Pregnant Mare Serum Gonadotropin Injection Research Articles

Influence of repeated estrus synchronization treatment on hormone secretion, growth, and development of dairy goats.

In large-scale intensive farms, dairy goats often undergo frequent estrus synchronization (ES) treatment, which may result in a decline in reproductive performance; however, the underlying mechanism remains unclear. The present study aimed to investigate the effect of pregnant mare serum gonadotropin (PMSG) and progesterone (P4)-mediated ES treatment on fertility in dairy goats, while also identifying key metabolic and endocrine mechanisms that influence reproductive performance in does subjected to repeated ES treatment. Forty-eight Saanen does were randomly assigned to two groups (24 goats each) that received ES treatments either thrice fortnightly (3-PMSG) or once (1-PMSG) simultaneously with the third ES treatment of the 3-PMSG group during the breeding season. ES treatment was performed via the intravaginal insertion of a controlled internal drug release (CIDR) device impregnated with 300 mg P4, followed by 300 IU PMSG injections 48 h before CIDR withdrawal. Blood was collected to detect the level of hormones and blood biochemical indices. Additionally, estrus rate, fecundity rate, body weight, size, and lactation performance were measured. The results showed that repeated ES treatment markedly decreased the estrus rate and fecundity rate of goats. Among the does in all groups, there was no substantial difference in follicle stimulating hormone, luteinising hormone, gonadotropin-releasing hormone, melatonin, growth hormone, PMSG, total cholesterol, total protein, and glucose levels, as well as the body weight, body size, and lactation performance. Repeated ES treatment elevated estrogen (E2) levels 36, 48, and 72 h post-CIDR removal; increased P4 upon CIDR insertion; and raised PMSG antibody levels 24, 48, and 72 h post-CIDR removal. The results suggest that elevated anti-PMSG levels are the primary reason for the decline in ES efficiency, and that high E2 and P4 levels at some time points also impair reproductive performance. These findings provide novel insights into the metabolic effects of repeated PMSG stimulation in goats, guiding future reproductive hormone use in breeding practices.

Quantitative Histomorphometry of Pre-ovulatory Follicles and Uterine Glands of Ongole-grade Heifer in Response to the Low Doses of PMSG Administration

Injection of female mammalian animals with gonadotropin prior to mating using superovulation dose increases estradiol 17beta (E2) synthesis and secretion and improves the growth and development of the uterus to support prenatal growth with the side effect of increasing litter size. This experiment was designed to study the effects of low doses of PMSG injection in heifers on the growth of granulose cells (GC) of pre-ovulatory follicles, E2 synthesis, and uterine glands growth and development. Nine Ongole-grade heifers were divided into three groups of doses of PMSG injection, i.e., 0 IU/kg BW, 0.5 IU/kg BW, and 1.0 IU/kg BW. The injection of pregnant mare serum gonadotropin (PMSG) was conducted at the early stage of the second follicle wave. Blood samples were collected at estrus (day 0) to measure plasma E2 concentrations. The ovary and uterus were collected for histological observation after slaughtering the animal. The results showed that non-superovulation doses of PMSG injections at the second follicle wave significantly increased the growth of GC that eventually produced a significantly higher plasma E2 concentration by 39.47% and 113.57%, in heifers receiving 0.5 IU/kg BW and 1.0 IU/kg BW (p<0.05), respectively. The increase in E2 synthesis and secretion significantly stimulated the growth and development of uterine glands (p<0.05), which was reflected in the improved measures of uterine glands. In conclusion, injections of heifers with low doses of PMSG at the second follicle wave could increase the growth of GC to produce a higher secretion of E2 that eventually improves the growth and development of uterine glands in the endometrium without the risk of multiple calving.

The Corticosterone-Glucocorticoid Receptor-AP1/CREB Axis Inhibits the Luteinizing Hormone Receptor Expression in Mouse Granulosa Cells.

Under stress conditions, luteinizing hormone (LH)-mediated ovulation is inhibited, resulting in insufficient oocyte production and excretion during follicular development. When the body is stressed, a large amount of corticosterone (CORT) is generated, which will lead to a disorder of the body’s endocrine system and damage to the body. Our previous work showed that CORT can block follicular development in mice. Since LH acts through binding with the luteinizing hormone receptor (Lhcgr), the present study aimed to investigate whether and how corticosterone (CORT) influences Lhcgr expression in mouse ovarian granulosa cells (GCs). For this purpose, three-week-old ICR female mice were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG). In addition, the treatment group was injected with CORT (1 mg/mouse) at intervals of 8 h and the control group was injected with the same volume of methyl sulfoxide (DMSO). GCs were collected at 24 h, 48 h, and 55 h after PMSG injection. For in vitro experiments, the mouse GCs obtained from healthy follicles were treated with CORT alone, or together with inhibitors against the glucocorticoid receptor (Nr3c1). The results showed that the CORT caused a downregulation of Lhcgr expression in GCs, which was accompanied by impaired cell viability. Moreover, the effect of the CORT was mediated by binding to its receptor (Nr3c1) in GCs. Further investigation revealed that Nr3c1 might regulate the transcription of Lhcgr through inhibiting the expression of Lhcgr transcription factors, including AP1 and Creb. Taken together, our findings suggested a possible mechanism of CORT-induced anovulation involving the inhibition of Lhcgr expression in GCs by the CORT–Nr3c1–AP1/Creb axis.

Superovulatory responses using pregnant mare serum gonadotropin hormone in Murrah buffalo cows

Superovulation plays an important role in embryo transfer program. A preliminary study carried out in the Research Institute for Animal Production evaluated superovulatory responses in Murrah buffalo cows using pregnant mare serum gonadotropin (PMSG) hormone. The three buffalo cows were estrus synchronized using 5 ml prostaglandin (PGF) twice, with an interval of 11 days. PMSG was injected intra-muscularly 3000 IU on day-10 after estrus. Prostaglandins were administered 48 hours after PMSG injection. Fixed-Time artificial insemination (FTAI) was carried out at 72 hours after the last PGF treatment. Administration of hCG 2 ml/head was given at the time of FTAI. A non-surgery flushing was performed on day 6 after FTAI. Parameters observed using ultrasonography (USG) were diameter of follicle (DFL), total follicle (TFL), and number embryos (NE). Data were analyzed descriptively. The mean of DFL before PMSG treatment was 8.2 mm and after PMSG treatment was 12.5 mm. The mean of TFL before PMSG treatment was 7.7 and after PMSG treatment was 16.1. The NE obtained was one degenerative embryo. Superovulation using PMSG increased TCL and DFL. It can be concluded that the Murrah buffalo cows superovulated by PMSG showed a good response but no transferable embryo was found.

Determining the Effects of Serial Injections of Pregnant Mare Serum Gonadotropin on Plasma Testosterone Concentrations, Testicular Dynamics, and Semen Production in Leopard Geckos (Eublepharis macularius).

Simple SummaryCurrent estimates have determined that one in five reptile species are already threatened with extinction. The aim of this study was to determine if pregnant mare serum gonadotropin could be used in leopard geckos (Eublepharis macularius) to stimulate the production of sperm and increase their testosterone concentrations. This information may aid in the development of breeding programs for endangered gecko species in the future that will be useful for conservation efforts. Our results demonstrated that this hormone did stimulate sperm production and increased testicular size in leopard geckos. However, it did not increase testosterone concentrations under the current conditions between October–December in the Northern hemisphere. Ultimately, future studies are needed to further characterize the annual reproductive cycle of leopard geckos.Reptiles are highly susceptible to anthropogenic activities as a result of their narrow geographical ranges and habitat specialization, making them a conservation concern. Geckos represent one of the mega-diverse reptile lineages under pressure; however, limited assisted reproductive technologies currently exist for these animals. Exogenous pregnant mare serum gonadotropin (PMSG) has been found to exhibit follicle stimulating hormone-like action and has been routinely used to alter reproductive hormones of vertebrates in assisted reproductive protocols. The purpose of this study was to determine the effects of serial injections of 20 IU and 50 IU PMSG on circulating testosterone concentrations, testicular dynamics, and semen production in a model species of gecko. Twenty-four captive-bred, adult, male leopard geckos (Eublepharis macularius) were divided into three treatment groups and administered a once-weekly injection of either PMSG or saline for a total of nine weeks. Ultrasonographic testicular measurements, electrostimulation for semen collection, and venipuncture were performed on days 0, 21, 42, and 63. Right unilateral orchidectomies and epididymectomies were performed in all animals on day 63; tissues were submitted for histopathology. PMSG treated geckos had significantly higher testicular volumes and weights, spermatozoa motility, and spermatozoa concentrations compared with controls. However, there were no significant differences in testosterone concentrations by treatment or time. Under the conditions outlined, PMSG is effective at stimulating spermatogenesis and increasing testicular size, but not effective at increasing testosterone concentrations in the leopard gecko between October–December in the Northern hemisphere.

Improvement of First-Service Conception Rate in Dairy Cows after the Injection of Pregnant Mare Serum Gonadotropin Six Days Postpartum

BACKGROUND: Early resumption of ovarian activity after parturition could positively influence the fertility of dairy cows. Ovulation of the first postpartum follicular wave and formation of corpus luteum that secretes progesterone can sub-stantially benefit the resumption of ovarian cyclicity in cows. In this regard, the presence of a dominant follicle in the ovary before day 10 postpartum could enhance ovulation rate in the first postpartum follicular wave. Pregnant mare serum gon-adotropin (PMSG) is one of the hormones with the potential to improve the growth of follicles. OBJECTIVES: Accordingly, the present study was conducted to evaluate the effect of PMSG injection six days after parturition on reproductive parameters in dairy cows. METHODS: The subjects were randomly assigned to the two groups of control (N=74) and treatment (N=79). Cows in the control group received no treatment, while animals in the treatment group received 500 IU PMSG on day six postpartum. In both experimental groups, cows were subjected to Presynch-Ovsynch protocol 30-35 days postpartum. The subjects that experienced estrus during the Presynch-Ovsynch protocol were inseminated. On the other hand, the cows that were not in estrus during this period were subjected to fixed-time insemination at the end of the Presynch-Ovsynch protocol. Pregnancy was diagnosed 45 days after artificial insemination using the rectal examination. RESULTS: In the control group, 66.22% of cows were inseminated based on estrus detection and 33.78% of cows were inseminated at fixed times. In the intervention group, 68.35% of cows were inseminated based on estrus detection and 31.65% of cows were inseminated at fixed times. The first-service conception rate (FSCR) was 27.03% and 48.1% in the control and treatment groups, respectively (p =0.01). CONCLUSIONS: The present study showed that the injection of 500 IU PMSG six days after parturition could enhance FSCR in dairy cows.

Histochemical Study of the Rat Uterine Glycoconjugate Alteration following Treatment with Exogenous Gonadotropic Hormones during the Implantation Period.

One of the female causes of infertility is anovulation which is treatable with gonadotropin hormones. These hormones affect the molecular organization of the uterus such as glycoconjugates that are the first site of contact between the blastocyst and the uterus. The objective of this project was to study the alteration of glycoconjugates on the uterine apical, Golgi zone, and basement membrane of epithelial cells and the uterine gland after hyperstimulation with pregnant mare serum gonadotropin (PMSG) (4, 8, 16, 24, and 40 IU), during the implantation period. Injection of PMSG (in experimental groups) and injection of distilled water (in the control group) were followed by HCG administration (10 IU), mating, isolation of positive vaginal plug rats, and killing at 5.5 days of pregnancy. Histochemistry was done on the pregnant uterine horns with the use of WGA, DBA, PNA, ConA, SBA, and UEA lectins. The intensity of the immunohistochemical staining was scored, and quantitative data were generated. 4 IU did not show any significant differences with the control, 8 IU had less effect on the alteration of the Golgi zone, and apical and basement membrane glycoconjugates and 40 IU had the least effects on the alteration of uterine gland glycoconjugates. Also, 24 IU had the most effect on the alteration of uterine glycoconjugates. Understanding of the effects of gonadotropin hormones at the uterine level in implantation time helps to optimize hormonal manipulation for improving the outcome of assisted reproductive procedures. It seems that the optimal dose for superovulation and less alteration in uterine glycoconjugates of rats at implantation time were induced by the administration of 8 IU PMSG.

Oxytocin and cabergoline alleviate ovarian hyperstimulation syndrome (OHSS) by suppressing vascular endothelial growth factor (VEGF) in an experimental model.

Ovarian hyperstimulation syndrome (OHSS) is a life-threatening complication of ovarian stimulation in reproductive medicine. Here, we aimed to investigate the role of oxytocin (OT) and cabergoline in the prevention and alleviation of the OHSS in an animal model. Thirty-five female immature Wistar rats were randomly assigned to five groups. The control group (n = 7) received saline only for five consecutive days. Remaining twenty-eight rats received 10IU of pregnant mare serum gonadotropin (PMSG) followed by 30IU of human chorionic gonadotropin (hCG) to induce OHSS. Group 2 (n = 7) was managed with no additional intervention after the induction of OHSS. Group 3 (n = 7) received 100μg/kg cabergoline 2h before the PMSG injection for four consecutive days and 2h before the hCG injection on the fifth day. Group 4 (n = 7) and group 5 (n = 7) received 80μg/kg and 160μg/kg OT after induction of OHSS, respectively. Oxytocin was administered 2h before the PMSG injection for four consecutive days and 2h before the hCG injection on the fifth day. Body and ovary weight, vascular permeability (VP), VEGF expression in the ovaries, and levels of VEGF in the peritoneal fluids were examined in all animals. Cabergoline and OT reduced body weight, ovary weight, and VP compared to that of the OHSS group (p < 0.05). VEGF expressions in ovaries and peritoneal VEGF levels were decreased in cabergoline and OT groups compared to that of the OHSS groups (p < 0.001 for cabergoline and OT-80μg/kg; p < 0.00001 for OT-160μg/kg). However, there was no statistically significant difference in these parameters between the OT and cabergoline groups. Both OT and cabergoline were active in the alleviation of OHSS through suppression of VEGF and VP. Overall, we conclude that OT is effective for downregulation for VEGF and improvement in vascular permeability in OHSS.

Topical application of cervix with hyaluronan improves fertility in goats inseminated with frozen-thawed semen.

ObjectiveArtificial insemination plays an important role in genetic improvement in the goat farming system. The aim of this study was to investigate the effect of cervical application of hyaluronan (HA) on the fertility in goats after cervical artificial insemination using frozen-thawed (F-T) semen.MethodsAfter oestrous synchronisation with progesterone sponges and pregnant mare serum gonadotropin injection, both nulli- and multi-parous goats, were randomly allocated to 2 groups, and were inseminated with 0.25 mL of F-T semen (150×106 spermatozoa) twice at 52 h and 56 h after sponge removal. Prior to the insemination, goats in Group 1 only were given topical cervical HA application at 48 h after sponge removal. Site of insemination was recorded as os-cervix or intra-cervix or intra-uterus. Pregnancy was tested ultrasonographically 42 days after insemination. The data on pregnancy rates and percentage of animals according to the site of semen deposition were compared by Chi-square analysis.ResultsThe overall pregnancy rate was significantly (p<0.004) higher in goats with prior application to the cervix with HA (63.3%) than without (36.0%). Same pattern was observed in the pregnancy rates of nulli- and multi-parous goats in both the groups. Percentage of nulliparous goats according to the site of insemination in the HA group did not differ between first and the second insemination. However, in multiparous goats the percentage of animals inseminated intra-cervically was significantly increased (p≤0.05) between the first and the second inseminations.ConclusionThe results suggest that significantly higher fertility rate in the “HA goats” compared to the “without HA” group was because of deeper insemination facilitated by topical cervical application of HA. The deeper insemination into the cervical canal increase the rate of fertilisation when the cervical artificial insemination is performed.

Merino ewe lambing after stimulation with prostaglandin and PMSG and insemination with sperm of grey Karakul rambs

Aim. To determine the effectiveness of a two-time treatment with prostaglandin F-2α (PGF2α) followed by the injection of pregnant mare serum gonadotropin (PMSG) for stimulation of sexual activity in sheep in the second half of the estrous season in the scheme of increasing the lambing rate. Methods. Thirty-seven 2.5-yearold ewes of the Askanian Merino breed, which were kept together from the age of one year, were used. In September 2018, 28 animals of this group lambed and had sucklings until November 20. Hormonal stimulation was started on December 7, 2018 by injecting all ewes with 1 ml of synthetic PGF2α and 1 ml of Prozerin. The second injection of PGF2α was given 10 days after the fi rst one. Simultaneously with the second treatment with PGF2α, the animals were injected with 500 IE of PMSG (Sergon, Czech Republic) and 2.5 ml of SuperAminoC (South Korea). The presence of estrus was not detected, and the ewes were forcibly inseminated with freshly obtained sperm of 4 grey rams of the Askanian Karakul breed in the morning for three days, beginning with the fi rst day after the PMSG injection. During the fi rst treatment with PGF2α and 3 days before the second one, the genitals of 12 ewes were sonographically examined. Results. In total, 45.9 % of stimulated animals lambed; the total fecundity was 75.7 %, and the average prolifi cacy – 1.65 ± 0.18 lambs per ewe. Among the animals, which before the experiment had the sucklings, 57.1 % lambed, among those, who did not have the suckling, – 11.1 %. After using the sperm of ram 17314, 77.8 % of ewes lambed, for rams 73697, 73792 and 73796 – 30.0, 44.4 and 33.3 %, respectively. Among the 19 lambs analyzed by wool color, 10 lambs were completely black, 9 were black with white spots. Among the spotted lambs, the color on the back, forearms and hips of one lamb had an additional shade, close to gray. Ultrasound testing showed that the ewes, which before the experiment had sucklings, showed better genital reactivity. According to the distribution of lambs by wool color, an assumption about possible partial incompatibility of the genotypes of Askanian Merino ewes and grey Karakul sheep was made that requires further study. Conclusions. The scheme of stimulating sexual activity based on two-time treatment with PGF2α followed by an injection of PMSG is capable of ensuring lambing of more than 70 % of stimulated sheep with the prolifi cacy of 1.65 lambs per ewe. A factor, increasing the effectiveness of the scheme, is the presence of sucking lambs of ewes before the stimulation starts.

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 (14hr light) until late September (12hr light). ES treatment was performed via intravaginal insertion of a controlled internal drug release (CIDR) device impregnated with 300mg progesterone (P4), followed by 300IU PMSG injections 48hr 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.

Improving the Quality of Oocytes of Old and Productive Ages White Rats (Rattus norvegicus) Using Pregnant Mare Serum Gonadotrophin

Abstract This study was conducted to improve the quality of oocytes in old-female rats by using pregnant mare serum gonadotrophin (PMSG). Female rats at productive age were used as a control. The experimental rats were injected with 4 doses of PMSG i.e., 0, 2.5, 5.0, and 10 IU PMSG. After 2 weeks of acclimation to the experimantal condition, the experimental rats were injected with PGF2α at a dose of 25µg/g BW two times with 2 days interval to synchronize estrous cycle. PMSG injections were conducted at the same time with the second PGF2α injection. After PMSG injection, the experimental rats were divided into two groups rats, i.e., rats without mating for masurement of oocyte qualities and rats mated for measurement of offspring qualities. Therefore, 16 experimental rats from each age group were sacrifized for maeasurement of oocyte qualities. The other 16 experimental rats for each age group were mated for measurement of offspring qualities. Parameters measured were hematological profile, uterine and ovarian weights, the qualities of oocytes, the qualities of the offsspring born by using swimming test and rat maze test. The collected data were analyzed by analysis of variance (ANOVA) and continued with Duncan test with a 95% confidence level. The data were analyzed using SSPS. The results showed that the improvement in the quality of oocytes in old female rats using the PMSG hormone in this study showed an increase in the quality of oocytes in old and productive age rats. The highest number of oocyte quality was found in rats of productive age (3.25) and significantly different (P&lt; 0.05) from the other oocyte qualities. The quality of offspring born to old age and productive age rats injected with PMSG were improved. It was concluded that the improvement of oocyte quality by using PMSG also improves offspring qualities.

Production of lambs’ resilience to Haemonchus contortus

Haemonchus (H.) contortus infection in sheep is a major health problem in tropical and sub-tropical regions that cause great economic losses. Our previous studies have shown that improved uterine environments during pregnancy can improve postnatal growth and health performance of the offspring, indicated by lower mortality and morbidity. In the present experiment, we evaluated the resistance and resilience to H. contortus of lambs born to ewes injected with pregnant mare’s serum gonadotropin (PMSG) prior to mating. Improvement of the uterine environment was conducted by increasing endogenous secretion of estrogen and progesterone as pregnancy hormones during pregnancy by injecting the ewes with PMSG prior to mating. A total of 16 lambs, regardless of sex, at the age of 5 months were assigned into a 2 × 2 factorial experiment with 4 replications. The first factor was PMSG injection, consisting of two levels, i.e., lambs born to ewes without PMSG injection (Non-PMSG lambs) and those born to PMSG-injected ewes (PMSG lambs). The second factor was the infection of lambs with H. contortus at the age of 5 months, consisting of two levels, i.e., lambs without infection (Non-infected lambs) and lambs infected with H. contortus (Infected lambs). Non-infected lambs were administered with distilled water in a capsule without infective larvae. Infected lambs were individually infected with a single dose containing 1200 L3 of H. contortus. Compared to non-PMSG lambs, PMSG lambs tended to have better prenatal growth indicated by greater birth weights (P = 0.06). The improved prenatal growth during pregnancy improved the postnatal growth and health performance of the lambs. Three months after infection of H. contortus, non-PMSG lambs and PMSG lambs had similar worm counts. However, the PMSG lambs showed significantly higher resilience to H. contortus as indicated by the lower fecal egg counts 6–10 weeks after infection. The higher resilience of the PMSG lambs was shown by the positive growth rate during infection, while non-PMSG lambs had a negative growth rate after infection. Prior to infection, PMSG lambs showed a higher segmented neutrophil percentage with lower lymphocyte numbers. Three months after infection, PMSG lambs had significantly higher lymphocyte and thrombocyte numbers as well as mean corpuscular hemoglobin concentration (MCHC) with lower neutrophil and monocyte numbers. The conclusion of this study is that the improvement of the uterine environment during pregnancy could be used to produce superior offspring with high resilience to the infection of H. contortus.

Electroacupuncture facilitates implantation by enhancing endometrial angiogenesis in a rat model of ovarian hyperstimulation.

Controlled ovarian hyperstimulation (COH) impairs the synchronized development of endometrium and embryo, resulting in the failure of embryo implantation. Here, we investigated what effects electroacupuncture had on embryo implantation in COH rats. Female rats were randomly assigned to four groups: normal (N), model (M), electroacupuncture (EA), and electroacupuncture pretreatment (PEA). Rats in groups M, EA, PEA were injected with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin to establish the COH model. Rats in group EA received electroacupuncture treatment from the PMSG injection day to the 3rd day of pregnancy (D3), while those in group PEA received electroacupuncture treatment for 3 days before the PMSG day and continuing to D3. Furthermore, another 30 female rats who received the same treatment as the rats in group PEA were injected with siVEGFR2 into uterine lumen. The endometrial microvascular density (MVD) and the expression levels of vascular endothelial growth factor-A, angiopoietin-1, and fibroblast growth factor-2 were significantly lower in groups M than in groups N and PEA. The percentage of dolichos biflorus agglutinin positive uterine natural killer cells in groups N, EA and PEA was higher than that in group M. After the siVEGFR2 injection, the protein expression levels of vascular endothelial growth factor receptor 2 (VEGFR2), PI3K, p-AKT and p-ERK, the embryo number and the MVD were significantly reduced. In conclusion, electroacupuncture can facilitate embryo implantation in COH rats by activating the VEGFR2/PI3K/AKT and VEGFR2/ERK signaling pathways which have a positive relationship with endometrial angiogenesis.

Optimum dose and time of pregnant mare serum gonadotropin injections in Kacang goats to increase endogenous secretion of estrogen and progesterone without superovulation response

The present study was conducted to determine what the optimum dose and time of pregnant mare serum gonadotropin (PMSG) injections in Kacang does to increase endogenous secretion of estrogen and progesterone without increasing the number of ovulating follicles above the normal range. Forty-eight Kacang does were assigned to a 3×4factorial randomized experimental design with 4 replications. The first factor was the dose of PMSG injection, and it consisted of three levels: 0, 7.5, and 15IU/kg body weight (BW). The second factor was the time of PMSG injection at the four stages of follicular development: the pre-luteolytic, luteolytic, pre-antral, and antral stages. The experimental goats were injected twice with prostaglandin to synchronize their estrous cycles. The parameters measured included the number and diameter of the dominant follicles and serum estrogen concentrations during the pre-ovulatory period and the number and diameter of corpora lutea and serum progesterone concentrations during the post-ovulatory period. The diameter of each dominant follicle and corpus luteum was also calculated. The results showed that an increased dose of PMSG injection significantly increased the numbers, total diameters, and diameter of each dominant follicle and corpus luteum, as well as serum estradiol and progesterone concentrations (P<0.05). The time of PMSG injection significantly affected all parameters (P<0.05), except for the diameter of each dominant follicle and corpus luteum. There was a significant effect of the dose and time of injection of PMSG on all parameters, except on the diameter of each dominant follicle and corpus luteum. Average pre-ovulatory serum estrogen concentrations and post-ovulatory serum progesterone concentrations in does injected with 7.5IU/kg BW at the luteolytic stage increased by 133.68% and 545.11%, respectively. Injection of PMSG during the pre-antral and antral stages also increased estradiol and progesterone secretion to a lesser degree without increasing the number of ovulating follicles and corpora lutea. In conclusion, the results of the present study indicate that injection of 7.5IU PMSG/kg BW at the luteolytic stage could be used to increase the secretion of pregnancy hormones without increasing the number of ovulating follicles and corpora lutea above the normal ranges.

Estrus induction and fertility response following different treatment protocols in Murrah buffaloes under field conditions.

Aim:The aim of this study was to evaluate the efficacy of three different treatment protocols for estrus induction and conception rate in postpartum anestrus buffaloes during breeding season under field conditions.Materials and Methods:The 47 postpartum anestrus buffaloes of the 2nd to 6th parity were divided into three groups. Group 1 (n=16): Buffaloes received cosynch treatment, that is, buserelin acetate 10 µg on day 0 and 9, cloprostenol 500 µg on day 7 followed by fixed-time artificial insemination (FTAI) at the time of second buserelin acetate and 24 h later. Group 2 (n=15): Buffaloes received norgestomet ear implant subcutaneously for 9 days, estradiol benzoate 2 mg on the day of implant insertion (day 0), pregnant mare serum gonadotropin (PMSG) 400 IU and cloprostenol 500 µg on day 9 followed by AI at 48 and 72 h after implant removal. Group 3 (Cosynch-plus, n=16): Buffaloes received Cosynch protocol as per Group 1 except an additional injection of PMSG 400 IU (i.m.) was given 3 days before the start of protocol and FTAI done at the same time of Group 1. Pregnancy diagnosis was performed after 45 days of AI.Results:The estrus induction response following the treatment was 81.3%, 100%, and 93.7% in Group 1, 2, and 3, respectively. The buffaloes of Group 1, 2, and 3 expressed intense (38.4%, 60% and 46.6%, respectively) and moderate estrus (46.1%, 26.6%, and 40%, respectively). The conception rates in Group 1, 2, and 3, at FTAI and overall including subsequent estrus were 37.5% and 62.5%, 53.3%, and 66.6%, 56.3%, and 75%, respectively.Conclusion:All the three treatment protocols can be effectively used for induction of estrus with acceptable conception rate in postpartum anestrus buffaloes during breeding season under field conditions. However, Cosynch-plus (similar to Cosynch protocol except addition of PMSG, 400 IU 3 days before the start of first buserelin acetate administration) protocol results comparatively better pregnancy rate.

In vitro maturation of immature rat oocytes under simple culture conditions and subsequent developmental ability.

Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative method to produce numerous matured oocytes. Furthermore, establishment of an in vitro maturation system with simple culture conditions is cost effective and leads to easy handling of oocytes. This study examined developmental ability of rat germinal vesicle (GV) oocytes maturing in vitro under simple culture conditions. Significantly different numbers of ovulated oocytes reached the second metaphase of meiosis (MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/SsNSlc (2.2) rats in whom superovulation was induced by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. However, similar numbers of GV oocytes were obtained from ovaries of PMSG-injected Wistar (27.7), F344 (34.7), and BN (24.7) rats. These GV oocytes were cultured in vitro in HTF, αMEM, and a 1:1 HTF + αMEM or TYH + αMEM mixture. High proportions of Wistar and F344 oocytes that matured to MII in αMEM were parthenogenetically activated by strontium chloride treatment (78% and 74%, respectively). Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injection and embryo transfer to foster mothers. Although BN oocytes cultured in αMEM could be parthenogenetically activated and developed into offspring, the success rate was lower than that for Wistar and F344 oocytes. This study demonstrated that numerous GV oocytes were produced in rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed to maintain valuable rat strains experiencing reproductive difficulties.

GONAD REMATURATION ON Pangasionodon hypophthalmus FEMALE THROUGH INJECTION OF PREGNANT MARE SERUM GONADOTROPIN AND HUMAN CHORIONIC GONADOTROPIN

The success of spawning is influenced by internal and external factors. One of the factors that affect the var iabi li ty of Pangasianodon hypophthalmus female reproductive is the change of seasons that cause disrupted continuity of the seed availability, especially in the dry season. In the present study, combination of PMSG (pregnant mare serum gonadotropin) + HCG (hormone chorionic gonadotropin) hormone injections was done to induce gonad development. The treatments in this study were without hormone injections as control (A), injection of PMSG 10 IU/kg + HCG 10 IU/kg (B), and injection of PMSG 20 IU/kg HCG + 10 IU/kg (C). Injections were conducted at intervals of two weeks as many as six times. The results showed that gonad maturation generally occurs 2-4 weeks after estradiol-17 peak. PMSG + HCG hormone injections gave a significant effect on increasing the quantity and quality of eggs production. The fecundity in the A, B, C treatments, were 233,700±220,676; 300,305±24,581 and 488,433±142,228; respectively. Number of larvae produced from the A, B, C treatments, were 156,979±170,838; 229,997±18,081 and 362,713±101,850; respectively. Combination of PMSG 20 IU/kg + HCG 10 IU/kg hormone injection gave the best result on fecundity and the number of larvae production.

Fertilizability of Superovulated Eggs by Estrous Stage-independent PMSG/hCG Treatment in Adult Wistar-Imamichi Rats

We investigated the fertilization and developmental ability of superovulated eggs obtained from adult Wistar-Imamichi (WI) rats, by using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) treatment. Female WI rats, 11–13 weeks of age, were divided into four groups by estrous stage (metestrus [ME], diestrus [DE], proestrus [PE], or estrus [E]). PMSG (150 IU/kg) and hCG (75 IU/kg) were injected at an interval of 48 or 55 h and the female rats were mated with mature male rats. The ovulated eggs were collected 20, 24, and 27 h after hCG injection. Regardless of the estrous stage at the time of PMSG injection, the treated rats mated and ovulated similar to the untreated spontaneously ovulated rats (S group). Although the proportion of fertilized eggs in the E- and PE-treated groups was less than the S group 20 h after hCG injection, the proportion was not different among all treated and S groups 24 h after hCG injection. The proportion of fertilized eggs using in vitro fertilization and the proportion of offspring obtained from 2-cell stage embryo transfer did not differ among the treated and S groups. In comparison with PMSG/hCG-treated immature rats, mating and ovulation rate of adult rats were significantly higher. The proportion of fertilized eggs obtained from mated rats did not differ between immature and adult rats. These results demonstrate that adult WI rats are good egg donors for reproductive biotechnological studies using unfertilized or fertilized eggs.

214 SUPEROVULATION IN A/J MICE USING A COMBINATION OF GONADOTROPINS AND THE PTEN INHIBITOR bpV(pic)

Strain and individual differences in superovulation constitute a serious problem in mice. Gonadotropins stimulate the maturation of developing ovarian follicles, but not primordial follicle activation (PFA), which is negatively controlled by the Phosphatase and Tensin Homologue Deleted from Chromosome 10 (PTEN). PTEN inhibitors may increase the number of developing follicles by promoting PFA. If so, subsequent gonadotropin injections may make more follicles ovulate. This study tested whether PTEN inhibitors promote superovulation by gonadotropins and examined the fertilizability of ovulated oocytes in vitro. Method: Immature females of the low responder A/J mouse strain were used. Based on preliminary results regarding peaks in the daily changes in the ovary weight/body weight ratio and ovarian anti-Müllerian hormone protein content after injections of the PTEN inhibitor, dipotassium bisperoxo (picolinato) oxovanadate (V) [bpV(pic)], the number of ovulated oocytes was examined using four combinations of the bpV(pic) dose and interval between the bpV(pic) and pregnant mare serum gonadotropin (PMSG) injections: 1) 3 μg and PMSG on Day 3; 2) 3 μg and PMSG on Day 4; 3) 30 μg and PMSG on Day 1; and 4) 30 μg and PMSG on Day 2. Ovulation was induced by hCG 48 h after PMSG injection. In vitro fertilization of the obtained oocytes was performed using TYH medium and epididymal sperm from adult ICR male mice (Day 1). Subsequently, the oocytes were cultured in KSOMaa+0.1% bovine serum albumin for 4 days. The numbers of two-cell embryos and blastocysts were recorded on Days 2 and 5, respectively. Results: The average number of oocytes collected in treatments 1 to 4 was 1) 7.2 ± 0.7 v. 9.4 ± 1.6 [control v. bpV(pic), mean ± s.e.m., n = 5]; 2) 7.8 ± 0.7 v. 9.0 ± 1.4; 3) 9.8 ± 1.6 v. 11.2 ± 1.0; and 4) 9.4 ± 1.0 v. 7.4 ± 1.9. More oocytes tended to be collected in the bpV(pic) groups in treatments 1 to 3, but the differences were not significant on ANOVA. The corresponding percentages of two-cell embryos on Day 2 after insemination were 1) 78.7 ± 7.7% v. 83.7 ± 5.6%; 2) 76.0 ± 4.2% v. 65.2 ± 4.2%; 3) 53.5 ± 13.0% v. 68.6 ± 9.1%; and 4) 78.2 ± 9.7% v. 55.2 ± 5.7%. The respective percentages of blastocysts on Day 5 after insemination were 1) 62.7 ± 6.5% v. 69.5 ± 8.1%; 2) 67.9 ± 7.7% v. 41.6 ± 5.6%; 3) 74.9 ± 11.3% v. 79.2 ± 7.8%; and 4) 78.0 ± 3.8% v. 60.5 ± 4.3%. On weighted ANOVA with angular transformations, the two-cell embryo and blastocyst rates were significantly lower in bpV(pic) groups than in control groups for treatments 2 and 4. Discussion: More oocytes tended to be collected in the bpV(pic) groups in 3 of the 4 experimental treatments. Different injection intervals between bpV(pic) and PMSG influenced both the superovulation efficiency and fertilizability of the oocytes. This new method using both PTEN inhibitors and gonadotropins is a promising method for improving superovulation efficiency, although the optimal injection scheme should be determined. This work was supported by a grant from the Ministry of Health, Labour, and Welfare of Japan.