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The aim of this study was to evaluate the superestimulatory and superovulatory responses of cattle treated with corifollitropin-alpha, a long-acting human recombinant FSH (rhFSH). In the first and second experiments, we used Nelore (Bos indicus) heifers previously submitted to follicular wave suppression by active immunization against GnRH. In Experiment 1 (a dose-response study), heifers (n = 20) were randomly allocated into five groups, which received placebo (saline) or a single sc dose of 7.5, 15.0, 22.5 or 30.0 μg rhFSH. The heifers were subjected to daily ovarian scan and blood sampling during 11 days. We observed group, time, and group x time effects (P< 0.0001) for both average follicle size and circulating FSH concentrations, with a strong correlation (R = 0.82, P< 0.0001) between the area under curve (AUC) for both parameters. The peak concentration of FSH 24h after treatment and average follicle size at all timepoints, however, were similar (P> 0.05) between groups 22.5 and 30.0 μg. In Experiment 2, heifers (n = 18) were allocated into three groups, which received (0h) either placebo (control), 25 μg rhFSH or 130 mg pFSH (Folltropin). There was no difference (P> 0.05) in average follicle size at any moment, as well as in intrafollicular E2 at 120h or in plasma P4 seven days later between groups rhFSH and pFSH. In Experiment 3, cycling Nelore heifers (n = 20) were subjected to a wave synchronization protocol and superovulated (day 0) using a standard pFSH protocol (120 mg split in eight decreasing im doses) or with a single sc injection of 20 μg rhFSH. The number of follicles >7 mm on day 4 did not differ (P= 0.4370). Heifers receiving rhFSH had greater average follicle size on day 4 (P= 0.0005), ovulation rate (P< 0.0001), and number of CL (P= 0.0155), as well as a trend towards a greater number of ova (P= 0.07) and viable embryos (P= 0.0590). In Experiment 4, superovulation was induced with a single sc injection of 25 μg rhFSH in Girolando and Nelore cows and heifers (n = 20). None of the embryo yield endpoints differed between the two breeds (P> 0.05). In conclusion, cattle superstimulation and superovulation can be successfully induced with a single dose of a long-acting rhFSH (corifollitropin-alpha). • Cattle respond to superstimulation with corifollitropin-alpha in a dose-dependent manner. • Ovarian superovulation can be induced with 20–25 mcg of corifollitropin-alpha. • Corifollitropin-alpha is detected in circulating serum up to 11 days after treatment. [ABSTRACT FROM AUTHOR]

The study was planned to evaluate the effect of two superstimulation protocols (2-Day vs. 3-Day) on in vivo maturation of oocytes in buffaloes. Buffaloes (n=8) were superstimulated twice using 2x2 design at an interval of 30-35 days using 200 mg and 300 mg FSH divided into four and six doses (each dose of 50 mg) given over 2 and 3 days at 12 hourly intervals, respectively. Prostaglandin F2a analogue administered (500 µg Cloprostenol; i/m) at 72 and 84 hrs after start of superstimulatory treatment. Oocytes were retrieved from >6 mm follicles size at 48h after the last FSH injection by ovum pick up. Oocyte maturation was assessed by the degree of cumulus cell expansion and by Orcein staining of denuded oocytes. OPU of 60 and 89 follicles resulted in recovery of 38 and 50 cumulus oocyte complexes (COCs) in 2-Day and 3-Day group, respectively. Significantly higher proportion of expanded oocytes was recovered in 3-Day protocol compared to 2-Day protocol (36.0% vs. 13.2%) and higher number of compact COCs were recovered in 2-Day protocol. In 3-Day protocol, significantly higher percentage of oocytes retrieved was at Metaphase-II stage compared to 2-Day protocol (40% vs. 15.8%, respectively). The preliminary trial demonstrated that 3-day superstimulatory protocol yielded higher percentage of in vivo matured oocytes than 2-Day protocol in buffaloes. [ABSTRACT FROM AUTHOR]

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. • Ewes were subjected to an ovarian stimulation treatment varying only the coasting time (12, 36, or 60 h). • The G60-oocytes had more large follicles and oocytes with a larger diameter. • The G60-oocytes showed a better configuration of chromatin condensation pattern. • The G60-oocytes had a greater expression of BMP15, MATER, ZAR1, PTGS2, and HAS2. • 60 h of coasting positively affects the quality of COCs from superstimulated ewes. [ABSTRACT FROM AUTHOR]

In bovine in vitro embryo production (IVP), FSH has been used to improve the quality and developmental capacity of oocytes to reach the blastocyst stage. The objective of this study was to evaluate in Bos taurus indicus heifers the effect of FSH incorporated in Pluronic® F127 on the quality of oocytes collected by follicular aspiration. A total of 30 cyclic Brahman heifers were selected and assigned into one of three experimental groups (control group, FSH-SC and FSH-PF127). Heifers in the FSH-SC group received a single dose of 50 mg FSH subcutaneously and heifers in the FSH-PF127 group were treated with 50 mg FSH incorporated in Pluronic® F127. The rate of viable oocytes was higher for the FSH-PF127 group compared to the control and FSH-SC groups. The administration of a single dose of 50 mg of FSH in Pluronic® F127 influenced the oocyte quality and the rate of viable oocytes of Brahman breed heifers. [ABSTRACT FROM AUTHOR]

In bovine in vitro embryo production (IVP), FSH has been used to improve the quality and developmental capacity of oocytes to reach the blastocyst stage. The objective of this study was to evaluate in Bos taurus indicus heifers the effect of FSH incorporated in Pluronic® F127 on the quality of oocytes collected by follicular aspiration. A total of 30 cyclic Brahman heifers were selected and assigned into one of three experimental groups (control group, FSH-SC and FSH-PF127). Heifers in the FSH-SC group received a single dose of 50 mg FSH subcutaneously and heifers in the FSH-PF127 group were treated with 50 mg FSH incorporated in Pluronic® F127. The rate of viable oocytes was higher for the FSH-PF127 group compared to the control and FSH-SC groups. The administration of a single dose of 50 mg of FSH in Pluronic® F127 influenced the oocyte quality and the rate of viable oocytes of Brahman breed heifers.

The aim of this study was to compare the efficacy of two doses (700 and 1000 IU) of the native eCG (N-eCG) and a synthetic eCG like glycoprotein (S-eCG) on ovarian superstimulation and embryo production in llamas. Females were examined daily by transrectal ultrasonography until a follicle ≥ 7 mm was observed and a GnRH analogue was injected (i.v.) (Day 0). On Day 3, transrectal ultrasonography was performed to confirm the absence of follicles > 5 mm and animals were randomly divided into four groups (n=10/group): N-eCG700, N-eCG1000, S-eCG700 and S-eCG1000 which received 700 or 1000 IU (i.m.) of N-eCG or S-eCG, accordingly. On Day 7, 112.5 μg of cloprostenol (i.m.) was injected in all llamas. On Day 10, the number of follicles ≥7 mm, mean diameter and their vascularization were determined by ultrasonography. Then, llamas were injected with a GnRH analogue and mated. Natural mating was repeated 24 h later. On Day 18, the number of corpora lutea (CL), their diameter and vascularization were assessed and embryo recovery was performed by uterine flushing. On Day 10, there were no significant differences in the number of follicles ≥ 7 mm, neither in their diameter or vascularization between groups. Similarly, no significant differences were observed in the number of CL on Day 18, neither in their diameter or vascularization between groups. In conclusion, the results of the present study indicate that S-eCG produced by recombinant technology has the same biological activity as N-eCG in llamas. • Embryo recovery rate was similar after native eCG or synthetic eCG treatment. • Superstimulatory response was similar after native eCG or synthetic eCG treatment. • No significant differences between doses of synthetic eCG were observed. [ABSTRACT FROM AUTHOR]

The effect of heat stress with melatonin treatment on the superovulatory responses and embryo characteristics in Thai-Holstein crossbreeds under heat stress conditions was examined. Six non-lactating cows (replication = 4; n = 24) were assigned to one of 2 treatments in double cross-over design. All cows were superstimulated with traditional treatment. Melatonin group (n = 12): cows received intramuscularly injection 18 mg/50 kg. simultaneously with GnRH injection, while those in the control group (n = 12) received none. Bloods samples were taken to determine lipid peroxidation (MDA) and the activity of the antioxidant enzymes (superoxide dismutase; SOD). The experiment was conducted from April to September, which determined severe heat stress (the mean temperature-humidity index above 77). The results revealed that numbers of large follicles and corpora lutea were higher in the melatonin group than in the control group (p < 0.01). Numbers of recovered ova/embryos, fertilized ova, and transferable embryos were higher in the melatonin group (p < 0.01); meanwhile, more degenerated embryos were found in the control group (p < 0.01). Increased activity of the antioxidant enzymes SOD after melatonin administration decreased MDA levels (p < 0.05). In summary, melatonin administration benefited the ovarian response and embryo quality in superstimulated Thai-Holstein crossbreed affected by heat stress.

The present study investigated the effect of superstimulation to improve in vitro embryo production in the Gulf area, where the temperature is high. Holstein cows were classified into the control and superstimulation groups. Superstimulation was induced with a single intramuscular injection of pregnant mare serum gonadotropin (PMSG; 2500 IU) on day 14 of the estrus cycle (day 0; estrus). The development of follicles was evaluated by ultrasonography of the ovaries daily. At 40 h after the PMSG injection, oocytes were collected by the ovum pick-up (OPU) technique. OPU was performed at the same stage of the estrus cycle in the control group as in the superstimulation group. The number of follicles with a diameter of more than 6 mm and the number of retrieved cumulus-oocyte complexes were significantly higher in the superstimulation group than in the control group. Furthermore, the maturation rate was higher in the superstimulation group than in the control group. Cloned embryos were produced by somatic cell nuclear transfer using matured oocytes. The cleavage and blastocyst formation rates were significantly higher in the superstimulation group than in the control group. In conclusion, a single injection of PMSG can facilitate the efficient production of cloned cow embryos.

The conventional follicle‐stimulating hormone (FSH) treatment for bovine superstimulation involves multiple intramuscular injections, which is stressful for animals and onerous. We herein investigated whether a single epidural injection of porcine FSH (pFSH) can induce superovulation and peripheral concentrations of pFSH and steroid hormones after the treatment in Holstein dry cows. We intramuscularly administered pFSH twice daily to three cows for 3 days (control) or a single epidural pFSH administration (epidural). Numbers of follicles (≥10 mm in diameter) at estrus and corpora lutea at luteal phase were counted by ultrasonography. Blood was sampled from 0 to 104 h after the first pFSH administration and plasma pFSH, progesterone, androstenedione, testosterone, and estradiol‐17β concentrations were measured. Numbers of follicles (control: 18.3 ± 7.5, epidural: 15.7 ± 4.0; mean ± SD) and corpora lutea (control: 7.3 ± 4.2, epidural: 8.0 ± 2.6) were similar between both treatments. Plasma pFSH concentrations were higher in epidural than in control (p < 0.01). Although no significant differences were observed in progesterone, androstenedione, or estradiol‐17β concentrations between the groups, testosterone concentrations were slightly lower with the epidural treatment than with the control treatment (p = 0.08). In conclusion, superovulation was induced by a single epidural injection of pFSH, which achieved higher pFSH level than the multiple injections in Holstein dry cows. [ABSTRACT FROM AUTHOR]

We examined whether the use of in vivo-matured oocytes, collected by ovum pick-up (OPU) from superstimulated Japanese Black cows, can improve the productivity and quality of in vitro produced embryos. The cows were superstimulated by treatment with progesterone, GnRH, FSH and prostaglandin F2α according to a standardized protocol. The resulting in vivo-matured oocytes were collected by OPU and used subsequently for the other experiments. The immature oocytes from cows in the non-stimulated group were collected by OPU and then subjected to maturation in vitro. We found that the rate of normally distributed cortical granules of the matured oocyte cytoplasm in the superstimulated group was significantly higher than that in the non-stimulated group. The normal cleavage rate (i.e., production of embryos with two equal blastomeres without fragmentation) and freezable blastocyst rate were significantly higher in the superstimulated group than in the non-stimulated group. Among the transferable blastocysts, the ratio of embryos from normal cleavage was also significantly higher in the superstimulated group than in the non-stimulated group. For in vivo-matured oocytes, it was observed that the pregnancy rates were significantly higher when normally cleaved embryos were used for transfer. Taken together, these results suggest that high-quality embryos with respect to developmental kinetics can be efficiently produced with the use of in vivo-matured oocytes collected by OPU from superstimulated Japanese Black cows.

Letrozole is used for the treatment of subfertility in women undergoing ovarian superstimulation, but the mechanism of action has not been investigated critically. The objective was to test the hypothesis that treatment with letrozole will potentiate the superstimulatory response following gonadotropin treatment by increasing the number of follicles present at ovarian follicular wave emergence in cattle. In Experiment 1, ovarian follicular wave emergence was synchronized among beef heifers (n = 8) by transvaginal ultrasound-guided follicle ablation. On Day 0 (wave emergence), a letrozole-releasing device (LRD) was placed intravaginally for 5 days, followed again by transvaginal follicle ablation on Day 5. The number of follicles ≥3 mm was recorded by transrectal ultrasonography on Days 0 and 6.5 (i.e., pre- vs. post-LRD treatment). In Experiment 2, non-lactating dairy cows were assigned randomly to one of two groups (n = 15/gp) after follicle ablation-induced synchronization of wave emergence (Day 0), and given either an LRD or sham device for 5 days. Superstimulatory treatment was initiated on Day 0, consisting of 8 doses of 50 mg of porcine FSH im at 12 h intervals, and luteolytic doses of prostaglandin on Days 3 and 3.5. The LRD/sham devices were removed on Day 3.5, GnRH was given im on Day 5, estrus response was determined on Days 5 and 6, and the ovarian response was recorded by ultrasonography on Days 0, 3.5, 5, 6.5, and 12. In Experiment 1, no difference was detected in the number of antral follicles at wave emergence pre- vs. post-LRD treatment (23.2 ± 3.2 vs. 23.5 ± 3.8 follicles; P = 0.67; mean ± SEM). In Experiment 2, the interval from prostaglandin treatment to estrus was longer (50.3 ± 1.1 vs. 40.7 ± 2.0 h; P < 0.001) and less variable (residuals: 3.1 ± 0.5 vs. 6.7 ± 0.9 h; P < 0.01) in the LRD vs. sham group. The proportion of ovulations (number of CL on Day 12 over the number of follicles ≥3 mm on Day 0) did not differ (0.65 ± 0.02 vs. 0.70 ± 0.02; P = 0.15) nor did the number of CL on Day 12 (15.9 ± 2.5 vs. 19.0 ± 2.0; P = 0.32) between the LRD and sham groups. In summary, treatment with letrozole did not increase the number of antral follicles at wave emergence or the superstimulatory response, but increased precision in the interval to estrus and may be useful for artificial insemination at a fixed time in superstimulatory protocols. • Treatment with an aromatase inhibitor delayed the onset of estrus and decreased the variation in the onset of estrus. • Treatment with an aromatase inhibitor did not affect the number of antral follicles at wave emergence. • Co-treatment with an aromatase inhibitor and follicle stimulating hormone did not potentiate the superstimulatory. [ABSTRACT FROM AUTHOR]

Objective Superstimulatory treatment of one-month-old lambs can achieve synchronous development of numerous growing follicles. However, these growing follicles cannot complete maturation and ovulation. Oocyte maturation and competence are acquired during follicular development, in which granulosa cells play an essential role. Methods In this study, we applied RNA sequencing to analyze and compare gene expression between prepubertal and adult superstimulated follicle granulosa cells in sheep. Results There were more than 300 genes that significantly differed in expression. Among these differently expressed genes, many extracellular matrix genes (EGF containing Fibulin Like Extracellular Matrix Protein 1, pentraxin 3, adrenomedullin, and osteopontin) were significantly down-regulated in the superstimulated follicles. Ingenuity pathway and gene ontology analyses revealed that processes of axonal guidance, cell proliferation and DNA replication were expressed at higher levels in the prepubertal follicles. Epidermal growth factor, T-Box protein 2 and beta-estradiol upstream regulator were predicted to be active in prepubertal follicles. By comparison, tumor protein P53 and let-7 were most active in adult follicles. Conclusion These results may contribute to a better understanding of the mechanisms governing the development of granulosa cells in the growing follicle in prepubertal sheep.

Aim: To evaluate the follicular dynamics, superovulatory response, and embryo recovery following superstimulatory treatment initiated at estradiol-17β induced follicular wave emergence and its comparison with conventional superstimulatory protocol in buffaloes. Materials and Methods: Six normal cycling pluriparous buffaloes, lactating, 90-180 days post-partum, and weighing between 500 and 660 kg were superstimulated twice with a withdrawal period of 35 days in between two treatments. In superstimulation protocol-1 (estradiol group) buffaloes were administered estradiol-17β (2 mg, i.m.) and eazibreed controlled internal drug release (CIDR) was inserted intravaginally (day=0) at the random stage of the estrous cycle. On the day 4, buffaloes were superstimulated using follicle stimulating hormone (FSH) 400 mg, divided into 10 tapering doses given at 12 hourly intervals. Prostaglandin F2α analogs (PGF2α) was administered at day 7.5 and day 8, and CIDR was removed with the second PGF2α injection. In superstimulation protocol - 2 (conventional group) buffaloes were superstimulated on the 10th day of the estrous cycle with same FSH dose regimen and similar timings for PGF2α injections. In both groups, half of the buffaloes were treated with luteinizing hormone (LH) 25 mg and other half with 100 ug buserelin; gonadotrophin releasing hormone (GnRH) analog at 12 h after the end of FSH treatment. All buffaloes in both protocols were inseminated twice at 12 and 24 h of LH/GnRH treatment. Daily ultrasonography was performed to record the size and number of follicles and superovulatory response. Results: Significantly higher number of small follicles (8 mm), corpora lutea, and transferable embryos was higher in buffaloes superstimulated at estradiol-induced follicular wave compared to the conventional protocol: Further the percentage of transferable embryos was significantly higher in buffaloes administered with LH compared to GnRH.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120 mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300 IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P > 0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80 mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80 mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes. [ABSTRACT FROM AUTHOR]

Background: Prolongation of superstimulatory treatment appears to be associated with a greater superovulatory response and with greater oocyte maturation in cattle. A genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells collected from ovarian follicles after differing durations of the growing phase induced by exogenous FSH treatment. Cows were given a conventional (4-day) or long (7-day) superstimulatory treatment (25 mg FSH im at 12-h intervals; n = 6 per group), followed by prostaglandin treatment with last FSH and LH treatment 24 h later. Granulosa cells were harvested 24 h after LH treatment. Results: The expression of 416 genes was down-regulated and 615 genes was up-regulated in the long FSH group compared to the conventional FSH group. Quantification by RT-PCR of 7 genes (NTS, PTGS2, PTX3, RGS2, INHBA, CCND2 and LRP8) supported the microarrays data. Multigene bioinformatic analysis indicates that markers of fertility and follicle maturity were up-regulated in the long FSH group. Conclusion: Using the large gene expression dataset generated by the genomic analysis and our previous associated with the growth phase and gene expression changes post LH, we can conclude that a prolonged FSH-induced growing phase is associated with transcriptomic characteristics of greater follicular maturity and may therefore be more appropriate for optimizing the superovulatory response and developmental competence of oocytes in cattle. [ABSTRACT FROM AUTHOR]

Contents: Follicular fluid (FF) accumulates in the antrum of the ovarian follicle. In addition, FF provides the microenvironment for oocyte development, oocyte maturation and competence, which are acquired during follicular development. Superstimulatory treatment of 1‐month‐old lambs can achieve synchronous development of numerous growing follicles. However, these growing follicles are unable to completely mature and ovulate. Furthermore, the oocytes exhibit lower competence compared with those of ewes. In this study, we utilized an isobaric tag for relative and absolute quantification (iTRAQ)‐based proteomics analysis and compare protein composition between pre‐pubertal and adult superstimulated follicle FF in sheep. In total, 243 differentially expressed proteins were identified, including 155 downregulated and 88 upregulated between lamb and ewe. Gene ontology (GO) and KEGG pathway analysis indicated that the differentially expressed proteins are involved in signal transduction, anatomical structure development, stress response, metabolic pathways, and the complement and coagulation cascades. Many of the proteins known to affect follicle development were observed in lower abundance in FF of lamb (e.g. ADAMTS9, CD14, CTNNB1, FST, GCLC, HSPG2, IGFBP2, IGFBP6, INHBA, PRL, PAPPA, POSTN, PRDX1, SERPINA1, SOD3, STC1, VEGFC, etc.). However, a higher abundance was observed for proteasome proteins. Inadequate amounts of these proteins in FF may be lead to the unique characteristics of follicular development in lamb. These differentially expressed proteins illuminate the age‐dependent changes in protein expression in the follicle microenvironment. [ABSTRACT FROM AUTHOR]

Hereford heifers were assigned randomly to three superstimulation groups and given FSH for 4 days in the short FSH group (n = 5) and FSH starvation group, (n = 5) or for 7 days in the long FSH group (n = 4). In vivo oocyte maturation was induced with LH given 12 hours after the last FSH treatment in short and long FSH groups and 84 hours after the last FSH treatment in the FSH starvation group. The ovaries were removed by colpotomy 18 to 20 hours after LH treatment to aspirate cumulus–oocyte complexes (COCs) from follicles 8 mm or greater. Cumulus–oocyte complexes were graded morphologically, and oocytes were processed for either mitochondrial staining or for ATP assay. Collection efficiency was similar among treatment groups, but a greater proportion of COCs were expanded (P < 0.01) and oocyte ATP content of the expanded COC tended to be greater (P < 0.09) in the long FSH group than other two groups. Oocytes in the FSH starvation group had a greater proportion (P = 0.01) of mitochondrial clusters (i.e., fewer scattered individual mitochondria). Individual mitochondria and mitochondrial clusters in oocytes from the long FSH and FSH starvation groups had twice the relative staining intensity (P < 0.01) compared to oocytes from the short FSH group. In summary, the long FSH superstimulation protocol yielded a greater proportion of expanded COCs that had oocytes with a scattered mitochondrial population and a greater ATP content than other two protocols. FSH starvation of 84 hours yielded a high proportion of grade 4 COCs characterized by a greater proportion of mitochondrial clusters within the oocyte. [ABSTRACT FROM AUTHOR]

The objective of this study was to develop a superovulatory program based on synchronization of follicular waves with GnRH which could be applied regardless of the stage of the oestrous cycle. 36 heifers were subjected to this experiment and GnRH (Cystorelin, 200 µg) was applied between Days 0 and 7 (n = 15), 8 and 12 (n = 8) or 13 and 20 (n = 13) of the oestrous cycle. Four days after GnRH treatment, all follicles ≥ 6 mm of heifers (n) were either punctured (n = 21) or left intact (n = 15). All heifers were superstimulated from Day 6 to Day 10 after GnRH treatment with 320 mg Folltropin-V. In parallel, 21 heifers were superstimulated in a conventional manner (Days 8 to 12) and were used as controls. The homogeneity of follicular inventories among Stage-groups occurred within 4 days of GnRH treatment for follicles ≥ 7 mm but only 2 days after follicular puncture for follicles 4 to 6 mm. In response to the follicular puncture, the mean number of follicles 4 to 6 mm increased in heifers of the punctured group (P < 0.01). Following the superstimulation, the follicular (P < 0.01) and ovulatory (P < 0.01) responses were higher in the punctured group than in the nonpunctured group. The in vivo production of transferable embryos in the punctured group was similar to that of the nonpunctured group but it was lower (P < 0.01) than in heifers of the control group. In conclusion, results from the present study indicate that regardless of the stage of the oestrous cycle, the homogeneity of follicular inventories following the follicular synchronization is obtained using GnRH treatment and follicular puncture. The in vivo production of embryos was severely compromised in the present study with heifers. Causes of such reduction in the in vivo production of embryos are still unknown.

Response to superovulatory, embryo yields and quality, were evaluated after increasing exposure time to exogenous progesterone during superstimulation, with or without the addition of a GnRH agonist. Thirty-four ewes from the Santa Inês breed were synchronized with an intravaginal progesterone device (CIDR). The animals were randomly divided into three groups: exposure to progesterone for 14 days (Control, n = 12); 12 h extension of progesterone exposure (12hP4 group, n = 11); and 12 h extension, associated with the administration of 25 μg of gonadorelin acetate (12hP4GnRH group, n = 11). From the 12th to the 15th day of protocol, 133 mg of pFSH were administered through eight decreasing doses. Artificial inseminations were performed 36 and 48 h after device withdrawal, using frozen/thawed semen. The number of ewes on estrus and the time to onset of estrus after withdrawing device were similar among between all groups ( P > 0.05). All groups presented high superstimulatory response (averages ranging from 14.33 to 16.18 follicles ≥4 mm) ( P > 0.05). All groups also showed a large quantity of CL (averages ranging from 11 to 12) ( P > 0.05). The longer exposure to progesterone no reduced degenerated embryo proportion (Control = 30%; 12hP4 = 7% and 12hP4GnRH = 10%; P > 0.05). The fertilization rate was significantly higher in 12hP4GnRH when compared to Control and to 12hP4 (77%, 34% and 41% respectively). These results suggest the association of longer progesterone exposure plus GnRH administration is an alternative to increase oocyte fertilization rates when fixed time insemination is used. [ABSTRACT FROM AUTHOR]

Our objective was to study the effect of superstimulation protocols on nuclear maturation of the oocyte and the distribution of lipid droplets in the ooplasm. Heifers (n = 4 each group) during the luteal phase were either treated with FSH for 4 days (Short FSH), FSH for 4 days followed by 84 h of gonadotropin free period (FSH Starvation) or for 7 days (Long FSH) starting from the day of wave emergence. In all groups, LH was given 24 h after induced luteolysis (penultimate day of FSH) and cumulus-oocyte complexes were collected 24 h later. Oocytes were stained for nuclear maturation (Lamin/chromatin) and lipid droplets (Nile red). The Long FSH group had a greater proportion of mature oocytes (metaphase II) compared with heifers in the Short FSH and FSH Starvation groups (59/100 vs 5/23 and 2/25, respectively; P < 0.01). On average across all groups, oocytes contained 22 pL of lipids (3.3% of ooplasm volume) distributed as 3000 droplets. Average volume of individual lipid droplets was higher in the FSH Starvation (11.5 ± 1.5 10 -- 3 pL, P = 0.03) compared with the Short and Long FSH groups (7.2 ± 0.6 10 -- 3 and 8.0 ± 0.8 10 -- 3 pL, respectively). In conclusion, both FSH Starvation and Short FSH treatments yielded a lower proportion of mature oocytes compared with the Long FSH treatment. Furthermore, FSH starvation led to an accumulation of larger lipid droplets in the ooplasm, indicating atresia. Our results indicate that a longer superstimulation period in beef cattle yields higher numbers and better-quality oocytes. [ABSTRACT FROM AUTHOR]

Effectiveness of in vitro production of embryos (IVP) is limited among other factors by the recovery rate of oocytes. Gonadotropin superstimulation can improve the recovery rate of oocytes. The effect of FSH treatment on follicular population and recovery rate of oocytes at ovum pick-up (OPU) in the growing phase of the 1st as well as the 2nd follicular wave after superstimulation was the object of our experiment. Twelve unpregnant milking cows (15-20 kg milk per day) housed on a dairy farm were used in the experiment. The cows bearing corpus luteum were synchronized by PGF2 (day 0) and they were treated by FSH (Folicotropin inj. sicc. ad us vet., Spofa Prague, Czech Republic, single doses 80, 80, 80, 80, 40 and 40 UI) in 12 h intervals on days 12, 13 and 14. Transvaginal ultrasonographic puncture of oocytes in cows bearing a new corpus luteum was performed on day 7 (OPU 1, various phase of the follicular wave, removal of the dominant follicle) and it was repeated on days 10 (OPU 2, growing phase of the follicular wave - control), 16 (OPU 3, growing phase of the first follicular wave after superstimulation) and 20 (OPU 4, growing phase of the second follicular wave after superstimulation). All follicles > 2 mm were punctured. The ovarian follicles (ultrasonographically) and numbers and qualities of obtained oocytes (microscopically) were evaluated during and immediately after OPU. Follicular population was divided to small (FS, 2-5 mm), medium (FM, 5-9 mm) and large (FL, > 9 mm) follicles. Oocytes were classified as 1st (intact cumulus, > 3 layers of cumulus cells), 2nd (complete 1-3 layers of cumulus cells), 3rd (incomplete layers of cumulus cells, expanded cumulus mass) and 4th (absence of corona cells, degenerated oocytes) classes. Although we found the least of FS (x = 1.0) during OPU 3, significantly more FM (x = 24.7) and FL (x = 3.1) follicles were found at this procedure in comparison with others. Likewise a significantly higher number of oocytes (x = 8.1) was obtained at OPU 3 in comparison with OPU 1 and OPU 2. Significantly higher number of FM (x = 6.1) was found and non-significantly higher number of oocytes was obtained at OPU 4 in comparison with OPU 1 and 2. The results show that administration of FSH increases the number of follicles and the number of collected oocytes in the growing phase of the 1st follicular wave after superstimulation, nevertheless a higher number of follicles and a higher recovery rate of oocytes can be expected in the growing phase of the 2nd follicular wave after superstimulation as well.

Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4 , NANOG , and SOX2 genes is not affected. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the follicular dynamics, superovulatory response, and embryo recovery following superstimulatory treatment initiated at estradiol-17β induced follicular wave emergence and its comparison with conventional superstimulatory protocol in buffaloes. Materials and Methods: Six normal cycling pluriparous buffaloes, lactating, 90-180 days post-partum, and weighing between 500 and 660 kg were superstimulated twice with a withdrawal period of 35 days in between two treatments. In superstimulation protocol-1 (estradiol group) buffaloes were administered estradiol-17β (2 mg, i.m.) and eazibreed controlled internal drug release (CIDR) was inserted intravaginally (day=0) at the random stage of the estrous cycle. On the day 4, buffaloes were superstimulated using follicle stimulating hormone (FSH) 400 mg, divided into 10 tapering doses given at 12 hourly intervals. Prostaglandin F2α analogs (PGF2α) was administered at day 7.5 and day 8, and CIDR was removed with the second PGF2α injection. In superstimulation protocol - 2 (conventional group) buffaloes were superstimulated on the 10th day of the estrous cycle with same FSH dose regimen and similar timings for PGF2α injections. In both groups, half of the buffaloes were treated with luteinizing hormone (LH) 25 mg and other half with 100 ug buserelin; gonadotrophin releasing hormone (GnRH) analog at 12 h after the end of FSH treatment. All buffaloes in both protocols were inseminated twice at 12 and 24 h of LH/GnRH treatment. Daily ultrasonography was performed to record the size and number of follicles and superovulatory response. Results: Significantly higher number of small follicles (<8 mm) was present at the time of initiation of superstimulatory treatment in the estradiol group compared to the conventional group (12.5±0.80 vs. 7.3±1.21, respectively, p=0.019), however, the number of ovulatory size follicles (≥8 mm) did not differ significantly between the respective groups (15.5±1.24 vs. 12.2±1.30; p=0.054). Total embryos and transferable embryos recovered were non-significantly higher in the estradiol group compared to the conventional group (5.83±0.86 vs. 4.67±1.16, p=0.328, and 3.67±0.93 vs. 2.67±0.68, p=0.437, respectively). The significant higher proportion of transferable embryos were recovered in buffaloes treated with LH compared to GnRH (73.3% vs. 48.5%; p=0.044). Conclusion: The average number of ovulatory size follicles (>8 mm), corpora lutea, and transferable embryos was higher in buffaloes superstimulated at estradiol-induced follicular wave compared to the conventional protocol: Further the percentage of transferable embryos was significantly higher in buffaloes administered with LH compared to GnRH. [ABSTRACT FROM AUTHOR]

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 β 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 G q/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. [ABSTRACT FROM AUTHOR]

To evaluate follicular dynamics and oocyte recovery, eight buffaloes (n=8) were superstimulated with 200 mg or 300 mg FSH, given in divided doses over 2 and 3 days respectively. On the day of OPU, follicles > 6 mm were significantly higher (11.38±1.22 vs. 7.50±0.68; p=0.015) in 3 Days compared to 2 Days protocol. On an average, 4.75±1.08 and 6.25±1.01 oocytes per buffalo were recovered from 2 Days and 3 Days protocols, respectively. It was concluded that 3 Days superstimulatory protocol enhanced the availability of > 6 mm follicles to be aspirated followed by better oocyte recovery rate as compared to 2 Days protocol. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The review presents an overview of studies that examined the effects of follicular aging and maternal aging in the bovine model. The first of three main sections is a discussion of the developmental competence of oocytes from (1) the ovulatory follicle of 2-wave and 3-wave estrous cycles, (2) dominant follicles that develop under high or low LH pulse frequency, and (3) natural versus FSH-stimulated ovulatory follicles. The second section highlights the effects of maternal aging. Maternal aging in cattle is associated with (1) elevated circulating FSH concentrations, (2) reduced response to superstimulatory treatment, and (3) markedly decreased early embryonic development in cows >12 year of age. The third and final section on superstimulation protocols addresses the effects of the duration of FSH stimulation and withdrawal (i.e., FSH “starvation” or “coasting”) on oocyte competence. Ovarian superstimulation for 4 days altered the expression of genes related to angiogenesis, and activated oxidative stress-response genes. Extending the duration of FSH stimulation from 4 to 7 days resulted in a greater and more synchronous ovulatory response and optimal oocyte maturation. The highest rates of blastocyst development in vitro were obtained when FSH support was discontinued for 44 to 68 h and granulosa cell SMAD7 mRNA was predictive of this period. Longer periods of FSH starvation resulted in a loss of oocyte competence or ovulatory capability. By extending the bovine model to the transcriptome level, new approaches and treatments may be devised to resolve subfertility in women and animals. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Multiple metabolic and hormonal factors can affect the success of protocols for ovarian superstimulation. In this study, the effect of acute feed restriction and increased LH content in the superstimulatory FSH preparation on numbers of ovulations, fertilization, and embryo quality in lactating dairy cows was evaluated. Two experiments were performed using a Latin square design with treatments arranged as a 2x2 factorial: feed restriction (FR; 25% reduction in dry matter intake) compared with ad libitum (AL) feeding, combined with high (H) versus low (L) LH in the last 4 injections of the superstimulatory protocol. As expected, FR decreased circulating insulin concentrations (26.7 vs. 46.0μU/mL). Two analyses were performed: one that evaluated the complete Latin square in experiment 2 and a second that evaluated only the first periods of experiments 1 and 2. For both analyses, follicle numbers, ovulation rates, and corpora lutea on d 7 were not different. In the first period analysis of experiments 1 and 2, we observed an interaction between feed allowance and amount of LH on fertilization rates, percentage of embryos or oocytes that were quality 1 and 2 embryos, and number of embryos or oocytes that were degenerate. Fertilization rates were greater for the AL-L (89.4%) and FR-H (80.1%) treatments compared with the AL-H (47.9%) and FR-L (59.9%) treatments. Similarly, the proportion of total embryos or oocytes designated as quality 1 and 2 embryos was greater for AL-L (76.7%) and FR-H (73.4%) treatments compared with AL-H (35.6%) and FR-L (47.3%) treatments. In addition, the number of degenerate embryos was decreased for AL-L (1.3) and FR-H (0.4) treatments compared with the AL-H (2.6) and FR-L (2.3) treatments. Thus, cows with either too low (FR-L) or too high (AL-H) insulin and LH stimulation had lesser embryo production after superstimulation because of reduced fertilization rate and increased percentage of degenerate embryos. Therefore, interaction of the gonadotropin content of the superstimulatory preparation with the nutritional program of the donor cow needs to be considered to optimize success of ovarian superstimulatory protocols. [ABSTRACT FROM AUTHOR]

The present study aimed to establish an efficient system for the production of female embryos from dairy cows by in vitro fertilization (IVF) using X-sorted sperm and in vivo-matured oocytes collected by ovum pick up (OPU). Nonlactating Holstein cows (n = 36) were administered a controlled intravaginal progesterone-releasing (controlled internal drug release) device (d 0), underwent dominant follicle ablation (DFA) or ovulation by administration of 100 μg of GnRH on d 5, and were superstimulated with FSH and PGF2α, following standard procedures. Controlled internal drug release devices were removed on the evening of d 8 or on the morning of d 9, depending on the experiment. For LH surge induction, 200 μg of GnRH was administered on the morning of d 10 (0 h). In experiment 1, the peak (48.1%) of ovulating follicles was detected at 29 to 32 h after GnRH injection (0 h), and the range in the timing of the initiation of ovulation was less by timing from GnRH administration (30.0 ± 2.8 h) rather than by timing the onset of estrus (32.7 ± 4.7 h). Only 0.9% of total ovulated follicles were recorded before 26 h after GnRH injection. Therefore, OPU was carried out at 26 h and IVF occurred at 30 h after GnRH in experiments 2 and 3. In experiment 2, 83.3 ± 10.8% of oocytes with expanded cumulus cells had extruded the first polar body at 30 h after GnRH injection. The aim of experiment 3 was to compare the effect of either DFA or GnRH-induced LH surge before superstimulation on the efficiency of embryo production by IVF following superstimulation. Progesterone concentrations from d 10 to 12 in the DFA group were lower than those in the GnRH group. A greater proportion of recovered oocytes with expanded cumulus cells from ≥8-mm follicles was observed in the DFA group than in the GnRH group (95.9 and 77.4%, respectively). Blastocyst rates in the DFA and GnRH groups (58.0 and 52.8%, respectively) did not differ from those of oocytes collected from nonstimulated OPU and matured in vitro (49.9%). However, the proportion of high-quality blastocysts was higher in the DFA group compared with the GnRH group (54.9 vs. 21.5%). Our results demonstrate that high rates of good-quality blastocysts can be produced by IVF with X-sorted frozen sperm using in vivo-matured oocytes collected by OPU from cows after DFA and superstimulation combined with ovulation induction. [ABSTRACT FROM AUTHOR]

In the present study, we tested the hypotheses that oocyte competence is compromised by a longer duration of follicular growth and that it is not affected by FSH starvation. Cows were allocated to short FSH (n = 14), FSH starvation (n = 13) and long FSH (n = 13) groups. The first two groups were given eight doses of FSH, whereas the third group was given 14 doses of FSH, starting from the day of wave emergence (Day 0). A progesterone-releasing device (controlled internal drug release; CIDR) was placed intravaginally at the start of the experiment in all groups. The short FSH group was given prostaglandin (PG) F2α on Day 3, whereas the two other groups received PGF2α on Day 6. In all cows, the CIDR was removed at the time of PGF treatment; porcine (p) LH was given 24 h after CIDR removal and cows were inseminated 24 and 36 h later. Reproductive tracts were collected 4 days after insemination and ova and/or embryos were cultured for ≥6 days. The FSH starvation group had fewer ovulations (P = 0.001), and ova and/or embryos (P < 0.05). No difference in embryo quality was detected between long and short FSH groups at 7, 9 or 10 days after artificial insemination. In conclusion, oocyte competence was not altered by the duration of the follicular growth phase in superstimulated cows, whereas FSH starvation substantially reduced the ability of superstimulated follicles to ovulate. How can we improve oocyte competence after superstimulation? To answer this question, we tested two durations (4 and 7 days) of follicular growth with or without FSH support at end of the treatment (i.e. FSH starvation). The duration of follicular growth did not affect oocyte competence; however, FSH starvation compromised ovulation capability. Therefore, follicular age does not alter oocyte competence but follicles are dependent of gonadotropins to ovulate. [ABSTRACT FROM AUTHOR]

The effect of heat stress with melatonin treatment on the superovulatory responses and embryo characteristics in Thai-Holstein crossbreeds under heat stress conditions was examined. Six non-lactating cows (replication = 4; n = 24) were assigned to one of 2 treatments in double cross-over design. All cows were superstimulated with traditional treatment. Melatonin group ( n = 12): cows received intramuscularly injection 18 mg/50 kg. simultaneously with GnRH injection, while those in the control group ( n = 12) received none. Bloods samples were taken to determine lipid peroxidation (MDA) and the activity of the antioxidant enzymes (superoxide dismutase; SOD). The experiment was conducted from April to September, which determined severe heat stress (the mean temperature-humidity index above 77). The results revealed that numbers of large follicles and corpora lutea were higher in the melatonin group than in the control group ( p < 0.01). Numbers of recovered ova/embryos, fertilized ova, and transferable embryos were higher in the melatonin group ( p < 0.01); meanwhile, more degenerated embryos were found in the control group ( p < 0.01). Increased activity of the antioxidant enzymes SOD after melatonin administration decreased MDA levels ( p < 0.05). In summary, melatonin administration benefited the ovarian response and embryo quality in superstimulated Thai-Holstein crossbreed affected by heat stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ratchamak, Thananurak, Boonkum, Semaming and Chankitisakul.)

The present study investigated the effect of superstimulation to improve in vitro embryo production in the Gulf area, where the temperature is high. Holstein cows were classified into the control and superstimulation groups. Superstimulation was induced with a single intramuscular injection of pregnant mare serum gonadotropin (PMSG; 2500 IU) on day 14 of the estrus cycle (day 0; estrus). The development of follicles was evaluated by ultrasonography of the ovaries daily. At 40 h after the PMSG injection, oocytes were collected by the ovum pick-up (OPU) technique. OPU was performed at the same stage of the estrus cycle in the control group as in the superstimulation group. The number of follicles with a diameter of more than 6 mm and the number of retrieved cumulus-oocyte complexes were significantly higher in the superstimulation group than in the control group. Furthermore, the maturation rate was higher in the superstimulation group than in the control group. Cloned embryos were produced by somatic cell nuclear transfer using matured oocytes. The cleavage and blastocyst formation rates were significantly higher in the superstimulation group than in the control group. In conclusion, a single injection of PMSG can facilitate the efficient production of cloned cow embryos., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Son, Jeong, Hossein, Yu, Olsson, Kang, Kim, Bae, Tinson, Singh, Rajesh, Noura and Hwang.)

Abstract: Three experiments were designed to evaluate the superovulatory response of beef cows following two intramuscular (IM) administrations 48 h apart of Folltropin-V diluted in reduced concentrations of hyaluronan (Split-single IM administrations; Experiment 1—300 mg Folltropin-V on the first day and 100 mg 48 h later; Experiment 2—200 mg Folltropin-V on the first day and 100 mg 48 h later). In Experiments 1 and 2, superovulatory response and ova embryo/embryo production did not differ between donors receiving twice daily IM of Folltropin-V over 4 days and those given a Split-single IM administration of Folltropin-V diluted in 10 mg/mL hyaluronan solution. Experiment 3 compared Split-single IM administration of Folltropin-V diluted in two hyaluronan concentrations (5 or 10 mg/mL) with Folltropin-V diluted in saline and administered twice-daily over 4 days. Beef cows (17 Angus and 12 Simmental) were randomly assigned to one of three treatment groups to be superstimulated three times in a cross-over design, so that all cows received all treatments. A total dose of 300 mg Folltropin-V was divided into twice-daily IM over 4 days, or in two IM treatment 48 h apart (200 mg on first day and 100 mg 48 h later) in the hyaluronan groups. Mean (± SEM) numbers of transferable embryos did not differ among treatment groups (Control: 4.0 ± 0.8; 10 mg/mL hylauronan: 5.0 ± 0.9; 5 mg/mL hyaluronan: 6.1 ± 1.3). We concluded that the Split-single IM administration of Folltropin-V diluted in either concentration of hyaluronan resulted in a comparable superovulatory response to the traditional twice-daily protocol. [Copyright &y& Elsevier]

Abstract: The objective was to evaluate the developmental competence of cumulus-oocyte complexes (COC) collected by follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly to two groups (n = 16 per group) and treated, at the time of ovarian follicular wave emergence, with either: 1) 25 mg of FSH im, twice daily for 4 d; or 2) 1000 IU of eCG as a single i.m. dose. The start of gonadotropin treatment was considered Day 0. Both groups were given 5 mg of Armour Standard LH im on Day 6, and COC were collected by follicle aspiration on Day 7. Expanded COC collected from FSH- (n = 157) and eCG-treated llamas (n = 151) were fertilized in vitro using epididymal sperm, and presumptive zygotes were in vitro cultured in SOF medium for 8 d. The FSH and eCG treatment groups did not differ with respect to: the number of follicles ≥7 mm (16.0 ± 2.7 vs 14.0 ± 1.9, respectively; P = 0.5); the number of COC collected (11.5 ± 1.9 vs 9.7 ± 1.2; P = 0.4); the number of expanded COC (9.8 ± 1.4 vs 9.4 ± 1.2; P = 0.8); or the percentage of presumptive zygotes which developed into 2 to 8 cell stage embryos (65.3 vs 63.1), morulas (46.2 vs 42.5), or blastocysts (23.1 vs 20.5; P > 0.05). In conclusion, FSH and eCG treatments were equally effective for recovery of a high number of expanded COC which were used directly for in vitro fertilization. Furthermore, rate of embryo development was not significantly affected by the gonadotropin treatment used. [Copyright &y& Elsevier]

Abstract: Llamas are monotocous and the length of their gestation period varies between 342 and 350 days. Thus the average number of offspring any female can produce throughout her reproductive life is very limited to spread a desired genome. The multiple ovulation and embryo transfer (MOET) technique allows an alternative to this limitation and reduces the generation interval. The objective of this study was to evaluate embryo recovery in superstimulated llamas which had previously been hormone-treated to inhibit follicular growth. A total of 50 female llamas were monitored daily via rectal palpation and ultrasound and divided according to their ovarian follicular growth into four phases. The females in each phase were then randomly divided into two groups: A (n =20) received a single dose of 1mg of estradiol benzoate (EB) on the first day of the treatment+100mg of progesterone (P4) i.m. for 5 days with 5 animals per phase and B (n =20) received 1mg EB at onset+150mg P4 i.m. for a period of 5 days with 5 animals per phase. Group C (n =10) or control did not receive any prior hormonal treatment and the females were in follicular phase I. All groups were monitored daily and, in the presence of ovarian follicles smaller than the dominant size at the end of treatment, all were superstimulated with 1000IU eCG. For plasma progesterone concentration recording, daily blood samples were collected from days −1 to 5 in the treated females in Group A and B. No significant differences were observed regarding the inhibition of follicle growth and in the plasma progesterone concentrations between Group A and B. The ovarian response to superstimulation was 56.2%, 71.4% and 90%, with the average number of dominant follicles produced per female being 4.4±0.9; 4.8±0.7 and 4.6±0.6 in Groups A, B and C, respectively. The embryo recovery rate was 77.7%; 90% and 66.7% and the average number of embryos recovered per female was 2.9±0.9; 2.6±0.9 and 2.4±0.8 for Groups A, B and C, respectively. In Groups A and B, the static follicular phase (III) seemed to be ideal for initiating the assisted reproductive technique of MOET. Although prior administration of P4+EB seems to have no effect on the number of females that responded to the superstimulation treatments, the number of embryos recovered showed a tendency to be higher when ovarian follicle growth inhibition was performed beforehand. [Copyright &y& Elsevier]

The article focuses on a study which investigated whether superovulation (Sov) subsequent to induction of follicular wave emergence could be utilized as an alternative to conventional protocol in Ongole cows. Sov treatment included 200 milligrams (mg) NIH-FSH-β1 twice daily and the induction of luteolysis at 48 and 60 hours after treatment initiation. The study found that the ovulation rate was higher in ablation group compared to the steroid group. It also discovered that there is no difference in the total and viable embryos recovery among the groups. Study authors concluded that follicular wave emergence induction prior to Sov is safe in zebu donors.

Abstract: Two experiments were done to test the hypotheses that aging in cattle is associated with a reduced number of follicles recruited into an ovarian follicular wave, and a reduction in the ovarian response to gonadotropin treatment. Older cows (13–16 years of age) and their daughters (3–6 years of age) were treated with FSH for ovarian superstimulation four times over two consecutive years (31 and 33 superstimulations in old and young cows, respectively, experiments and years combined). In Experiment 1, ovulation was induced using LH. In Experiment 2, cumulus-oocyte complexes were collected by ultrasonographic-guided follicle aspirations before expected ovulations. The ovarian follicular and ovulatory responses were monitored daily by ultrasonography. Fewer 2–5mm follicles (P <0.01) were detected at the expected time of follicular wave emergence in older cows than in their daughters. After superstimulation, older cows had fewer follicles ≥6mm (P <0.01), and tended (P =0.1) to have fewer ovulations than their daughters (32±4 versus 40±3, respectively). There was a positive correlation in the response of individual cows to successive superstimulatory treatments (r >0.8; P <0.0001) and the number of detected ovulations from one year to the next (r =0.6; P =0.04). In conclusion, aging was associated with fewer 2–5mm follicles at follicular wave emergence and a lesser follicular and ovulatory response after superstimulatory treatment. The follicular and ovulatory response after superstimulation was repeatable within individuals, regardless of age. [Copyright &y& Elsevier]

Abstract: The responsiveness of the hypothalamo-pituitary axis to steroid treatments for ovarian synchronization and the ovarian superstimulatory response to exogenous FSH was compared in 13–14 year old cows and their 1–4 year old young daughters. We tested the hypotheses that aging in cattle is associated with: (1) decreased follicular wave synchrony after estradiol and progesterone treatment; (2) delayed LH surge and ovulation in response to exogenous preovulatory estradiol treatment; (3) reduced superstimulatory response to exogenous FSH. Higher plasma FSH concentrations (P <0.01), and a tendency (P =0.07) for fewer 4–5mm follicles at wave emergence were observed in old cows (n =10) than in young cows (n =9). The suppressive effect of estradiol/progesterone treatment on FSH was similar between old and young cows. Although the preovulatory LH surge in response to estradiol treatment was delayed in old than young cows (P =0.01), detected ovulation times were not different. No difference in ovarian superstimulatory response was detected between age groups, but old cows (n =8) tended (P =0.10) to have fewer large follicles (≥9mm) 12h after last FSH treatment than in young cows (n =7). We concluded that pituitary and ovarian responsiveness to estradiol/progesterone synchronization treatment was similar between old and young cows, but aging was associated with a delayed preovulatory LH surge subsequent to estradiol treatment. Old cows tended to have fewer large follicles after superstimulatory treatment than young cows. [Copyright &y& Elsevier]

Abstract: Compared to Bos taurus breeds, Bos indicus breeds of cattle present several differences in reproductive physiology. Follicular diameter at deviation and at the time of ovulatory capability are smaller in B. indicus breeds. Furthermore, B. indicus breeds have a greater sensitivity to gonadotropins, a shorter duration of estrus, and more often express estrus during the night. These differences must be considered when setting up embryo transfer programs for B. indicus cattle. In recent studies, we evaluated follicular dynamics and superovulatory responses in B. indicus donors with the objective of implementing fixed-time AI protocols in superstimulated donors. Protocols using estradiol and progesterone/progestrogen releasing devices to control follicular wave emergence were as efficacious as in B. taurus cattle, allowing the initiation of superstimulatory treatments (with lower dosages of FSH than in B. taurus donors) at a self-appointed time. Furthermore, results presented herein indicate that delaying the removal of progesterone/progestogen-releasing devices, combined with the administration of GnRH or pLH 12h after the last FSH injection, results in synchronous ovulations, permitting the application of fixed-time AI of donors without the necessity of estrus detection and without compromising the results. [Copyright &y& Elsevier]

Abstract: The aim of the present study was two-fold. First, to evaluate the effect of exogenous progesterone on ovarian follicular dynamics in order to assess its ability to synchronize ovarian activity in the vicuna. Secondly, to evaluate the ovarian response to the treatment with eCG through the observation of the structures developed in the ovaries. Follicular dynamics was monitored daily by transrectal ultrasonography in 12 adult, non-pregnant vicunas. Plasma progesterone and estradiol-17β concentrations were measured in blood samples collected daily. In experiment 1, intravaginal devices containing 0.33g of progesterone were inserted into the vagina and kept in place for 5 days (treatment group, n = 8). After progesterone withdrawal, five animals were further monitored in order to evaluate the efficacy of the CIDR® to synchronize the emergence of a dominant follicle. In experiment 2, four females received 750IU of eCG IM. Two were previously monitored ultrasonographically to confirm the absence of a dominant follicle at the beginning of the superstimulatory treatment (group A). The other two animals had a CIDR® inserted into the vagina for 5 days and the superstimulatory treatment was applied 24h after device withdrawal (group B). Females from both groups were surgically explored 96h after eCG injection; the ovaries were exposed and the number of newly formed structures produced by each ovary was counted. Peak progesterone concentrations (25.9 ± 5.29nmoll-1, mean ± S.E.M.) were attained on day 1 after device insertion, remained high until the day of device withdrawal (9.7 ± 1.98nmoll-1) and decreased to 5.5 ± 1.13nmoll-1 the day after. There was no follicle development to the state of dominance after device insertion. Moreover, mean follicle diameter steadily decreased after insertion of the device until the minimum mean value (1.85 ± 0.17mm) was recorded on day 5 (P = 0.006). Similarly, plasma concentrations of estradiol-17β remained below 35pmoll-1 during the period of progesterone treatment in all animals and the mean estradiol-17β declined with the lowest value (22.1 ± 2.19pmoll-1) being recorded on day 4 after device insertion. After superstimulation of follicular development with eCG, the total number of follicles that developed was 33 in group A and 58 in group B and the mean number of newly developed ovarian structures per female was 22.75 ± 4.26. In conclusion, progesterone released by the CIDR® exerts a negative effect on ovarian follicular development and function suggesting intravaginal devices could be used to synchronize the beginning of follicular waves during a superstimulatory treatment. There was also a tendency for greater ovarian follicular development when the animals were previously treated with progesterone. [Copyright &y& Elsevier]

We tested the hypotheses that: (1) the superstimulatory response is related to the intrinsic number of follicles recruited into a follicular wave; and (2) the number of follicles recruited into a wave is correlated to the number of follicles recruited into the successive wave. A positive correlation will form the basis of a test for predicting the superstimulatory response. Cows (n=141) were treated with estradiol and progesterone to synchronize follicular wave emergence (first synchronization) and ranked according to the number of follicles ≥2 mm at wave emergence to select the upper and lower 10% of the herd. Follicular wave emergence was synchronized again in the high-end (n=16) and low-end (n=20) groups (second synchronization), and cows were treated with FSH twice daily for 3 days. High-end cows had a greater number of follicles (P<0.001) than low-end cows at the time of wave emergence after both the first and second synchronizations in the 2–3 and 4–6 mm categories. The numbers of 2–3 and 4–6 mm follicles at wave emergence after the first and second synchronizations were positively correlated (P<0.001; r=0.77 and 0.71, respectively). Endogenous FSH peak at the time of wave emergence was higher in the low-end group than in the high-end group. Superstimulatory treatment resulted in more than double the number of follicles (P<0.003) in the 5–7 mm and ≥8 mm categories in the high-end group than in the low-end group (16.8±2.2 versus 8.1±0.9 and 22.7±4.1 versus 9.7±1.6, respectively). The number of follicles ≥5 and ≥8 mm at the end of superstimulation was positively correlated (P<0.001) with the total number of follicles ≥2 mm at the time of wave emergence after both the first (r=0.64 and 0.54, respectively) and second (r=0.65 and 0.5, respectively) synchronizations. Based on the results of this study, the superstimulatory response can be predicted by the number of follicles ≥2 mm at wave emergence. For practical purposes, practitioners can expect the number of follicles ≥5 mm after ovarian superstimulation to be approximately 71% of the number of follicles ≥2 mm at the time of wave emergence. Results validated the proposed simple ultrasound-based test for predicting the superstimulatory response of individual cows. [Copyright &y& Elsevier]

Treatments with follicle stimulating hormone (FSH) to enhance ovarian follicular development before ovum pick-up (OPU) are important for improving in-vitro embryo production (IVEP) procedures in cows, however, their procedural efficacy needs to be evaluated. The objective of the present study was to use meta-analytic methods to determine the effects of FSH treatments prior to OPU when there is progestin-priming on ovarian functions and IVEP in parous Bos taurus cows (n = 243). The literature was systematically reviewed and data from eight experiments, with 23 treatment means including 448 OPU sessions, were used for analyses. All eight experiments included a group of cows in which there was no FSH treatment (CON), and treatment with FSH before OPU to conduct IVEP. Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. Models included fixed effects of treatment (i.e., CON and FSH) and random effect of the experiment, and data were weighted using the inverse of the SEM squared to account for precision of each experiment. Number of medium sized follicles, quality of oocytes, and transferable embryos were greater (P < 0.05) in FSH-treated as compared with cows of the CON group, without indications of publication bias or heterogeneity of data. Taken together, the results from the present study lead to the recommendation for treatments with FSH prior to OPU for IVEP when there was progestin-priming imposed to obtain a large number of transferable embryos for economic sustainability of commercial embryo transfer programs in parous non-lactating Bos taurus cows. [ABSTRACT FROM AUTHOR]