Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG).

BACKGROUND: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). RESULTS: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24 h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by > 2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses. CONCLUSIONS: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

Superovulation with human chorionic gonadotropin (hCG) trigger and gonadotropin releasing hormone agonist (GnRHa) trigger differentially alter essential angiogenic factors in the endometrium in a mouse ART model†.

Gonadotropin-releasing hormone agonists (GnRHa) are used as an alternative to human chorionic gonadotropin (hCG) to trigger ovulation and decrease the risk of ovarian hyperstimulation syndrome. GnRHa is less potent at inducing ovarian vascular endothelial growth factor (VEGF), but may also affect endometrial angiogenesis and early placental development. In this study, we explore the effect of superovulation on endometrial angiogenesis during critical periods of gestation in a mouse model. We assigned female mice to three groups: natural mating or mating following injection with equine chorionic gonadotropin and trigger with GnRHa or hCG trigger. Females were killed prior to implantation (E3.5), post-implantation (E7.5), and at midgestation (E10.5), and maternal serum, uterus, and ovaries were collected. During peri-implantation, endometrial Vegfr1 and Vegfr2 mRNA were significantly increased in the GnRHa trigger group (P < 0.02) relative to the hCG group. Vegfr1 is highly expressed in the endometrial lining and secretory glands immediately prior to implantation. At E7.5, the ectoplacental cone expression of Vegfa and its receptor, Vegfr2, was significantly higher in the hCG trigger group compared to the GnRHa group (P < 0.05). Soluble VEGFR1 and free VEGFA were much higher in the serum of mice exposed to the hCG trigger compared to GnRHa group. At midgestation, there was significantly more local Vegfa expression in the placenta of mice triggered with hCG. GnRHa and hCG triggers differentially disrupt the endometrial expression of key angiogenic factors during critical periods of mouse gestation. These results may have significant implications for placental development and neonatal outcomes following human in vitro fertilization.

Onset of oestrus and periovulatory events in sheep exposed to 5 and 14 days of CIDR treatment with and without eCG.

The present study supports that 5-day short-term CIDR treatments without administration of eCG are equally effective for inducing oestrus behaviour, preovulatory LH discharge and ovulation in sheep than classical protocols based on 14-day treatments plus eCG at CIDR withdrawal. However, the implementation of a 5-day protocol without eCG for fixed-time artificial insemination would be adapted to a later timing of ovulation (p < .05).

Evaluation of different hormonal treatments on oestral and ovarian responses in Moroccan Beni Arouss goats during anoestrus and breeding season.

The efficacy of eight combinations of fluorogestone acetate (FGA, 20 or 40 mg as intravaginal device during 11 days), equine chorionic gonadotropin (eCG, 300 or 500 UI injected 48 hr before FGA removal) and prostaglandin F2α (cloprostenol, 0 or 50 µg injected 48 hr before FGA removal) aiming at induction and synchronization of oestrus and ovulation was evaluated during the anoestrus season in spring and during the breeding season in autumn in adult Beni Arouss goats. Oestrous behaviour was recorded between 12 and 60 hr after FGA removal. Blood samplings allowing to assess onset of the pre-ovulatory LH surge and increase of progesterone as sign of an active corpus luteum were performed, respectively, between 20 and 60 hr and 3, 5, 8 and 15 days after FGA removal. No season-related differences (spring vs. autumn) were observed for oestrous response (95% vs. 93%), pre-ovulatory LH surge (94% vs. 84%) and luteal response after 3-8 and 11-15 days post-treatment (respectively 92% vs. 66% and 92% vs. 98%). The onset of oestrus (21 [13-53] vs. 32 [12-54] hr) and LH surge (26 [20-60] vs. 38 [22-60] hr) occurred significantly later in autumn. FGA (40 vs. 20 mg) in autumn significantly delayed the onset of oestrus (36 [16-54] vs. 23 [12-47] hr) and LH surge (44 [26-58] vs. 33 [22-60] hr). Significant treatment-related differences were recorded for onset of LH surge (earliest for 20 mg FGA, 300 IU eCG, 50 µg PGF2α ) and onset of luteal phase (latest for 40 mg FGA, 300 IU eCG, 50 µg PGF2α ). In conclusion, the hormone combinations tested appeared equally effective in terms of oestrous and ovulation rates. Season has influenced significantly the onset of oestrus and LH surge, and the high dose regimen of FGA delayed the ovarian response in autumn.

Repeated pregnant mare serum gonadotropin-mediated oestrous synchronization alters gene expression in the ovaries and reduces reproductive performance in dairy goats.

This study aimed to elucidate the effects of repeated pregnant mare serum gonadotropin (PMSG) treatment for oestrous synchronization (ES) on ovarian gene expression and reproductive parameters in Xinong Saanen dairy goats, the dominant breed of dairy goat in China. The experiment was carried out at the Research Station of Northwest A&F University (NWAFU), China (34°16'N, 108°4'E). Forty-one does were randomly assigned to groups receiving ES treatments thrice every fortnight (3-PMSG group; n = 19), or ES treatment only once simultaneously with the third ES treatment in the 3-PMSG group (1-PMSG group; n = 22) during middle of the breeding season from late July (14 hr light) until late September (12 hr light). ES treatment was performed via intravaginal insertion of a controlled internal drug release (CIDR) device impregnated with 300 mg progesterone (P4), followed by 300 IU PMSG injections 48 hr before CIDR withdrawal. Oestrus was monitored using vasectomized bucks. Ovaries of three goats in oestrus from both groups were harvested for morphological examination and RNA sequencing (RNA-Seq). Then, all the oestrous goats in the 1-PMSG (n = 21) and 3-PMSG (n = 11) groups were artificially inseminated twice. The 3-PMSG group showed reduced oestrous rate (57.89%), pregnancy rate (31.58%) and litter size (1.17) compared, respectively, with 95.45%, 68.18% and 1.67 for 1-PMSG group (p < 0.05). However, no differences were found in the ovarian morphology between the 1-PMSG and 3-PMSG groups (p > 0.05). RNA-Seq revealed 114 differentially expressed genes (DEGs) in the ovaries of the 3-PMSG group, among which GCG, FSTL3, TET3 and AQP3 were deemed novel and promising candidate genes for regulating fertility. The present study indicates that the three-time PMSG treatment dysregulated several ovarian genes, thereby reducing reproductive performance.

What is the impact of short- and long-term supplementation of either cabergoline or clarithromycin on resolving rat ovarian hyperstimulation syndrome (OHSS) model?

This study aimed to evaluate the effects of coasting, cabergoline and clarithromycin in a rat ovarian hyperstimulation syndrome (OHSS) model. The 42 female Wistar rats were divided into seven groups: control, OHSS (was given 10 IU of pregnant mare serum gonadotropin for 4 consecutive days from day 22 and 30 IU hCG on the fifth day to induce OHSS ), coasting (hCG was applied on the 27th day after gonadotropin injections and the rats were decapitated on the 28th day), Cabergoline (100 mg/kg/d) and clarithromycin (100 mg/kg/d) were given (on the 26th day) with a short-term supplementation (on the 26th day) and long-term supplementation (from the 22nd to the 26th day) groups. The rats were decapitated on the 27th day. Cabergoline and clarithromycin significantly lowered VEGF-2 levels. Clarithromycin significantly reduced IL-1b and TNF-a and significantly increased IL-10 levels. Clarithromycin may be an effective drug for the treatment of OHSS. Impact statement What is already known on this subject? Ovarian hyper-stimulation syndrome (OHSS) is a self-limited disease, in which vascular endothelial growth factor (VEGF) plays the most important role and has a large clinical spectrum related with increased capillary permeability and fluid retention. Some treatment methods that block VEGF over-expression are used in treatment of OHSS. Clarithromycin is known to suppress the production of some pro-inflammatory molecules such as VEGF, IL-8, IL-1, IL-6 and TNF-a. In our study, we compared the efficacy of coasting, short- and long-term supplementation of clarithromycin and cabergoline on correcting OHSS parameters in an experimental study. What do the results of this study add? As a result of our study, we found that OHSS parameters improved better in early prophylactic treatment regimens. We have shown that clarithromycin may be a more effective treatment agent than coasting and cabergoline. What are the implications of these findings for clinical practice and/or further research? Although our study is important in that it is the first pilot study to show that clarithromycin is effective in the treatment of OHSS, there is a need for larger clinical trials.

Superovulatory response and embryo quality in Katahdin ewes treated with FSH or FSH plus eCG during non-breeding season.

The aim of this study was to evaluate the effect of a co-treatment of follicle-stimulating hormone (FSH) plus equine chorionic gonadotrophin (eCG) on serum insulin and insulin-like growth factor 1 (IGF-1) concentrations, superovulatory response, ovulatory rate, and number and production of embryos in Katahdin breed ewes during the non-breeding season. Twenty Katahdin ewes were synchronized with progestagens (CIDR) and assigned to two superovulation treatments (n = 10): (1): ewes treated with 200 mg ewe-1 of FSH from day 5 to 8 after CIDR insertion at decreasing doses every 12 h (FSH group) and (2) ewes treated as FSH group plus 300 IU of eCG on day 5 after CIDR insertion (FSH + eCG group). Estrous behavior was monitored and direct mating was performed. On days - 7 (CIDR insertion), 0 (CIDR withdrawal), and 7 (embryo recovery), blood samples were collected to determine serum hormone concentrations. Co-treatment with eCG (FSH group) did not affect (P > 0.05) serum hormone levels. Superovulation response, ovulation rate, recovery rate, fertilization, and number of embryos were also similar (P > 0.05) between treatments. Compared with FSH group, FSH + eCG ewes had lower (P < 0.05) number of transferable embryos and higher (P < 0.05) number of oocyte and a tendency to increase the number of degenerated embryos (P = 0.07). Overall results suggest that the administration of eCG is not beneficial either to improve the ovulatory response or the amount of transferable embryos in Katahdin ewes superovulated with a protocol using progesterone and FSH at decreasing doses.

Superovulation alters DNA methyltransferase protein expression in mouse oocytes and early embryos.

PURPOSE: DNA methylation is an epigenetic mechanism that plays critical roles during mammalian oocyte and preimplantation embryo development. It is achieved by adding a methyl group to the fifth carbon atom of cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites using DNA methyltransferase (DNMT) enzymes for de novo and maintenance methylation processes. DNMT1, DNMT3A, and DNMT3B play important roles in establishing methylation of developmentally related genes in oocytes and early embryos. The purpose of this study is to identify the effect of superovulation on the expression and subcellular localizations of these three DNMT enzymes in the mouse oocytes and early embryos. METHODS: Three groups composed of control, normal dose [5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG)], and high dose [7.5 IU PMSG and 7.5 IU hCG] were created from 4-5-week-old female BALB/c mice. The relative expression and subcellular localizations of the DNMT proteins in the control and experiment groups have been characterized by using immunofluorescence staining subsequently analyzed in detailed. RESULTS: DNMT1, DNMT3A, and DNMT3B protein expression in the germinal vesicle and metaphase II oocytes and in one-cell and two-cell embryos differed significantly when some of the normal- and high-dose groups were compared with the control counterparts. CONCLUSION: This study has demonstrated for the first time that superovulation alters expression levels of the DNMT proteins, a finding that indicates that certain developmental defects in superovulated oocytes and early embryos may result from impaired DNA methylation processes.

Equine Chorionic Gonadotropin (eCG) improves bucks' semen quality during the nonbreeding season.

In most goat breeds, testosterone serum concentration and semen quality decrease during the nonbreeding season. However, bucks reproductive activity may be stimulated with the administration of equine chorionic gonadotropin (eCG). Therefore, the aim of this study was to determine whether the repeated administration of eCG stimulates the reproductive status of bucks during the nonbreeding season. The study was performed with 19 bucks that were assigned to a group that was treated with eCG (GeCG) and an untreated control group (GCon). The GeCG bucks received an initial dose of 800 IU of eCG (Day 0), followed by four doses of 500 IU administered every 5 days beginning on Day 5. Serum testosterone and anti-eCG antibody concentrations, testicular and seminal traits were determined until Day 60. Testosterone concentration (from Day 3 to 21: p < 0.0001), anti-eCG titre (from Day 12 to 44: p ≤ 0.01), percentage of motile spermatozoa (Day 6: p = 0.006 and 14: p = 0.001) and of spermatozoa with progressive motility (Day 6: p = 0.01 and 14: p = 0.002) and the percentage of spermatozoa with functional membrane (Day 6: p = 0.02 and 22: p = 0.008) were higher in GeCG than in GCon bucks. Also in frozen-thawed samples, the percentage of motile spermatozoa tended to be higher in GeCG than that of GCon bucks (p = 0.07). In conclusion, the administration of eCG during the nonbreeding season stimulated the secretion of testosterone and improved fresh and possibly frozen-thawed semen quality. However, it also resulted in an increase in anti-eCG antibody titre.

Effectiveness of controlled internal drug release device treatment to alleviate reproductive seasonality in anestrus lactating or dry Barki and Rahmani ewes during non-breeding season.

This study aimed to evaluate the effectiveness of hormonal treatments on ovarian activity and reproductive performance in Barki and Rahmani ewes during non-breeding season. Forty-eight multiparous ewes, 24 Barki and 24 Rahmani ewes were divided into two groups, 12 lactating and 12 dry ewes for each breed. Controlled internal drug release (CIDR) device was inserted in all ewes for 14 days in conjunction with intramuscular 500 IU equine chronic gonadotrophin (eCG) at day of CIDR removal. Data were analysed using PROC MIXED of SAS for repeated measures. Breed, physiological status and days were used as fixed effects and individual ewes as random effects. Barki ewes recorded higher (p < .05) total number of follicles, number of large follicles, serum estradiol concentration and estradiol: progesterone (E2 :P4 ) ratio compared to Rahmani ewes. Lactating ewes recorded higher (p < .05) number of small follicles and lower concentration of total antioxidant capacity (TAC) compared to dry ewes. Number and diameter of large follicles recorded the highest (p < .05) values accompanied with disappearance of corpora lutea at day of mating. Serum progesterone concentration recorded lower (p < .05) value at day of mating and the highest (p < .05) value at day 35 after mating. CIDR-eCG protocol induced 100% oestrous behaviour in both breeds, but Rahmani ewes recorded longer (p < .05) oestrous duration compared to Barki. Conception failure was higher (p < .05) in Barki compared to Rahmani ewes. In conclusion, CIDR-eCG protocol was more potent in improving ovarian activity in Barki compared to Rahmani ewes, but this protocol seems to induce hormonal imbalance in Barki ewes that resulted in increasing conception failure compared to Rahmani ewes.

Hoxa-10 and Cyclin D3 Overexpression in the Decidual Reaction in a Superovulation Protocol in Young Adult C57BL/6J Mice.

Following the performance of a superovulation protocol, multiple nodules were observed bilaterally in the uterine horns of 31 of 276 (11.2%) C57BL/6 J female mice aged 8.5 ± 0.6 (mean and standard error of mean) weeks. These lesions prevented embryo collection, and the uterine decidual reaction was suspected. Samples of pathological uteri (n = 20) and the normal genital tracts of donors treated with a similar superovulation protocol (control group, n = 10) were collected. Immunohistochemistry was performed to evaluate pancytokeratin, desmin, vimentin, progesterone receptor (PR), estrogen receptor α (ERα), Ki-67, cyclin D3 and c-Myc expression, as well as quantitative polymerase chain reaction to assess cyclin D3, Hoxa-10 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA expression. The uterine decidual reaction presented a high degree of structural organization and specifically affected the antimesometrial region of the endometrium. The abnormal decidual cells were large polygonal cells that were frequently polyploid or binucleated and strongly positive for desmin. Immunohistochemistry showed higher Ki-67 proliferation index and higher expression of PR and cyclin D3 in decidual cells in the antimesometrial aspect of the endometrium, compared to nondecidualized endometrial stromal cells in the mesometrial aspect of affected uteri, and compared to endometrial stromal cells in healthy uteri. High expression of cyclin D3 and Hoxa-10 mRNA was also observed in uteri affected by the decidual reaction. These results suggest that PR overexpression in endometrial stromal cells, likely due to high progesterone levels, triggers cyclin D3 and Hoxa-10 overexpression, which may be involved in the pathological mechanisms of the mouse uterine decidual reaction.

Manipulation of reproductive seasonality using melatonin implantation in Anglo-Nubian does treated with controlled internal drug release and equine chorionic gonadotropin during the nonbreeding season.

The objective of this study was to compare the efficiency of hormonal treatments on ovarian activity and reproductive performance in anestrous Anglo-Nubian does during the nonbreeding season (February to May). A total of 48 multiparous does were divided into 2 groups (24 lactating does and 24 dry does). In each group, animals were allocated randomly into 2 equal subgroups (12 does each). In the first subgroup, does received a single 18-mg melatonin implant for 42 d followed by a controlled internal drug release (CIDR) device for 19 d in conjunction with 500 IU of equine chorionic gonadotropin (eCG) i.m. on the day of CIDR device removal. The second subgroup received CIDR combined with eCG in parallel with the first subgroup. Melatonin implantation induced a luteotrophic effect, expressed as an increasing number of corpora lutea, increased serum progesterone concentration, and reduced estradiol concentration. Regardless of treatment, dry does showed greater value of progesterone concentration. With the advancement of day of treatment, number of total follicles, small follicles, and medium follicles tended to increase to the greatest values at the day of CIDR device insertion. Furthermore, at day of mating, the numbers of large follicles reached the greatest value, which was associated with the lowest value of the number of corpora lutea. At day of mating, serum progesterone concentration achieved the lowest value, which increased until d 56 of pregnancy. The estradiol:progesterone ratio showed the opposite trend. The detrimental effect of reproductive seasonality, expressed as cessation of estrus behavior and fertile mating during the nonbreeding season, was successfully alleviated by the CIDR-eCG protocol. Furthermore, melatonin implantation in conjunction with the CIDR-eCG protocol enhanced conception rate and fecundity at d 28 of pregnancy and prolificacy at d 56 of pregnancy compared with does that were not implanted. Interestingly, does that failed to conceive did not come to heat again. In conclusion, the beneficial effect of melatonin implantation in conjunction with the CIDR-eCG protocol on the luteotrophic effect was reflected in the increasing number of corpora lutea, increasing progesterone concentration, and decreasing estradiol concentration. Furthermore, conception rate, prolificacy, and fecundity were improved compared with does that were not implanted during the nonbreeding season.

Ewes carrying the Booroola and Vacaria prolificacy alleles respond differently to ovulation induction with equine chorionic gonadotrophin.

The magnitude of ovulation rate (OR) after hormonal induction in sheep should be considered when prolific genotypes are used. We investigated for the first time the effect of the Vacaria allele and its combined effect with the Booroola prolificacy mutation on OR after hormonal treatment during breeding and anoestrous season. A hundred forty-nine Ile de France crossbred ewes, raised in natural pastures in South Brazil, were used to evaluate the OR after treatment with progestagen (MAP) followed or not by equine chorionic gonadotrophin (eCG) treatment (MAP + eCG). During the breeding season, 96% MAP-treated ewes ovulated in comparison to 97% of MAP + eCG-treated females. The double heterozygous carriers (BNVN) presented the higher OR, followed by the single Vacaria (NNVN) and Booroola (BNNN) heterozygous females and least the wild-type (NNNN) ewes. During anoestrus, 96% eCG-treated ewes ovulated, in contrast to 6% treated with MAP alone. The OR of the gonadotrophin-treated females was higher in BNVN and BNNN than NNVN and NNNN ewes. An additive effect in the OR of the two mutations was observed since OR in double heterozygous ewes was similar to the sum of the effects of the alleles of the single heterozygous carrier ewes.

Lack of effects of an equine chorionic gonadotropin (eCG) administration between days 9 and 15 postpartum on reproductive performance in a Holstein dairy herd.

Recently, it has been demonstrated that administration of equine chorionic gonadotropin (eCG) in the postpartum period in dairy cows can enhance follicle growth, reduce the interval from calving to first ovulation and increase plasma estradiol concentrations, and, thus, could enhance reproductive performance in a dairy herd when administered on day 6 postpartum. The aim of this study was to investigate the effects of a single dose of eCG between days 9 and 15 postpartum on parameters of reproductive performance in dairy cows. German Holstein cows (n = 1937; primiparous cows: n = 748; pluriparous cows: n = 1189) in a commercial dairy farm were randomly assigned to three experimental groups. Animals within the group eCG received a single dose of 600 IU eCG intramuscularly (i.m.) between days 9 and 15 postpartum followed by an i.m. administration of 500 µg cloprostenol after 14 days. Those of treatment group PG received cloprostenol only between days 23 and 29 postpartum. Cows of the control group remained untreated. Starting on day 49 postpartum, cows were subjected to a Presynch-Ovsynch protocol and inseminated artificially. The impact of application time (days postpartum) of eCG on the intervals calving to first service and calving to conception was statistically not significant. Outcomes of reproductive performance (i.e. first service conception rate, proportion of pregnant cows until 100 and 150 days in milk [DIM], number of inseminations until 150 DIM, calving to first service interval and calving to conception interval) did not differ significantly between treatment group eCG and group PG compared to control group. Regarding postpartum eCG administration, significant interactions between treatment and parity, season, milk yield, and early puerperal disorders, respectively, could not be shown. In conclusion, an eCG treatment of dairy cows between days 9 and 15 postpartum to increase reproductive performance cannot be recommended under the given circumstances.

Administration of exogenous hormones in ovulatory and embryonic response in Pelibuey sheep.

The objective of this study was to evaluate the effect of two sources of commercial porcine pituitary-derived follicle-stimulating hormone (pFSH) and pFSH-porcine Luteinizing Hormone (pLH), including equine chorionic gonadotropin (eCG), in ovulatory and embryonic response in Pelibuey sheep. Twenty-four Pelibuey sheep were used and were assigned randomly to four treatments (n = 6): (T1; 200 mg pFSH-Folltropin® ); (T2; 200 mg pFSH + 300 UI eCG-Folligon® ); (T3; 250 UI pFSH/pLH-Pluset® ) and (T4; 250 UI pFSH/pLH + 300 UI eCG). The interval of hours from withdrawal of the device to the beginning of oestrus (BO) was lower (p < .05) in sheep treated with eCG (T2 = 8.0 ± 1.4 and T4 = 10.0 ± 2.8) than in those without eCG (T1 = 12.6 ± 0.6 and T3 = 20.6 ± 2.4). The ovulatory rate (OR) was higher (p < .05) in T1 = 15.5 ± 2.8 and T2 = 15.6 ± 1.4, compared to T3 = 8.1 ± 3.2 and T4 = 11.8 ± 2.8; a significant difference was not shown between them (T1 vs. T2 and T3 vs. T4) when including eCG. The number of non-fertilized oocytes (NFO) was lower (p Ë‚ .05) in T1 = 0.8 ± 0.4 and T3 = 1.8 ± 1.8, compared to those that included eCG (T2 = 6.3 ± 2.4 and T4 = 2.1 ± 1.2). The number of transferable embryos (TE) was higher (p < .05) when FSH was applied (T1 = 5.8 ± 1.1), compared with (T2 = 2.6 ± 1.1, T3 = 2.3 ± 1.4 and T4 = 2.8 ± 1.5). The commercial treatments (pFSH or pFSH-pLH) in combination with eCG did not improve OR, NFO and TE. However, the exclusive pFSH (Folltropin) treatment presented a higher OR, lower number of NFO and higher number of TE.

Distribution pattern of cytoplasmic organelles, spindle integrity, oxidative stress, octamer-binding transcription factor 4 (Oct4) expression and developmental potential of oocytes following multiple superovulation.

The aim of the present study was to determine the effects of repeated superovulation on oocyte quality and embryo developmental potential. Female Swiss albino mice were injected with 5IU pregnant mare's serum gonadotropin followed 48h by 10IU human chorionic gonadotropin. Mice were superovulated up to four times with a gap of 7 days between each superovulation cycle. Ovarian weight increased significantly with an increasing number of superovulation cycles. Although the first stimulation cycle resulted in a threefold increase in the number of oocytes, the number of oocytes decreased gradually after subsequent stimulations. Increased cytoplasmic fragmentation, abnormal mitochondrial distribution, aggregation of Golgi apparatus, spindle damage, increased intracellular oxidative stress and a decrease in expression of octamer-binding transcription factor 4 (Oct4) expression were observed in these oocytes. Further, embryos derived from mice subjected to multiple stimulation cycles exhibited a low blastocyst rate, decreased hatching rate and increased apoptosis in blastocysts. In conclusion, the present study demonstrates that repeated superovulation adversely affects mouse oocyte quality by altering the distribution of cytoplasmic organelles, increasing oxidative stress and decreasing Oct4 expression, resulting in poor developmental potential of the embryos.

Effect of combined exogenous progesterone with luteotrophic support via equine chorionic gonadotrophin (eCG) on corpus luteum development, circulating progesterone concentrations and embryo development in cattle.

The aim was to examine the effect of a single intramuscular (i.m.) injection of equine chorionic gonadotrophin (eCG) on Day 3 after oestrus on corpus luteum (CL) development, circulating progesterone and conceptus development in cross-bred beef heifers. In Experiment 1, heifers received: (1) saline, or a single i.m. injection of eCG on Day 3 at (2) 250IU (3) 500IU (4) 750IU or (5) 1000IU. Administration of eCG resulted in increased luteal tissue area and progesterone and oestradiol concentrations compared with controls. In Experiment 2, heifers received (1) a progesterone-releasing intravaginal device (PRID Delta) from Day 3 to 5 or (2) a PRID Delta from Day 3 to 5 plus a single injection of 750IU eCG on Day 3. In vitro-produced blastocysts (n=10 per recipient) were transferred on Day 7 and heifers were slaughtered on Day 14 to assess conceptus development. Administration of eCG reduced the number of short cycles (6.3% vs 31.3%) and increased mean luteal tissue weight (P=0.02). Insertion of a PRID Delta on Day 3 resulted in an elevation (P<0.05) in serum progesterone until removal on Day 5. Administration of eCG at the time of PRID Delta insertion resulted in higher progesterone levels (P<0.05) from Day 10 onwards. Conceptus dimensions were not affected. In conclusion, a single injection of eCG on Day 3 increased CL size and progesterone concentrations and, when given in conjunction with a progesterone-releasing device, appeared to reduce the number of short cycles, presumably due to its luteotrophic nature. The implications of the elevated oestradiol concentrations for embryo quality require further study.

Pre-Selection Test to Identify High Responder Donor Goats.

The aim of the study was to evaluate the feasibility of pre-selection of high or low responder does prior to the superovulatory protocols. Twenty Saanen does received 800 IU of equine chorionic gonadotropin (eCG) at the end of long-term progestogen treatment. Fourteen days later, a second progestogen protocol associated with a multiple-dose follicle stimulation hormone (FSH) treatment (5 IU/kg of FSH, in six decreasing doses between days 4 to 6 of the protocol) was administered. Transrectal ultrasound was used to assess the follicular status at the beginning of superovulatory treatments, at the oestrous onset and on the seventh day of the oestrous cycle for counting corpora lutea (CL). A significant lower number of CL was obtained in eCG-treated in comparision with FSH-treated does (p < 0.05). A quartic regression was able to explain the relationship between the number of CL in response to both treatments (r(2) =0.50; p < 0.05). Seventy per cent (14 of 20) of does maintained the same ovulatory response (high or low) after treatments. The Kappa (κ = 0.40; p < 0.05) and Spearman (rs = 0.39; p = 0.08) coefficients were able to show a relationship between treatments. Regarding the follicular status, there is a significant relationship between the number of small follicles (r = 0.71; r(2) =0.47; p < 0.01) and total follicles (r = 0.60; p < 0.01) at eCG and first FSH dose with the number of CL. Moreover, it was found a negative relationship between the presence of large follicles and the number of CL in response to eCG treatment (r = -0.44; p < 0.05), but not from FSH (p > 0.05). In conclusion, the screening test with eCG has the potential to identify Saanen does that will better respond to the superovulatory protocol with FSH. In addition, it highlighted the importance of an ultrasound evaluation prior to the beginning of superovulatory treatments with FSH to characterize the follicular status and identify the potential donors of high ovulatory response in MOET programmes in goats.

Fixed-time induction of ovulation in camels superovulated by different eCG modalities during the transition period in Egypt : Superovulation in camels during the transition period.

The current investigation aimed to establish a fixed-time induction of ovulation/ insemination protocol in camels superovulated by different equine chorionic gonadotropin (eCG) regimens during the transition period in Egypt (mid-October to mid-November). Seventeen pluriparous camels, Camelus dromedarius, were used. All females retained controlled intra-vaginal drug releasers (CIDRs) for 13 consecutive days, and at CIDR withdrawal, the camels were randomly divided into three groups. The control group (n = 5) received 1 ml saline intra-muscularly (i.m.), whereas remaining camels were superovulated by 2500 IU eCG either in a single shot (SS, n = 6) or in serial decreasing doses over 3 days (DD, n = 6). Ovarian dynamics were monitored by transrectal ultrasonography at 2-day intervals, and ovulation was induced by 5000 IU hCG i.m. The changes in reproductive hormones throughout the period of the study were determined. The results showed that mean values of total no. of follicles and size of dominant follicles remained low (P < 0.05) in all groups until day of CIDR removal. Thereafter, total follicle no. increased (P < 0.05) in both superovulated groups compared to the control, where the dominant follicles attained the highest (P < 0.05) diameter 12 days after the eCG treatment. Double-ovulation rate was higher (P < 0.05) in SS (50%) and DD (66.6%) groups compared to that of control (0.0%). However, 33.3% of the SS group developed large anovulatory follicles (ø > 25 mm), which did not respond to induction to ovulation. These results elucidate that eCG administration in serial decreasing doses generates a reliable superovulatory response in camels, and ovulation can be blindly induced 12 days after the gonadotropin treatment. This fixed-time hormonal protocol represents a sufficient alternative to conventional day-to-day ultrasonography and would have profound implication for enhanced fertility in dromedary camels by facilitating infield application of embryo transfer technique.

Preovulatory follicle diameter, growth rate and time of ovulation during induced oestrus using a CIDR® in trypanotolerant female Bos taurus N'Dama cattle.

The aim of this study was to assess the dose (300 to 600 IU) effects of equine chorionic gonadotropin (eCG) on the preovulatory follicle diameter, growth rate and time of ovulation characterized by echography. The eCG was injected at the end (D0) of the 7-day treatment with a controlled internal device release (CIDR®) and a PGF2α being injected 2 days before the removal of the CIDR® (d-2). The 120 N'Dama female were distributed into five experimental groups. The control group (n = 26) was treated with physiological saline at the removal of the CIDR®, while the animals in the four treated groups received, respectively, 300 IU (n = 25), 400 IU (n = 24), 500 IU (n = 22) and 600 IU (n = 23) of eCG. The diameter of the preovulatory follicle was significantly higher (P < 0.05) in the animals treated with 300 IU (10.1 ± 1.4 mm) than in untreated animals (9.3 ± 1.2 mm). Follicle growth rate was significantly (P < 0.05) higher in treated animals (1.0 ± 0.4 mm/day) than in the control group (0.9 ± 0.4 mm/day). The average interval between the time of eCG injection and ovulation was similar in the non-treated (83.7 ± 14.4 h) and treated animals (79.7 ± 11.9). Treated animals showed a significant increase in the percentage of ovulation (94.7 % compared to 73.1 %) (P < 0.01). Use of eCG contributed towards synchronising the time of ovulation between 72 to 96 h, which would facilitate the use of systematic insemination.