Reduced embryo yield obtained from superstimulated ewes with low circulating AMH concentration is improved by lengthening the FSH treatment.

The aim of the present study was to evaluate: 1) the association between AMH, AFC, superovulatory response and embryo yield in sheep; and 2) the effect of FSH treatment length during superstimulation of the first follicular wave on ovarian response and embryo yield, particularly in ewes with low and high AMH. The experiment was performed on 63 Polled Dorset ewes that received an ovarian superstimulatory treatment during the first follicular wave (Day 0 protocol). Ewes were administered a total dose of 240 mg of FSH distributed in six (6-dose regimen, n = 30) or eight (8-dose regimen, n = 33) decreasing doses administered 12 h apart. On Day -9 (random stage of the estrous cycle) and Day 0 (day of the first FSH dose) ovarian ultrasonography was performed and blood samples were collected for AFC and AMH determinations, respectively. A weak positive correlation between AMH and small AFC (follicles <4 mm) was observed (r = 0.23; P = 0.07), and AMH concentration was positively correlated (r = 0.29; P < 0.05) with the number of corpora lutea (CL) determined at embryo collection (i.e., 6 d after insemination). The length of FSH treatment tended (P = 0.06) to affect the ovarian response, such that the number of CL was greater in 8-dose than 6-dose treated ewes, while no differences (P > 0.10) in embryo yield outcomes were observed. For further analysis, ewes were classified into low (<7 ng/mL) and high (>10 ng/mL) serum AMH. In high AMH ewes, there were no differences (P > 0.05) in the number of CL nor embryo yield between the 6-dose and 8-dose treatment (e.g., 7.8 ± 2.4 and 8.3 ± 2.5 transferable embryos, respectively; P = 0.92). Conversely, for low AMH ewes, fertilized ova and embryo yield were greater (P ≤ 0.05) for ewes receiving the 8-dose than the 6-dose superstimulatory treatment (e.g., 8.4 ± 2.8 vs. 2.7 ± 0.9 transferable embryos, respectively, P ≤ 0.05). In conclusion, embryo production in poor responding ewes with low low circulating AMH is improved by extending the superstimulatory treatment length from 6 to 8 FSH doses.

The coasting time affects the quality of cumulus-oocyte complexes in superstimulated ewes.

We hypothesized that the coasting time may be beneficial to the quality of cumulus-oocyte complexes recovered from live ewes, as reported in cattle. The present study assessed the effect of coasting times on the quantity and quality of cumulus-oocyte complexes (COCs) in sheep. All ewes were subjected to the "Day 0 protocol", followed by an ovarian stimulation (80 mg of pFSH in three decreasing doses), varying only the coasting time [12 (G12), 36 (G36), or 60 h (G60]. In Experiment 1, data regarding follicular population was assessed. In Experiment 2, the COC quality was checked by their morphology, brilliant cresyl blue (BCB) test, evaluation of chromatin condensation pattern, and oocyte diameter. In Experiment 3, genes related to oocyte developmental competence were evaluated in BCB + COCs. The oocytes in the G60 group had more (P < 0.05) large follicles than the other groups and oocytes with a greater diameter than the G12. Oocyte morphology was similar (P > 0.05) among groups, as well as the BCB + COCs quantity. The G60-oocytes presented a better (P < 0.05) configuration of chromatin condensation compared with the other groups and a greater (P < 0.05) gene expression of BMP15, MATER, ZAR1, and PTGS2 compared with G12, and PTGS2 and HAS2 compared with G36 group. In conclusion, 60 h of coasting time positively affects the quality of COCs recovered after subjecting ewes to the "Day 0 protocol" and ovarian superstimulation. Implementing the appropriate coasting time to a given protocol can positively impact the in vitro embryo production outcomes in sheep.

Ovarian responses and FSH profiles at superovulation with a single epidural administration of gonadotropin in the Thai-Holstein crossbreed.

The conventional method of ovarian superstimulation requires multiple injections of gonadotropins which is time-consuming and may be stressful for the cows. This study was designed to determine whether a single epidural injection of FSH (EI group) would induce the superovulatory response in the Thai-Holstein crossbreed and evaluate FSH plasma hormone concentrations. Eight cows (replication = 3; n=24) were assigned to one of 2 treatments in switch back design. Control group (n=12): cows were received 400 mg FSH twice daily by intramuscularly for 4 days (80, 80, 60, 60, 40, 40, 20 and 20 mg), EI group (n=12): cows were received 400 mg FSH by single epidural injection. Data were collected in term of ovarian follicle responses, superovulatory responses, ova/embryo collection. FSH concentrations were examined using ELISA. The total follicular responses during oestrus were not different between treatments; however, the large follicles were less frequent (P < 0.01) while the medium follicle sizes were higher (P < 0.05) in the EI group. The plasma concentration of FSH in EI was dramatically increased within 2 hours before decreasing sharply thereafter (P < 0.01) and did not remain above baseline after 10 hours of administration. The embryo quality was better in the control than the EI groups (P < 0.05). Interestingly, the number of ovulation cysts in the EI group was 50%. The ovarian responses and embryo quality in the cows with cysts were worse compared with the non-cyst groups (P < 0.05). In conclusion, alternative protocols decreased the superovulatory response and increased poor embryo quality in Thai-Holstein crossbred. Also, the incidence of ovarian follicular cysts is higher in the EI group.

Ovarian responses and FSH profiles at superovulation with a single epidural administration of gonadotropin in the Thai-Holstein crossbreed

Abstract The conventional method of ovarian superstimulation requires multiple injections of gonadotropins which is time-consuming and may be stressful for the cows. This study was designed to determine whether a single epidural injection of FSH (EI group) would induce the superovulatory response in the Thai-Holstein crossbreed and evaluate FSH plasma hormone concentrations. Eight cows (replication = 3; n=24) were assigned to one of 2 treatments in switch back design. Control group (n=12): cows were received 400 mg FSH twice daily by intramuscularly for 4 days (80, 80, 60, 60, 40, 40, 20 and 20 mg), EI group (n=12): cows were received 400 mg FSH by single epidural injection. Data were collected in term of ovarian follicle responses, superovulatory responses, ova/embryo collection. FSH concentrations were examined using ELISA. The total follicular responses during oestrus were not different between treatments; however, the large follicles were less frequent (P < 0.01) while the medium follicle sizes were higher (P < 0.05) in the EI group. The plasma concentration of FSH in EI was dramatically increased within 2 hours before decreasing sharply thereafter (P < 0.01) and did not remain above baseline after 10 hours of administration. The embryo quality was better in the control than the EI groups (P < 0.05). Interestingly, the number of ovulation cysts in the EI group was 50%. The ovarian responses and embryo quality in the cows with cysts were worse compared with the non-cyst groups (P < 0.05). In conclusion, alternative protocols decreased the superovulatory response and increased poor embryo quality in Thai-Holstein crossbred. Also, the incidence of ovarian follicular cysts is higher in the EI group.

Ovarian responses and FSH profiles at superovulation with a single epidural administration of gonadotropin in the Thai-Holstein crossbreed

The conventional method of ovarian superstimulation requires multiple injections of gonadotropins which is time-consuming and may be stressful for the cows. This study was designed to determine whether a single epidural injection of FSH (EI group) would induce the superovulatory response in the Thai-Holstein crossbreed and evaluate FSH plasma hormone concentrations. Eight cows (replication = 3; n=24) were assigned to one of 2 treatments in switch back design. Control group (n=12): cows were received 400 mg FSH twice daily by intramuscularly for 4 days (80, 80, 60, 60, 40, 40, 20 and 20 mg), EI group (n=12): cows were received 400 mg FSH by single epidural injection. Data were collected in term of ovarian follicle responses, superovulatory responses, ova/embryo collection. FSH concentrations were examined using ELISA. The total follicular responses during oestrus were not different between treatments; however, the large follicles were less frequent (P < 0.01) while the medium follicle sizes were higher (P < 0.05) in the EI group. The plasma concentration of FSH in EI was dramatically increased within 2 hours before decreasing sharply thereafter (P < 0.01) and did not remain above baseline after 10 hours of administration. The embryo quality was better in the control than the EI groups (P < 0.05). Interestingly, the number of ovulation cysts in the EI group was 50%. The ovarian responses and embryo quality in the cows with cysts were worse compared with the non-cyst groups (P < 0.05). In conclusion, alternative protocols decreased the superovulatory response and increased poor embryo quality in Thai-Holstein crossbred. Also, the incidence of ovarian follicular cysts is higher in the EI group.(AU)

Development of a GnRH-PGF2α Based Synchronization and Superstimulation Protocol for Fixed-Time Mating in Llama Embryo Donors.

The aim of the present study was to evaluate the application of a GnRH-PGF2α based synchronization and superstimulation protocol for fixed-time natural mating in llama embryo donors. All females (n = 8) received 8 µg IM of GnRH analog (GnRHa; buserelin) on day 0, regardless of follicular status. After eight days, another GnRHa dose was administered followed by 250 µg IM PGF2α (cloprostenol). A dose of 1000 IU IM of equine chorionic gonadotrophin (eCG) was applied on day 12 and a new dose of PGF2α was administered on day 13. All embryo donors were mated with a male of proven fertility followed by a GnRHa dose on day 18. 24 h later, mating was repeated with a different male. Transcervical uterine flushing for embryo recovery was carried out on all females on day 26. Recipient females received one dose of GnRHa (day 0) two days after the first mating of embryo donor females. A 75% (6/8) of embryo donors responded to the superstimulation treatment with a range of 2 to 5 corpus luteums (CLs) on embryo recovery day. A total of 24 CLs were registered, with a mean of 4 ± 0.9 CLs per female. Embryo recovery rate was 66.7% (16/24), with a range of 0 to 4 embryos and a mean of 2.7 ± 1.5 embryos per female. Regarding quality of the recovered embryos, 56.2% were grade I, 6.2% were grade II and 37.5% were grade V (untransferable; arrested morulae). Grade I and II embryos (n = 10) were transcervically transferred into recipient females (n = 10) six days after inducing their ovulation. At 24 days after embryo transfer (ET), a 50% pregnancy rate was registered. In conclusion, a group of llama embryo donors can be synchronized and superstimulated using a fixed-time mating protocol based on GnRHa, PGF2α, and eCG without the necessity of using ultrasonography in the field.

Strategies to increment in vivo and in vitro embryo production and transfer in cattle.

Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and re-synchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.

Strategies to increment in vivo and in vitro embryo production and transfer in cattle

Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and resynchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.(AU)

Strategies to increment in vivo and in vitro embryo production and transfer in cattle

Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and resynchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.

Pattern of follicular development in sheep subjected to ovarian superstimulation after follicular ablation by laparoscopic ovum pick-up / Padrão de desenvolvimento folicular em ovelhas submetidas à superestimulação ovariana após ablação folicular por laparoscopia

The aim of this study was to determine the pattern of follicular development in ewes following ablation of all follicles associated with ovarian superstimulatory treatment. After confirmation of reproductive cyclicity using ultrasonography and progesterone levels, five adult ewes of undetermined breed received a vaginal pessary containing 60mg of medroxyprogesterone, maintained for 10 days, with the application of 100ug cloprostenol on the fourth day. On the tenth day, 300UI equine chorionic gonadotropin (eCG) was applied together with 80mg FSH, and all visible follicles were aspirated by laparoscopic ovum pick-up (LOPU). Following LOPU, ovarian structures were assessed using sonography every 8 hours for a period of 64 hours, and the number and diameter of small (< 2.5mm), medium-sized (2.5-4.5mm), and large (>4.5mm) follicles were recorded. The average diameter of small follicles reduced from 5.6±1.5mm initially to 0.8±1.3mm by 56 hours. Population size of the medium-sized follicles during the observation period followed a parabolic distribution wherein the theoretical maximum size emerged at 34.6 hours with no difference at (P>0.05) 24 (4.6±1.5) and 48 hours (4.8±2.3). It was concluded that in ewes subjected to ovarian superstimulation immediately following LOPU, follicular development is characterized by a new wave of follicle growth with a predominance of medium-sized follicles (2.5-4.5mm) between 24 and 48 hours, and a theoretical population maximum occured at 34.6 hours.(AU)

O objetivo deste estudo foi determinar o padrão do desenvolvimento folicular em ovelhas após a ablação de todos os folículos, associado a tratamento superestimulatório ovariano. Após confirmação da ciclicidade reprodutiva por avaliação ultrassonográfica e dosagem de progesterona, cinco ovelhas adultas, sem raça definida, ao início do tratamento, receberam pessário vaginal contendo 60mg de acetato de medroxiprogesterona. Após quatro dias, foi administrado 100µg cloprostenol sódico e, no décimo dia, realizou-se a remoção do pessário e aspiração de todos os folículos ovarianos visíveis, guiada por laparoscopia, além da administração de 300UI eCG associada a 80mg FSH. Ao final do tratamento, a cada 8 horas, foi realizada avaliação ultrassonográfica das estruturas ovarianas por um período de 64 horas, onde o número e o diâmetro de folículos pequenos ( < 2,5mm), médios (2,5 a 4,5mm) e grandes (>4,5mm) foram contabilizados. O número de folículos pequenos, que inicialmente apresentava média 5,6±1,5, foi reduzido para 0,8±1,3 a partir de 56 horas, folículos grandes somente foram vistos a partir de 32 horas (0,2±0,4); a população de folículos médios, durante o período de observação, apresentou uma distribuição parabólica cujo vértice, considerado o ponto teórico de maior concentração, foi às 34,6 horas, não havendo diferença (P>0,05) nessa população de folículos, entre 24 e 48 horas (4,6±1,5 vs 4,8±2,3). Conclui-se que, em ovelhas submetidas à superestimulação ovariana iniciada imediatamente após remoção de todos os folículos visíveis à laparoscopia, o desenvolvimento folicular é caracterizado pela emergência de nova onda de crescimento, com predomínio de folículos médios (2,5 a 4,5mm) entre 24 e 48 horas, sendo o ponto teórico de maior ocorrência às 34,6 horas.(AU)

Pattern of follicular development in sheep subjected to ovarian superstimulation after follicular ablation by laparoscopic ovum pick-up / Padrão de desenvolvimento folicular em ovelhas submetidas à superestimulação ovariana após ablação folicular por laparoscopia

The aim of this study was to determine the pattern of follicular development in ewes following ablation of all follicles associated with ovarian superstimulatory treatment. After confirmation of reproductive cyclicity using ultrasonography and progesterone levels, five adult ewes of undetermined breed received a vaginal pessary containing 60mg of medroxyprogesterone, maintained for 10 days, with the application of 100ug cloprostenol on the fourth day. On the tenth day, 300UI equine chorionic gonadotropin (eCG) was applied together with 80mg FSH, and all visible follicles were aspirated by laparoscopic ovum pick-up (LOPU). Following LOPU, ovarian structures were assessed using sonography every 8 hours for a period of 64 hours, and the number and diameter of small (4.5mm) follicles were recorded. The average diameter of small follicles reduced from 5.6±1.5mm initially to 0.8±1.3mm by 56 hours. Population size of the medium-sized follicles during the observation period followed a parabolic distribution wherein the theoretical maximum size emerged at 34.6 hours with no difference at (P>0.05) 24 (4.6±1.5) and 48 hours (4.8±2.3). It was concluded that in ewes subjected to ovarian superstimulation immediately following LOPU, follicular development is characterized by a new wave of follicle growth with a predominance of medium-sized follicles (2.5-4.5mm) between 24 and 48 hours, and a theoretical population maximum occured at 34.6 hours.

O objetivo deste estudo foi determinar o padrão do desenvolvimento folicular em ovelhas após a ablação de todos os folículos, associado a tratamento superestimulatório ovariano. Após confirmação da ciclicidade reprodutiva por avaliação ultrassonográfica e dosagem de progesterona, cinco ovelhas adultas, sem raça definida, ao início do tratamento, receberam pessário vaginal contendo 60mg de acetato de medroxiprogesterona. Após quatro dias, foi administrado 100µg cloprostenol sódico e, no décimo dia, realizou-se a remoção do pessário e aspiração de todos os folículos ovarianos visíveis, guiada por laparoscopia, além da administração de 300UI eCG associada a 80mg FSH. Ao final do tratamento, a cada 8 horas, foi realizada avaliação ultrassonográfica das estruturas ovarianas por um período de 64 horas, onde o número e o diâmetro de folículos pequenos ( 4,5mm) foram contabilizados. O número de folículos pequenos, que inicialmente apresentava média 5,6±1,5, foi reduzido para 0,8±1,3 a partir de 56 horas, folículos grandes somente foram vistos a partir de 32 horas (0,2±0,4); a população de folículos médios, durante o período de observação, apresentou uma distribuição parabólica cujo vértice, considerado o ponto teórico de maior concentração, foi às 34,6 horas, não havendo diferença (P>0,05) nessa população de folículos, entre 24 e 48 horas (4,6±1,5 vs 4,8±2,3). Conclui-se que, em ovelhas submetidas à superestimulação ovariana iniciada imediatamente após remoção de todos os folículos visíveis à laparoscopia, o desenvolvimento folicular é caracterizado pela emergência de nova onda de crescimento, com predomínio de folículos médios (2,5 a 4,5mm) entre 24 e 48 horas, sendo o ponto teórico de maior ocorrência às 34,6 horas.

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles<5 mm decreased during superstimulation suggesting that there was no continuous recruitment of small follicles, and the number of follicles>1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment. (AU)

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment.

Prostaglandin receptors (EP2 and EP4) and angiotensin receptor (AGTR2) mRNA expression increases in the oviducts of Nelore cows submitted to ovarian superstimulation.

Many peptides are responsible for the coordination of muscle contraction, secretion and ciliary beating of the oviduct epithelium to allow the transport of gametes and embryos, including vascular endothelial growth factors (VEGF), prostaglandins (PGs), endotelin-1 (ET-1) and angiotensin II (Ang II). The effect of reproductive biotechnologies used to improve embryo yield on oviduct gene expression is poorly understood. Thus, the aim of the present study was to evaluate the effect of ovarian superstimulation on the mRNA expression of the genes encoding the major peptides involved in oviduct contraction in bovine. Therefore, Nelore cows were submitted to P-36 (n=5) or P-36/eCG (n=5) ovarian superstimulatory protocols and a control group of cows was not submitted to any superstimulatory protocol (n=5). The relative expression of VEGF (VEGF, Flk1, Flt1), Ang II (AGTR2, ACE1), ET1 (ET1, ECE1) and PG pathway members (PGES, EP2, EP4, COX1, COX2) was analyzed using real time RT-PCR in each of oviduct segment (infundibulum, ampulla and isthmus). All target genes were expressed in the three segments of the bovine oviduct; however, specific genes were regulated by ovarian superstimulation: EP2 and EP4 receptors mRNA was affected by P-36/eCG protocol, in the ampulla and infundibulum, respectively; and AGTR2 mRNA was up-regulated by both the P-36/eCG and P-36 protocols in the isthmus. The upregulation of EP2, EP4 and AGTR2 expression in the superstimulated cows suggests a suitable effect of FSH and eCG on bovine oviduct physiology, coordinating the contraction in Nelore cows.

Ovarian superstimulation using FSH combined with equine chorionic gonadotropin (eCG) upregulates mRNA-encoding proteins involved with LH receptor intracellular signaling in granulosa cells from Nelore cows.

The LH plays a key role in controlling physiological processes in the ovary acting via LH receptor (LHR). In general, the effects of LHR on the regulation of granulosa cell differentiation are mediated mainly via the Gs-protein/adenylyl cyclase/cAMP system; however, the LHR activation could also induce phospholipase C (PLC)/inositol trisphosphate (IP3) via Gq/11 system. Additionally, the expression of G-proteins (GNAS, GNAQ, and GNA11) and PLC ß has been showed in bovine antral follicle, concomitant with an increase in LHR expression. To gain insight into the effects of superstimulation with FSH (P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the mRNA expression of proteins involved in LHR signaling in bovine granulosa cells, Nelore cows (Bos indicus) were treated with two superstimulatory protocols: P-36 protocol or P-36/eCG protocol (replacement of the FSH by eCG administration on the last day of treatment). Nonsuperstimulated cows were only submitted to estrous synchronization without ovarian superstimulation. The granulosa cells were harvested from follicles and mRNA abundance of GNAS, GNAQ, GNA11, PLCB1, PLCB, PLCB4, and adenylyl cyclase isoforms (ADCY3, ADCY4, ADCY6, ADCY8, and ADCY9) was measured by real-time reserve transcription followed by polymerase chain reaction. No differences on mRNA abundance of target genes were observed in granulosa cells of cows submitted to P-36 protocol compared with control group. However, the cows submitted to P-36/eCG protocol showed upregulation on the mRNA abundance of target genes (except ADCY8) in granulosa cells. Although the P-36 protocol did not regulate mRNA expression of the proteins involved in the signaling mechanisms of the cAMP and IP3 systems, the constant presence of GNAS, GNAQ, GNA11, PLCB1, PLCB3, PLCB4, and adenylyl cyclase isoforms (ADCY3, ADCY4, ADCY6, and ADCY9) mRNA and the upregulation of these genes in granulosa cells from cows submitted to P-36/eCG protocol reinforce the participation of Gq/11/PLC/IP3 signaling as well as Gs-protein/adenylyl cyclase/cAMP system on LHR pathways during bovine granulosa cell differentiation submitted to superstimulatory treatments.

Efficiency of superstimulatory protocol P-36 associated with the administration of eCG and LH in Nelore cows.

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos.

Donor category and seasonal climate associated with embryo production and survival in multiple ovulation and embryo transfer programs in Holstein cattle.

The present study investigated the effect of Holstein donor category (cows vs. heifers) and climate variation (hot vs. cooler season) on the efficiency of in vivo embryo production programs as well as embryo survival after transferred to Holstein recipient cows. A total of 1562 multiple ovulation (MO) procedures (cows: n = 609, and heifers: n = 953) and 4076 embryo transfers (ETs) performed in two dairy herds were evaluated. Donor cows had greater number of CLs (10.6 ± 0.6 vs. 7.5 ± 0.4; P < 0.0001) and ova/embryos recovered (7.6 ± 0.6 vs. 4.6 ± 0.4; P < 0.0001) compared with donor heifers. However, fertilization rate (47.9 vs. 82.4%; P < 0.0001) and proportion of transferable embryos (31.5 vs. 67.4%; P < 0.0001) were lower in donor cows than heifers, respectively. Regardless of donor category, the proportion of freezable embryos was less (P < 0.001) during hot season than in cooler season (21.4 vs. 32.8%). However, greater decline in the proportion of freezable embryos during the hot season was observed in cows (21.7 vs. 10.7%) compared with heifers (46.2 vs. 38.1%; P = 0.01). In contrast, the season on which the embryo was produced (hot or cool) did not affect pregnancy rate on Day 31 (30.5 vs. 31.7%; P = 0.45) and 45 (25.3 vs. 25.1%; P = 0.64) of pregnancy. Regardless of the season in which the embryos were produced, embryonic survival after transferring embryos retrieved from donor cows was greater on Days 31 (36.0 vs. 30.7%; P = 0.001) and 45 (28.3 vs. 23.1%; P = 0.001) of pregnancy when compared with embryos from donor heifers. In conclusion, MO embryo production efficiency decreased during the hot seasons both in cows and heifers; however, the decline was more pronounced in donor cows. Regardless of the embryo source, similar pregnancy rate was observed in the recipient that received embryos produced during the hot and cooler seasons. Curiously, embryos originating from donor cows had higher embryonic survival when transferred to recipient cows than embryos originating from heifers.

Comparação de diferentes técnicas de sincronização da emergência da onda folicular visando a superovulação em bovinos / Comparative technics of follicular wave emergence synchronization in the cattle superovulation

Foram avaliadas quatro técnicas de sincronização da onda folicular em protocolos de superovulação. Para tal, foram utilizadas 112 vacas doadoras, das raças Simental, Limousin e Red Angus, com escore corporal médio de 3,0. Os animais foram divididos aleatoriamente em cinco grupos experimentais de acordo com o método de sincronização da emergência da onda folicular. Foram realizadas 30 superovulações em cada grupo, considerando os seguintes protocolos: GI - grupo controle animais superovulados entre o 8o e o 12o dia do ciclo estral (dia zero = estro); GII animais que sofreram punção folicular no 9o dia (dia 0 = estro) e início do tratamento superovulatório no 11o dia; GIII animais que sofreram punção folicular em fase não conhecida do ciclo estral, associada ao uso de um dispositivo intravaginal contendo progesterona (P4) e tratamento superovulatório iniciado 48h após a punção, GIV animais que utilizaram implante intravaginal de progesterona colocadoem fase aleatória do ciclo estral, mantido por nove dias, associado à administração de 50 mg de P4 e de 2mg de benzoato de estradiol, sendo o tratamento superovulatório iniciado cinco dias após a colocação do dispositivo e GV animais que receberam implante intravaginal de P4 colocado em fase aleatória do ciclo estral e mantido por oito dias, associado à administração de 50mg de P4 e 2mg de 17â-estradiol, sendo o tratamento superovulatório iniciado quatro dias após a colocação do dispositivo. Nos grupos I, II, III, IV e V o total de estruturas coletadas e de embriões viáveis foram, respectivamente (13,53±9,23 vs 13,87 ± 7,85 vs 18,70 ± 10,88 vs 9,03 ± 4,97 vs 13,60 ± 8,39) e (8,43±5,68 vs 8,27 ± 7,06 vs 10,47 ± 8,19 vs 5,37 ± 2,92 vs 7,23 ± 5,30). Os resultados observados no GIII foram superiores ao GI, GII, GIV e GV (P< 0,05), respectivamente. Os grupos I, II e IV não apresentaram diferenças entre si (P> 0,05), enquanto o desempenho de GIV foi inferior (P<0,05). Os resultados permitem concluir que é possível sincronizar a emergência da onda folicular de vacas doadoras, com início da superovulação em qualquer momento do ciclo estral, e que o tratamento progestágeno associado à punção folicular oferece os melhores resultados.(AU)

We evaluated four different techniques of follicular wave synchronization by comparing total number of structures recovery, number of viable and degenerated embryos, number of oocytes recovery and cost of uterine flushing. One hundred twelve Simental, Limousin and Red Angus donators were randomly allocated in five treatment groups according to protocol used for follicular wave emergence synchronization. Thirty superovulations were performed in each group, the programs were: G1- Control, superovulation treatment between days 8 and 12 of the estrous cycle; G2- follicle aspiration on day 9 of the estrous cycle; G3- follicle aspiration plus intravaginal progesterone implant; G4- intravaginal progesterone implant plus estradiol benzoate and G5- intravaginal progesterone implant plus estradiol-17â. Artificial insemination was performed twice, 12 and 24 hours after detection of behavioral estrus. Embryo collection was performed on day 7 after inseminations. Total number of recovered structures (18,70 ± 10,88 vs 13,53 ± 9,23) and viable embryos (10,47 ± 8,19 vs 8,43 ± 5,68) were higher (P< 0,05) in G3 than in G1, respectively. No differences (P>0,05) were detected amongst groups 2, 5 and 1, while performance of G4 was the lowest (P<0,05). Results demonstrate to be possible synchronize follicular wave emergence by initiating superstimulation at any time of the estrous cycle. Additionally we verified that the program using progesterone associated to follicular ablation had the best results in synchronizing follicular wave emergence aiming superstimulation in cattle.(AU)

Comparação de diferentes técnicas de sincronização da emergência da onda folicular visando a superovulação em bovinos / Comparative technics of follicular wave emergence synchronization in the cattle superovulation

Foram avaliadas quatro técnicas de sincronização da onda folicular em protocolos de superovulação. Para tal, foram utilizadas 112 vacas doadoras, das raças Simental, Limousin e Red Angus, com escore corporal médio de 3,0. Os animais foram divididos aleatoriamente em cinco grupos experimentais de acordo com o método de sincronização da emergência da onda folicular. Foram realizadas 30 superovulações em cada grupo, considerando os seguintes protocolos: GI - grupo controle – animais superovulados entre o 8o e o 12o dia do ciclo estral (dia zero = estro); GII – animais que sofreram punção folicular no 9o dia (dia 0 = estro) e início do tratamento superovulatório no 11o dia; GIII – animais que sofreram punção folicular em fase não conhecida do ciclo estral, associada ao uso de um dispositivo intravaginal contendo progesterona (P4) e tratamento superovulatório iniciado 48h após a punção, GIV – animais que utilizaram implante intravaginal de progesterona colocadoem fase aleatória do ciclo estral, mantido por nove dias, associado à administração de 50 mg de P4 e de 2mg de benzoato de estradiol, sendo o tratamento superovulatório iniciado cinco dias após a colocação do dispositivo e GV – animais que receberam implante intravaginal de P4 colocado em fase aleatória do ciclo estral e mantido por oito dias, associado à administração de 50mg de P4 e 2mg de 17â-estradiol, sendo o tratamento superovulatório iniciado quatro dias após a colocação do dispositivo. Nos grupos I, II, III, IV e V o total de estruturas coletadas e de embriões viáveis foram, respectivamente (13,53±9,23 vs 13,87 ± 7,85 vs 18,70 ± 10,88 vs 9,03 ± 4,97 vs 13,60 ± 8,39) e (8,43±5,68 vs 8,27 ± 7,06 vs 10,47 ± 8,19 vs 5,37 ± 2,92 vs 7,23 ± 5,30). Os resultados observados no GIII foram superiores ao GI, GII, GIV e GV (P 0,05), enquanto o desempenho de GIV foi inferior (P<0,05). Os resultados permitem concluir que é possível sincronizar a emergência da onda folicular de vacas doadoras, com início da superovulação em qualquer momento do ciclo estral, e que o tratamento progestágeno associado à punção folicular oferece os melhores resultados.

We evaluated four different techniques of follicular wave synchronization by comparing total number of structures recovery, number of viable and degenerated embryos, number of oocytes recovery and cost of uterine flushing. One hundred twelve Simental, Limousin and Red Angus donators were randomly allocated in five treatment groups according to protocol used for follicular wave emergence synchronization. Thirty superovulations were performed in each group, the programs were: G1- Control, superovulation treatment between days 8 and 12 of the estrous cycle; G2- follicle aspiration on day 9 of the estrous cycle; G3- follicle aspiration plus intravaginal progesterone implant; G4- intravaginal progesterone implant plus estradiol benzoate and G5- intravaginal progesterone implant plus estradiol-17â. Artificial insemination was performed twice, 12 and 24 hours after detection of behavioral estrus. Embryo collection was performed on day 7 after inseminations. Total number of recovered structures (18,70 ± 10,88 vs 13,53 ± 9,23) and viable embryos (10,47 ± 8,19 vs 8,43 ± 5,68) were higher (P0,05) were detected amongst groups 2, 5 and 1, while performance of G4 was the lowest (P<0,05). Results demonstrate to be possible synchronize follicular wave emergence by initiating superstimulation at any time of the estrous cycle. Additionally we verified that the program using progesterone associated to follicular ablation had the best results in synchronizing follicular wave emergence aiming superstimulation in cattle.