Transvaginal aspiration, oocyte maturation and production of blastocysts and a pregnancy via ICSI in an endangered breed: Baudet du Poitou donkey.

Two Poitou donkey jennies were presented for clinical oocyte recovery and embryo production via intracytoplasmic sperm injection (ICSI). Both jennies underwent transvaginal ultrasound-guided follicle aspiration on two occasions. Recovered oocytes were held overnight then placed into maturation culture, using standard methods for mare oocytes. On the first replicate for both jennies, the oocytes were divided into two groups; one group was denuded and examined at 30 h culture (standard culture duration for mare oocytes) and the second was denuded and examined at 36 h culture. No oocytes with polar bodies were observed at either time. The oocytes were maintained in maturation culture until 46 h, at which time oocytes with polar bodies were observed. Semen was then prepared; oocytes underwent ICSI approximately 48 h after being placed into maturation culture. On the second replicate for both jennies, oocytes were cultured for maturation for 42 h, then denuded and subjected to ICSI at 46 h. Sperm preparation, injection and embryo culture were performed as for mare oocytes. Blastocyst rates per injected oocyte were 8/19 (42 %) overall, being 4/12 and 4/7 for the first and second TVAs, respectively. Blastocysts were vitrified. Three blastocysts were warmed and transferred to Poitou donkey jenny recipients. One embryonic vesicle was visualized on ultrasonography on embryo Day 12, which increased in size on Day 13 but was not present when examined on Day 14. These results demonstrate that oocyte recovery and ICSI are efficient for production of Poitou donkey blastocysts. To the best of our knowledge, this is the first report of production of blastocysts via ICSI in the Poitou donkey, and the first report of transfer of ICSI-produced embryos in the donkey. Further work is needed on factors affecting pregnancy after embryo transfer in the donkey.

Selecting oocyte donors based on anti-Müllerian hormone (AMH) concentrations: A critical analysis of using cutoff values as exclusion criterion for an in vitro embryo production program in Gir cattle.

The aims of this study were to determine anti-müllerian hormone (AMH) cutoff values for selecting Gir (Bos taurus indicus) oocyte donors and estimate the impact of using AMH concentrations as a selection criterion. In Exp. 1, Gir heifers (n=120) were sampled for AMH analysis and submitted to ovum pick-up and in vitro embryo production (OPU-IVEP). AMH cutoff values were calculated using ROC analysis or, alternatively, by the successive exclusion of heifers with the lowest AMH values. The correlations between AMH and OPU-IVEP outcomes were significant (P<0.001), though low or moderate (r= 0.34-0.52). We estimated an improvement (P<0.05) after the use of AMH cutoff values to select donors of +15.3% for total oocyes, +19.4% for viable COC, and +23.4% for blastocysts. This selection pressure, however, led to the exclusion of 32.8%, 37.9%, and 50.0% of the initial potential donors, respectively. In Exp. 2, we analyzed data from OPU-IVEP sessions of 658 Gir donors with known genomic values for predicted transmitting ability for milk (GPTAm) and age at first calving (GPTAafc). The selection based on the number of oocytes recovered had no effect (P>0.05) on the average GPTAm nor GPTAafc values of the remaining donors. In summary, plasma AMH ≥700 pg/mL is a cutoff value that can be used to select Gir heifers with a greater potential as oocyte donors. Nevertheless, this selection leads to the exclusion of up to 50% of potential donors. Finally, exclusion of poor responders had no effect on mean genomic estimates for milk production or age at first calving in the selected subset of donors.

Successful equine in vitro embryo production by ICSI - effect of season, mares' age, breed, and phase of the estrous cycle on embryo production.

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites.

Effects of donor age and reproductive history on developmental potential of ovum pickup oocytes in Japanese Black cattle (Wagyu).

The objectives of this study were to analyze the (1) effects of donor age and multiparity on development of in vitro fertilization (IVF) embryos after ovum pickup (OPU), (2) effects of repeated and consecutive OPU-IVF procedures on embryo development, and (3) embryo production from OPU-IVF in donors with differing embryo yields after multiple ovulation and embryo transfer technology (MOET) in Japanese Black cattle (Wagyu). Donors were pre-treated with low-dosage follicle-stimulating hormone (FSH; 200 IU total), and oocytes were collected via OPU and fertilized by IVF to generate blastocysts. The number of oocytes collected per OPU session per donor was lower in heifers (2-4 years old, 5.3 oocytes) than in primiparous and pluriparous cows (2-10 years old, 13.6-19.1 oocytes; P < 0.05). Rates of blastocyst development for oocytes from heifers (33.1%) were lower than for those from cows (2-10 years old, 44.1-54.3%; P < 0.05), and average blastocyst yield/OPU/animal was lower in heifers (3.7) than in 5-6 years old cows (10.1; P < 0.05). Donors undergoing five consecutive OPU-IVF sessions after low-dosage FSH showed similar oocyte retrieval (12.2-15.1 oocytes per OPU/animal), blastocyst development rates (35.6-45.0%), and embryo yield/OPU/animal (4.8-5.8; P > 0.05) across sessions. Additionally, embryo yield from OPU-IVF was significantly improved in animals with previous low embryo yield from MOET (5.9 vs. 2.6, respectively, P < 0.05). These results indicate that Wagyu cows with previous births can be more productive as OPU-IVF donors than heifers, and oocytes from donors undergoing to five consecutive OPU-IVF cycles are competent for embryo development without loss of embryo yield/OPU/animal. Moreover, OPU-IVF can be used for embryo production and breeding from all elite Japanese Black cattle, regardless of previous low embryo yield in routine MOET.

Effect of follicular wave synchronization using a progesterone-releasing intravaginal device-based protocol on in vitro embryo production in Bos taurus cows subjected to 14-day intervals ovum pick-up.

Follicular wave synchronization (FWS) before ovum pick-up (OPU) is one of the strategies used to improve the efficiency of in vitro embryo production (IVP). This study aimed to evaluate the effect of FWS on the total follicular number, cumulus-oocyte complex (COC) recovery, and in vitro embryo development in Angus cows (n = 33) subjected to OPU with 14-day intersession intervals. Additionally, it was also evaluated the presence of carryover effects given the short intersession interval used. The experiment was run as a 2-treatment (FWS vs. Control) x 2-period (1 vs. 2) crossover design. Animals in the FWS group received an intravaginal progesterone implant (1gr), estradiol benzoate (2 mg), and D-cloprostenol (150 µg) on day 0 and the OPU was performed on day 5. Control group animals did not receive any hormone treatment. The FWS increased the number of 6-10 mm follicles (P = 0.05), but it decreased the COC recovery rate (P < 0.01). The FWS did not affect the total or frozen embryo numbers (P = 0.49 and P = 0.17; respectively), but it increased the total blastocyst cell number (P < 0.01). A carryover effect was found on the total and < 6 mm follicles number (P = 0.10 and P < 0.01; respectively), and on the regular, atretic, viable, and total number of COC (P = 0.01, P = 0.08, P = 0.02 and P < 0.01; respectively). We concluded that the FWS increased the quality of embryos after OPU with 14-day intersession intervals in Angus cows and that this kind of OPU/IVP scheme enabled the existence of a carryover effect, especially on the follicle number and COC morphology.

Oocyte recovery after overnight ovary transport provides an alternative source of cumulus oocyte complexes that are competent to produce live piglets.

COVID-19 impacted abattoirs worldwide. The processing lines became a hotspot for the spread of COVID-19 resulting in plant restructuring and ultimately a critical loss of pig material for research. Commercial sources of pig oocytes are available but are costly and companies were already operating at a maximum capacity for supplying the oocyte needs around the United States. Here, we provide an alternative source of oocytes that are competent to produce live, healthy piglets.

Influence of endometritis on the follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle.

Uterine infections often lead to culling of valuable animals from a herd, resulting in genetic drain. The genetic potential of problematic females could be harvested by in-vitro embryo production. The objective of this study was to evaluate the effect of clinical endometritis on follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. The B-mode ultrasonography was performed to examine the uterus for the presence of pus. Based on the history and reproductive examination of cows, a total of twelve (n = 12) Sahiwal cattle were selected for the experiment: (1) healthy group (n = 6) and (2) clinical endometritis group (n = 6). The 1st ovum pick-up (OPU) was conducted on day 165 postpartum. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33,342. The results revealed that the number of medium-sized follicle (1.3 ± 0.1 versus 0.6 ± 0.1) and total number of follicles (9.1 ± 0.7 versus 6.6 ± 0.7) were higher (p < .05) in the healthy group as compared to clinical endometritis group, respectively. Similarly, the number of oocytes recovered (5.0 ± 0.4 versus 2.8 ± 0.4), oocytes with grade A, B and C (2.9 ± 0.3 versus 1.5 ± 0.3), proportion of oocytes with grade A or B (33 ± 0.0 versus 20 ± 0.1) and nuclear maturation (68 ± 0.1 versus 55 ± 0.1) were also higher (p < .05) in the healthy group as compared to clinical endometritis group, respectively. Perhaps, cleavage rate (55.1 ± 0.1 versus 46.2 ± 0.1) and blastocyst rate (29.7 ± 0.0 versus 26.3 ± 0.1) did not differ (p > .05) between the groups. Likewise, the expression level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in immature oocytes did not differ (p > .05) between both the groups. In conclusion, clinical endometritis has a negative effect on follicular dynamics, oocyte recovery, oocyte quality and nuclear maturation; furthermore, the developmental competence of COCs is not compromised by it.

Transvaginal ultrasound-guided oocyte retrieval in cattle: State-of-the-art and its impact on the in vitro fertilization embryo production outcome.

Transvaginal ultrasound-guided oocyte retrieval (commonly called OPU) and in vitro embryo production (IVP) in cattle has shown significant progress in recent years, in part, as a result of a better understanding of the full potential of these tools by end users. The combination of OPU and IVP (OPU-IVP) has been successfully and widely commercially used worldwide. The main advantages are a greater number of embryos and pregnancies per unit of time, faster genetic progress due to donor quick turn around and more elite sires mating combinations, larger spectrum of female age (calves, prepuberal, heifer, cow) and condition (open, pregnant) from which to retrieve oocytes, a reduced number of sperm (even sexed) required to fertilize the oocytes, among other benefits. OPU-IVP requires significant less donor preparation in comparison to conventional embryo transfer (<50% of usual FSH injections needed) to the extent of no stimulating hormones (FSH) are necessary. Donor synchronization, stimulation, OPU technique, oocyte competence, embryo performance, and its impact on cryopreservation and pregnancy are discussed.

Effects of short-term dietary supplementation on the number of ovarian follicles, quantity and quality of oocytes, and in vitro embryo production in Japanese Black cows.

This study aimed to test the hypothesis that short-term supplementation with a high-energy diet promotes embryo production following ovum pick-up (OPU) in Japanese Black cows. After a period of adaptation to the maintenance diet, a 200% maintenance diet was fed to the high-energy diet group (HD group, n = 6) for four weeks, and a maintenance diet was fed to the other group (MD group, n = 6). OPU-in vitro fertilization (IVF) procedures were performed on days 14, 21, and 28; follicles and oocytes were counted and morphologically graded, and cultivable oocytes were cultured for in vitro maturation, fertilization, and culture. The mean plasma insulin concentrations on days 14 and 21 were significantly higher in the HD group than in the MD group (P < 0.05). The number of follicles observed at OPU, recovered oocytes, cultivable (Grades 1 to 4) oocytes, and the rate of degenerated (Grade 6) oocytes in the HD group were significantly higher than those in the MD group (P < 0.05). The proportion of cleaved oocytes was lower in the HD group than in the MD group (P < 0.05); consequently, there was no significant difference in the number of blastocysts obtained between the HD and MD groups. The present findings suggest that high-energy diets can promote follicular growth in parallel with an increase in plasma concentrations of insulin, but have a detrimental effect on the quality of oocytes with the OPU-IVF procedure in Japanese Black cows.

Influence of ovarian follicular wave synchronization and single-dose eCG superstimulation on oocyte collection and in vitro embryo production in bison during the ovulatory and anovulatory seasons.

In an effort to develop an effective, minimum-handling protocol for the conservation of wood bison, the present study was designed to determine the effects of ovarian synchronization and superstimulation on cumulus oocyte complex (COC) collection and in vitro embryo production in wood bison during the ovulatory (Exp. 1) and anovulatory seasons (Exp. 2). We tested the hypotheses that COC collection and in vitro embryo production are 1) greater after follicular wave synchronization than at random stages of the follicular wave, 2) repeatable within individuals, 3) greater after ovarian superstimulation with a single dose of eCG than without treatment, and 4) greater during the anovulatory season than the ovulatory season. In Exp. 1, ultrasound-guided COC collection was performed on Day -1 in wood bison to induce follicular wave emergence the following day (Day = 0). Immediately after the COC collection on Day -1, bison were given a single im dose of 2500 IU eCG or saline (n = 6 per group). Subsequent COC collections were on Days 4 and 9. A similar design was used in Exp. 2, with an additional treatment group given 5000 IU eCG (n = 8 per group). In Exp. 1, compared to the saline-treated group, a single dose of 2500 IU eCG resulted in a greater number of ≥8 mm follicles at the time of the Day 4 COC collection (P = 0.03), but not at the Day 9. In Exp. 2, treatment with 5000 IU eCG resulted in a greater number of ≥8 mm follicles than 2500 IU eCG or the saline treatment (37.5 ± 6.9, 17.5 ± 2.0, 16.9 ± 2.0; P = 0.01, respectively). Although the number of embryos produced/COC submitted to IVM was not different among groups (mean = 18.6%), treatment with 5000 IU eCG produced more than twice as many embryos per bison as unstimulated bison (0.8 vs 1.9). In summary, embryo production rates were higher from COC collected subsequent to follicular wave synchronization vs random stages of the wave, and ovarian superstimulation with eCG resulted in a dose-related increase in the number of ≥8 mm follicles, COC collected, and embryos produced. Repeated COC collections after successive wave synchronization resulted in similar follicular counts and embryo production rates within individuals, and the greatest number of follicles aspirated, COC collected, and embryos produced was in the anovulatory season. We conclude that the minimum-handling COC collection protocols in the present study are effective and provide realistic options for embryo production in wild bison.

Predictors of the ovarian superstimulatory response and oocyte collection in prepubertal heifers.

The objectives were to investigate the relationships between antral follicle counts and plasma AMH and FSH at the time of follicular wave emergence in prepubertal calves, and to determine the effects of age and duration of gonadotropin treatment on the ovarian superstimulatory response in pre- and post-pubertal heifers. Hereford crossbred prepubertal (Replicate 1 and 2, n = 20) and post-pubertal heifers (Replicates 1, n = 8; Replicate 2, n = 8) were assigned randomly to 2 treatment groups and given FSH for either 4 or 7 d (25 mg pFSH im at 12-h intervals). Prepubertal heifers were first treated at 4 mo and again at 7 mo of age. Blood samples were collected immediately before the first FSH administration, that was initiated 36 h after follicular ablation. An LH treatment (12.5 mg im) was given 12 h after the last FSH injection. Follicular fluid and cumulus-oocyte complexes (COC) were collected 24 h after LH treatment. At wave emergence, the number of follicles ≥1 mm (AFC, 31.1 ± 4.0 vs 16.2 ± 1.8; P < 0.001) and the plasma concentrations of AMH (606.4 ± 90.5 vs 279.6 ± 28.3 pg/mL; P = 0.001) were higher at 4 than at 7 mo of age, while plasma FSH concentrations did not differ between ages. At oocyte collection, a higher number of follicles ≥6 mm were observed in prepubertal calves at 4 mo of age and post-pubertal heifers than in calves at 7 mo of age (32.4 ± 5.4 and 22.0 ± 2.3 vs 14.9 ± 2.0, respectively; P = 0.003). Intrafollicular concentrations of estradiol were lower (23.7 ± 4.5 vs 144.0 ± 29.5 ng/mL; P < 0.0001) and of progesterone tended to be higher (217.5 ± 29.3 vs 157.0 ± 33.9 ng/mL; P = 0.07) in the 7- than in the 4-d groups. A greater number of COC was collected from calves at 4 mo of age and heifers than the 7-mo-old calves (13.4 ± 2.6 and 6.0 ± 1.0 vs 5.8 ± 1.1, respectively; P = 0.008). Overall, the 7-d FSH treatment tended to result in a greater proportion of expanded COC than the 4-d treatment in calves (50.1 ± 7.7 vs 31.9 ± 6.8%; P = 0.07). In summary, there was a positive relationship between AFC and plasma AMH concentrations at the time of wave emergence. A higher AFC was observed in calves at 4- than 7-mo of age, which resulted in greater ovarian response to gonadotropin treatment. Following an exogenous LH stimulus, COC maturation rates were greater in the 7-d than in the 4-d FSH treatment groups, resulting in collection of a higher proportion of fully expanded COC.

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes in vitro.

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.

Exogenous progesterone-dependent modulation in the follicular dynamics of Bos indicus cattle undergoing repeated ovum pick-up sessions.

The objective of the present study was to evaluate the role of serum progesterone (P4) in follicular dynamics, oocytes' recovery and quality and their in vitro developmental competence during consecutive ovum pick-up (OPU) sessions in Bos indicus dairy cows. Wave-synchronized Sahiwal cattle (n = 20) were randomly divided into treatment (n = 10) and control (n = 10) groups. CIDR was used as a source of external progesterone in the treatment group. Four consecutive OPU sessions at 96-hr intervals were conducted and repeated ultrasonography at 12-hr intervals was done to monitor follicular dynamics. The viable oocytes were processed for IVC following IVM and IVF until day 7. The serum P4 concentrations in the P4 and control groups were recorded as 2.31 ± 0.059 versus.0.32 ± 0.065 ng/ml, respectively (p < .05). In the treatment group, the total number of recorded follicles was higher (p < .05; 12.05 ± 0.37 versus. 10.87 ± 0.40), whilst the growth rate (mm/day) of follicles was lower (p > .05). Per session recovered oocytes (5.31 ± 0.19 versus. 3.58 ± 0.21; p < .0001) and recovery rate (54.23 versus. 42.53%; p < .05) were also higher in the treatment group compared to control. Similarly, the viable oocytes (4.54 ± 0.187 versus. 3.06 ± 0.199) and the number of grade I and II oocytes per session (3.37 ± 0.196 versus. 2.06 ± 0.21) were higher (p < .05) in the treatment group compared with the control group. However, the nuclear maturation, cleavage, and blastocyst rate did not differ (p > .05) between the groups. Taken together, during OPU sessions, serum P4 improves oocytes' recovery and quality, whilst does not affect the in vitro developmental competence of recovered oocytes.

Intrafollicular oocyte transfer (IFOT): Potential feasibility in the ovine species.

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 µL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

Antral follicle count, oocyte production and embryonic developmental competence of senescent Nellore (Bos indicus) cows.

Information on the follicular population and oocyte quality of cows in the final period of reproductive life is scarce. The present study aimed to compare the antral follicle count (AFC), oocyte production and embryonic developmental competence of young versus long-lived and senescent Bos indicus beef cows. Nellore cows (Bos indicus) were classified into three groups according to age: young (4-9 years, n = 10), long-lived (14-17 years, n = 10) and senescent (17-23 years, n = 10). At a random time in the estrus cycle, the cows received cloprostenol sodium salt (0.5 mg, IM), estradiol benzoate (1 mg, IM) and an intravaginal P4 device (1.4 g). Five days later, the P4 devise was removed and oocyte collection (OPU1) was performed. A second OPU (OPU2) was performed 5 days after the first in order to aspirate only growing follicles. During each OPU, AFC and the number and quality of cumulus-oocyte complexes (COCs) were evaluated. Then, the COCs were placed in standard maturation medium (IVM), fertilized and incubated for 9 days. The data were subjected to ANOVA and Multinomial Logistic Regression. The AFC was smaller in long-lived and senescent cows in both OPU1 and OPU2 when compared to younger cows. There was no difference in AFC between OPU1 (19.9 ± 1.8) and OPU2 (17.6 ± 1.9) in young cows, however, more follicles were punctured in long-lived and senescent cows in OPU1 (12.0 ± 2.6 and 19.3 ± 4.6) than in OPU2 (9.2 ± 1.9 and 10.3 ± 2.3), respectively (P < 0.01). The numbers of COCs recovered from young cows (OPU1 = 14.2 ± 1.8; OPU2 = 8.4 ± 0.9) were higher than those obtained from long-lived cows (OPU1 = 5.9 ± 2.3; OPU2 = 4.3 ± 1.0) and senescent cows (OPU1 = 7.2 ± 3.0; OPU2 = 4.1 ± 1.7), respectively (P < 0.05). The cleavage rate did not differ between groups. However, the rate of blastocyst formation was higher for young (64.8%) and long-lived (65.0%) compared to senescent (16.5%) cows (P < 0.01). In conclusion our results indicate that the AFC is lower in long-lived and senescent cows compared with young cows. However, unlike in senescent cows, the embryonic development of long-lived cows is similar to that of young cows. This suggests that Nellore cows aged >17 years begin to have reduced embryonic development capacity due to ovarian aging.

Improvement in early antral follicle development and gene expression modulation prior to follicle aspiration in bovine cumulus-oocyte complexes by equine chorionic gonadotropin.

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at -80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0-5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

Reproductive seasonality influences oocyte retrieval and embryonic competence but not uterine receptivity in buffaloes.

Since buffaloes are a seasonal, polyestrous species, optimizing reproduction during the non-breeding season is a key factor in increasing the reproductive and productive efficiency of herds. Ovum pick-up associated with in vitro embryo production and embryo cryopreservation is an alternative to reduce seasonal impacts. We studied the effects of seasonality in buffalo oocyte donors and embryo recipients during the favorable and non-favorable breeding seasons. Donors were evaluated for oocyte recovery and blastocyst production rate as dFBS (donors in favorable breeding season) or dNBS (donors in non-favorable breeding season). Embryos produced from dFBS or dNBS were cryopreserved by vitrification or the slow-freeze method for direct transfer and transferred to recipients in the favorable (rFBS) or non-favorable breeding season (rNBS). The heifers or cows were subjected to a fixed-time embryo transfer protocol and conception rates were determined on day 30 and on day 60. The oocyte recovery was lower in dFBS than in dNBS (7.6 vs. 10.0 oocyte/OPU, p = 0.0262); while no difference was found comparing blastocyst production rate (23.7% vs. 30.9% of blastocysts, respectively). Embryos from dFBS resulted in greater (p = 0.0013) conception rates on day 30 compared to dNBS (46.5% vs. 22.4%, respectively), despite the breeding season. The rFBS and rNBS treatments had similar (p = 0.6714) conception rates on day 30 (38.0% vs. 33.0%, respectively), indicating similar uterine receptivity. However, heifers on FBS had higher (p = 0.0003) conception rates on day 30 than cows (73.9% vs. 13.3%, respectively) when receiving embryos from dFBS. Vitrification and direct transfer had similar (p = 0.1698) conception rates on day 30 (30.4% vs. 41.4%, respectively). In conclusion, in vitro-produced embryos derived from dFBS were more competent in establishing pregnancy than dNBS counterparts, independent of recipients' reproductive seasonality. Heifers achieved better conception rates than cows during the favorable breeding season when the embryo came from dFBS. Cryopreserved in vitro produced embryos represent a reliable alternative to reduce seasonal variations in buffalo reproduction. The data elucidate the seasonal effects on embryo competence and on recipients' uterine receptivity, affording new strategies to implement ovum pick-up associated with in vitro embryo production programs in buffalo herds.

Recovery of Equine Oocytes in Ambulatory Practice and Potential Complications.

Field collection of oocytes in mares using transvaginal follicular aspiration (TVA) for embryo production has the potential to revolutionate the equine industry. Protocols for TVA in specialized laboratory settings have been described in the scientific literature since the early 1980s. The objective of this study was to determine the success rate of TVA oocytes recovery under ambulatory conditions. A secondary goal of this study was to determine if TVA is associated with any health complications when performed by recently trained practitioners in the field. Follicles (n = 296) from 66 adult clinically healthy mares were aspirated over a period of 6 days. TVAs were performed by 22 veterinarians with 5-20 years of experience in equine and bovine reproductive medicine, but no previous experience in TVA. Oocytes (n = 145) were recovered. No short- or long-term systemic or local complications were observed following TVA in any of the mares used in this study. Fifty-six out of 66 mares became pregnant within 3 months following TVA. This study shows that with proper training, TVA can be successfully used to obtain equine oocytes with no health complications under field conditions in nonspecialized laboratory settings.

Effect of photoperiodicity and methods of follicular wave emergence on follicle turn-over, recovery and quality of oocytes, and early in-vitro developmental competence of embryos using ovum pick-up in Nili-Ravi buffaloes: Preliminary evidence.

The objectives of the present study were to evaluate the effects of photoperiodicity, gauge (G) of ovum pick-up (OPU) needle, and two methods of follicular wave emergence on follicular turn-over, oocyte recovery (OR), quality of the oocytes (OQ), and early in-vitro developmental competence of embryos in Nili-Ravi buffaloes (n = 20). In 1st experiment, buffaloes (n = 12; 4 buffaloes/season) were randomly assigned to optimize the OPU's (n) either with 17 G or 18 G needle in one of the following seasons: 1) peak breeding season (PBS; Sep-Nov; n = 31), 2) transition breeding season (TBS; Dec-Feb; n = 32), and 3) low breeding season (LBS; Apr-June; n = 32). During 2nd experiment, buffaloes (n = 8) were enrolled randomly in a 2 × 2 cross-over design to compare the two methods of wave emergence either using follicular ablation (FA; n = 4), or synchronization protocol (CIDR-EB; n = 4) during PBS. In FA method, the ovarian follicles were aspirated (week -1), and subsequently repeated OPU's (n = 55) were performed for 7 weeks. However, in CIDR-EB synchronized buffaloes, a progesterone device (CIDR) was inserted in the anterior vagina and a single dose of estradiol benzoate (2 mg) and prostaglandin (150 µg) were administered i.m. on d 1. The CIDR was removed on d 7 and repeated OPU's (n = 56) were performed. In both experiments, a 7-d resting period was provided between each OPU session. Data on the follicular turn-over, OR, OQ, and early stages of embryonic development were analyzed with mixed models using the PROC MIXED and GLIMMIX procedures of SAS. Results revealed that the number of medium sized follicles (LSM ± SEM) and rate of OR (%) were greater (P < 0.05) during PBS (2.76 ± 0.40; 61%) and TBS (1.73 ± 0.30; 54%) as compared to LBS (0.68 ± 0.30; 31%) in buffaloes, respectively. The OR rate was also greater (P < 0.05) using 17 G as opposed to 18 G needle (62% vs. 35%), however, there was no effect (P > 0.05) of season and G of needle on OQ in buffaloes. In experiment 2, the number of small (3.50 ± 0.63 vs.2.69 ± 0.60) sized follicles was higher (P < 0.05) in FA, whereas, medium (1.13 ± 0.45 vs. 1.59 ± 0.45) sized follicles were greater (P < 0.05) in CIDR-EB synchronized buffaloes. The OR rate (67% vs. 53%), and OQ (Grade I_+_II; 15% vs. 16% and Grade III_+_IV; 85% vs. 84%), remained similar (P > 0.05) in FA and CIDR-EB treated buffaloes, respectively. Similarly, rates of in-vitro maturation (66% vs. 59%), cleavage (42% vs. 53%), 4-cell (27% vs. 32%), 8-cell staged embryos (23% vs. 25%), and morula (13% vs. 8%) did not differ (P > 0.05) between FA and CIDR-EB methods of follicular wave emergence, respectively. Taken together, it is concluded that peak or transition breeding seasons are suitable to perform OPU using 17 G needle for maximum follicular turn-over, and OR in buffaloes. However, the two fundamental methods of synchronization of wave emergence resulted in similar efficiency for OPU derived in-vitro embryo production in Nili-Ravi buffaloes.