Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows.

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.

Influence of the length of progestagen treatment and the time of oestradiol benzoate application on the ovulatory follicle size and ovulation time in anoestrous and cyclic beef cows.

Previous research from our laboratory in beef cattle suggests that the pre-ovulatory follicle size, maturity and subsequent susceptibility to gonadotropin are influenced by the length of progestagen treatment in artificial insemination programme in beef cows. To test this hypothesis, two experiments were conducted. In experiment 1, 35 anoestrous beef cows received an intravaginal sponge containing 200 mg of medroxyprogesterone acetate. The treatment lasted for 7 (n = 12), 8 (n = 11) or 9 (n = 12) days. Half of the animals in each group were injected with 0.7 mg of oestradiol benzoate (EB) at device removal (0 h) and the other half 24 h later. In experiment 2, 38 cycling beef cows were treated with the same protocols as in experiment 1. Ultrasound examinations were performed to determine the follicular diameter at device removal (dominant follicle), interval to ovulation and ovulatory follicle diameter. The dominant follicle of anoestrous cows with progestagen for 7 days (8.4 ± 1.6 mm) resulted smaller (p < 0.05) than the cows treated for 8 (10.5 ± 1.6 mm) and 9 days (10.6 ± 1.2 mm). However, regardless of the length of the treatments, ovulation time after device removal was longer (p < 0.05) when EB was injected 24 h after withdrawal than at 0 h in anoestrous cows (EB0 = 52.7 ± 4.0 h; EB24 = 70.8 ± 6.2 h) and in cyclic cows (EB0 = 50.0 ± 21.0 h; EB24 = 73.0 ± 20.0 h). In anoestrous cows, the treatment with progestagen for 9 days and EB at 24 h increased the diameter of the ovarian follicle (p = 0.033) but did not affect the diameter of the ovulatory follicle in cyclic cows. In conclusion, increasing the length of progestagen treatment for 8 or 9 days compared to 7 days increased the diameter of the dominant follicle, in anoestrous and cyclic beef cows. Oestradiol benzoate administered at device removal resulted in a shorter interval from device removal to ovulation compared with EB injection 24 h after the end of a progestagen treatment.

Transvaginal follicular aspiration and embryo development in superstimulated early postpartum beef cows and subsequent fertility after artificial insemination.

This study was conducted to investigate in early postpartum suckled beef cows with and without FSH pre-stimulation: (i) the influence of the postpartum period on the number and quality of oocytes recovered by ovum pick-up (OPU), (ii) the overall efficiency of the OPU/IVP embryos from days 30 to 80 postpartum and (iii) if repeated OPU negatively affect fertility following a fixed-time artificial insemination protocol. After parturition suckled Angus cows (n = 30) were divided in three groups (n = 10 group(-1)). All cows were anestrous at the commencement of experimental treatments (30.0 +/- 3.2 days postpartum, mean +/- SD; range 25-34 days). Group 1 treatments included: dominant follicle ablation (DFA), FSH treatment and OPU procedure 5 days after DFA. A total of 9 mg FSH (Ovagen) was administered s.c. once a day over 2 days at equal doses (4.5 + 4.5mg). For fertility test the cows received an intravaginal progesterone treatment from Days 78 to 86 postpartum and were fixed-time artificially inseminated (FTAI) at 56 and 72 h after device removal. Group 2: as cited for Group 1 with no FSH treatment. In both groups, OPU was repeated four times (Days 35, 49, 63 and 77 postpartum) and the collected oocytes classified as viable were in vitro matured, fertilized and presumptive embryos cultured for 8 days. Group 3 (Control FTAI): cows that had not previously aspirations were FTAI as Groups 1 and 2. Pregnancy was diagnosed by means ultrasonography 39 days after FTAI. The numbers (mean +/- SEM) of follicles visible and aspirated at the time of OPU in FSH-treated cows were greater (P < 0.05) than in non-treated cows (10.6 +/- 0.6 and 8.4 +/- 0.4 vs. 8.0 +/- 0.5 and 4.6 +/- 0.3, respectively). Following FSH treatment, the number (mean +/- SEM) of recovered oocytes per cow per OPU session and percentage of viable oocytes were greater in the treated (P < 0.05) than in non-treated animals (3.0 +/- 0.1 and 39.5% vs. 1.5 +/- 0.1 and 30.0%). The cleavage and embryo development rates were similar (P > 0.05) for both groups (14.8 and 6.4% vs. 16.6 and 5.5%). After FTAI the pregnancy rates were not different (P > 0.05) among groups (70, 60 and 90% for Groups 1, 2 and 3, respectively). We can conclude that (1) FSH-treated suckled postpartum cows can be a source of oocytes for in vitro fertilization and (2) repeated DFA/OPU applied during postpartum period did not affect the subsequent fertility following FTAI.

Influence of different doses of progesterone treatments on ovarian follicle status in beef cows.

To determine a dose of progesterone (P4) that allow ovarian follicular wave control, Aberdeen Angus cows were randomly assigned into four groups: T600 (n=5), 600 mg of P4/day; T400 (n=5), 400 mg of P4/day; T200 (n=4), 200mg of P4/day and Control (n=4) (excipient only). Progesterone was injected from day 3 to 9 of estrous cycle. Ultrasonographies and blood sample collections were performed daily from day 2 to 10 and on day 15 of the estrous cycle. Additionally, an ultrasonographic study was conducted on day 13. Progesterone concentrations were different among all groups (P<0.01). The diameter of the dominant follicle was greater for control than for T200, T400 and T600 groups (P<0.01); there was no difference between T200 and T400 (P>0.05), but they had a greater diameter follicle than the T600 group (P<0.01). The growth rate of the dominant follicle between day 3 and 7 of estrous cycle was greater for control group (1.63+/-0.3 mmday(-1)) than for T200 (0.56+/-0.19 mmday(-1), P<0.05), T400 (0.6+/-0.23 mmday(-1), P<0.05) and T600 (0.11+/-0.13 mmday(-1), P<0.01) groups. The mean number of class I follicles (3-4mm) per day for the entire experimental period was less for the control group than for T200 (P<0.05), T400 and T600 (P<0.01) groups (3.7+/-1.3; 5.3+/-1.3; 6.6+/-1.8 and 8.1+/-1.9, respectively). The mean number for the T200 group was less than for T600 (P<0.05) and similar for T400 and T600 groups (P>0.05). The number of class III follicles was greater for control group than for the other groups (P<0.01). T200 and T400 groups had similar numbers of class III follicles (P>0.05) and both had greater numbers of follicles than the T600 group (P<0.05). The diameter of the corpus luteum of the T600 group (15.8+/-1.6 mm) was less than for control (21.0+/-2.5 mm, P<0.01), T200 (19.3+/-2.7 mm, P<0.01) and T400 (20.0+/-2.2 mm) groups (P<0.05). The mean diameter of corpus luteum of T200 was similar to T400 (P>0.05), but different from the control group (P<0.05). In conclusion, the daily intramuscular administration of 200mg or more of progesterone from day 3 to 9 of the estrous cycle indicates that plasma concentrations of progesterone can be used to modify the pattern of follicular development during the follicular wave. From day 5 of the estrous cycle, progesterone concentrations greater than 15 ng/ml (T600 group: 600 mg/day of progesterone from day 3 to 9 of the estrous cycle) inhibit dominant follicle development, increase the class I follicle populations (3-4 mm) and diminish the development of the corpus luteum.

Regulation of mouse embryo development by autocrine throphic factors.

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-F10 culture medium during 24 h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F10, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-F10 medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96 h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CM10 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process.

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Regulation of mouse embryo development by autocrine throphic factors.

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-F10 culture medium during 24 h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F10, CM5 or CM10 (49.66, 53.04, 60.00

respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-F10 medium (80.00

and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96 h of culture (66.96 vs. 52.41

and 70.43 vs. 55.17

, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59

, 67.62 vs. 52.41

and 73.33 vs. 55.17

, respectively). At 48h of culture, differences between CM5, CM10 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process.

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu).

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu)

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu)

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.(AU)

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu).

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.