Biochemical and steroid concentrations in follicular fluid and blood plasma in different follicular waves of the estrous cycle from normal and superovulated beef cows.

The objectives of the current study were to (i) define the changes in size and number of follicles populations, (ii) determine the follicular fluid (FF) biochemical and steroid concentrations collected from different-sized follicles (5-9 and ≥ 10 mm) and (iii) compare between biochemical and hormonal concentrations of FF with those in blood plasma in relation to the first two follicular waves of the estrous cycle (days 4 and 13) from normal and cows primed for superovulation. After estrus, cows (n=20) were assigned randomly to each of four treatment groups. Group 1: ovariectomy on day 4 (day 0 = ovulation). Group 2: FSH treatment and ovariectomy on day 4. Group 3: dominant follicle ablation (DFA) on day 8 and ovariectomy on day 13. Group 4: DFA on day 8, FSH treatment and ovariectomy on day 13. Blood samples were collected and FF was aspirated and pooled per follicle class within cow to determine glucose, urea, triglycerides, cholesterol, total protein, albumin, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transpeptidase, aspartate aminotransferase, creatin phosphokinase, estradiol-17ß and progesterone concentrations. Follicular class×follicular wave interaction was detected for albumin and lactate dehydrogenase. Results showed that FF concentrations of cholesterol increased from medium to large follicles and decreased for urea and aspartate aminotransferase. Tryglycerides and total protein were greater in the second than in the first follicular wave. FSH treatment decreased FF alkaline phosphatase, E2 and P4 concentrations. Quantitative differences between these fluids are discussed with respect to follicular development.

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.

Effect of estradiol benzoate used at the start of a progestagen treatment on superovulatory response and embryo yield in lactating and non-lactating llamas.

Two experiments were conducted to determine the effect of estradiol benzoate (EB) and intravaginal progestagen treatment on ovarian follicular dynamics and superstimulatory response in eCG-treated llamas. The purpose of Experiment 1 was to evaluate the effect of EB and progestagen treatment starting at different phases of dominant follicle (DF1) development on regression pattern and subsequent follicle wave emergence (WE2) in lactating and non-lactating llamas. Early lactating (n=24, 30+/-4 days postpartum) and non-lactating (n=24) females were assigned in equal numbers (n=8) to one of three groups according to the phase of DF1 (growing, static or regressing) determined by ultrasonography from day -3 to day 0. At day 0, llamas received an intravaginal sponge (MPA, 150 mg) and 5mg of MPA (i.m.). Half of the females (n=4) of each group were injected with 2mg (i.m.) of EB and half were not (control group). A 2 x 2 x 3 (lactational status, EB treatment and follicular phases) factorial design was used. Each sponge was removed 8 days later. Ovaries were monitored from day 0 to day 12. Daily blood samples were taken to determine 17beta-estradiol (E(2)) profiles from day 0 to day 8. The DF1 regression pattern was not affected (P>0.05) by the phase of follicle wave at the start of the treatment or any interactions among main effects. Follicle wave emergence in EB-treated llamas was delayed (P<0.05) by 2.3 days compared with non-treated llamas. Following EB treatment, plasma concentrations of E(2) were greater (P<0.05) from day 1 to day 5 in the treated than in non-treated females, but not from day 6 onward (P>0.05). Experiment 2 was designed to evaluate the effect of this treatment on the ovarian superovulatory response and embryo yield following eCG treatment administered on day of follicular wave emergence as determined in the Experiment 1. The same lactating (n=18, 61+/-4 days postpartum) and non-lactating (n=18) llamas at random stages of follicle wave were treated as those in Experiment 1 and received 1200IU of eCG at the time of WE2 (EB-treated=day 6.5 and non-treated=day 4.5). Llamas were mated 5 days after sponge withdrawal. A second mating was allowed 24h later. Embryos were collected between 7 and 8 days after the first mating and blood samples were taken to determine progesterone (P(4)) concentrations. The mean number of follicles on day of mating and the number of CL on day of embryo collection were not affected by lactational status, EB treatment or their interactions (P>0.05). Ovulation rate and mean (+/-SEM) number of recovered embryos for EB treatment group (67.5% and 2.4+/-0.4) were greater (P<0.05) than for no EB treatment (51.1% and 1.1+/-0.4). Plasma P(4) concentrations and number of CL per llama were correlated (r=0.49; P=0.014). In conclusion, progestagen plus EB treatment facilitates the prediction of the emergence of a new follicular wave approximately 6 days after treatment and resulted in a higher ovulation rate and embryo production in ovarian superstimulated llamas regardless of lactational status.

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.

Fixed-time artificial insemination in red deer (Cervus elaphus) in Argentina.

Synchronization of estrous and fixed-time artificial insemination (FTAI) was conducted during the reproductive season of 2008 (March-April) in a local red deer breeding farm in Argentina. Multiparous suckling hinds (n=38) were artificially inseminated following hormonal treatment (intravaginal sponge containing 100mg of medroxiprogesterone acetate). At the time of sponge removal (day 12) 250 IU of eCG and 500 microg of PGF2alpha were given to each hind. The FTAI was performed at 48-55 h after device removal with cryopreserved semen imported from New Zealand. Rectal-transcervical AI method (similar to that in cattle) was performed and semen was deposited within the uterine body (n=28) or the cervix (n=10). Pregnancy was diagnosed by means of ultrasonography 44 days after FTAI. The overall pregnancy rate was 36.8% (14/38). Percentage of does that became pregnant with intrauterine seminal deposition was 42.9% (12/28) whereas pregnancy rate in the hinds with intracervical AI was 20% (2/10; P=0.27).

Successful transfer of vitrified Ilama (Lama glama) embryos.

The exploitation of the domestic animals species of South American camelids is of great social importance for the native people living in the High Andes. The reproductive physiology of these species is a unique challenge in the development of advanced breeding techniques. At present, the cryopreservation of embryos has not been developed and very few investigations have been conducted. The objective of the present work was to evaluate the in vivo survival of vitrified llama embryos after transfer to recipient females. Donors females were treated with a CIDR-estradiol benzoate-eCG regimen and were mated naturally 6 days after CIDR withdrawal. One ovulatory dose (8 microg) of GnRH was administered immediately after mating. A second mating was allowed 24 h later. Embryo recovery was performed nonsurgically between 8 and 8.5 days after the first mating. Twenty-two ova/embryos were recovered from 12 donor females. Hatched blastocysts were exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol (P/V)) in three steps, and after loading into 0.25 ml straws, were plunged into liquid nitrogen. For embryo transfer, recipients animals were ovulation-synchronized using GnRH administered at the same time as donors. A total of eight vitrified-warmed embryos and 12 fresh embryos were nonsurgically transferred to four and six recipient females, respectively (two embryo per recipient). The pregnancy rates were 50 and 33.3% for recipients that had received vitrified embryos and fresh embryos, respectively. The results demonstrated the effectiveness of this simple vitrification method for cryopreservation of llama embryos.