transferred to the uteri of SV-free pseudopregnant recipients. Forty-seven young were obtained from these recipients (61.8% of development rate) and 46 young were successfully reared up to 10th week of age. All the recipients and the young were negative by testing SV antibody. These results indicate that the embryo transfer technique is useful for cleaning of SV-infected mice.

Superovulation in cattle: A combined treatment using syncromate B with either pregnant mare serum gonadotrophin or follicle stimulating hormone

A comparison between different superovulatory treatments in dairy cattle was carried out at a commercial embryo transfer unit in Israel. Both pregnant mare serum gonadotrophin (PMSG) and follicle stimulating hormone (FSH) were used, either alone or combined with Syncromate B (SMB). The use of PMSG + SMB significantly decreased the number of corpora lutea present at the time of embryo collection 7 d after insemination, as compared with other treatment regimens. Consequently, a significantly lower number of ova was found in those animals treated with PMSG + SMB. Better superovulatory responses were obtained when FSH, rather than PMSG, was used, regardless of whether they were administered alone or combined with SMB. It was clear that the use of SMB combined either with PMSG or FSH resulted in poorer responses than when either gonadotrophin was used alone.

Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice

Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.

Pseudorabies virus infection of six- and ten-day-old porcine embryos

Nine susceptible gilts were exposed to pseudorabies virus (PrV) by intrauterine inoculation immediately after breeding. Embryos were collected from each of three gilts on days 3, 6, and 10 following exposure to PrV. The number of embryos collected from each gilt was compared with the number of corpora lutea (CL). On days 6 and 10, there were substantially fewer embryos collected than there were CL. The embryos were examined for the presence of viral particles by electron microscopy. PrV was observed in embryos collected at 6 and 10 days following exposure of the gilts. The fluids used to flush the embryos from the uterus during collection were tested for PrV by virus isolation and direct fluorescent antibody procedures. PrV was isolated from the uterine-flush fluids of one of three gilts at each time of embryo collection.