Pregnant Mare Serum Gonadotropin Research Articles

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.

Gonadotropin regulates NUCB2/nesfatin-1 expression in the mouse ovary and uterus

NUCB2/nesfatin-1 is expressed in the hypothalamus and regulates food intake and energy metabolism. Recent studies showed that NUCB2/nesfatin-1 also plays a role in other organs. However, its expression pattern and function in female reproductive organs are unclear. Therefore, we investigated NUCB2/nesfatin-1 expression in the ovary and uterus of mice and determined whether it is regulated by gonadotropins and sex steroid hormones. NUCB2 mRNA and nesfatin-1 protein were detected in the ovary and uterus of mice. NUCB2/nesfatin-1 expression in both organs was highest in the estrus period of the estrus cycle. Administration of pregnant mare serum gonadotropin (PMSG) dose-dependently increased mRNA expression of NUCB2 in the ovary and uterus of mice. On the other hand, mRNA expression of NUCB2 in the uterus was dramatically decreased after ovariectomy and was not increased upon administration of PMSG. Injection of 17β-estradiol upregulated mRNA expression of NUCB2 in the uterus of ovariectomized mice, whereas injection of progesterone did not. These results suggest that NUCB2/nesfatin-1 expression in the ovary and uterus of mice is regulated through the hypothalamus-pituitary-ovary axis and that NUCB2/nesfatin-1 is a local regulator of ovarian steroidogenesis and uterine function.

Effect of Shoutai Pills on Th1/Th2 Cytokines in Serum and Endometrium of Rats with Stimulated Ovulation.

In our previous study, we found that Shoutai pills could improve the embryo implantation rate as well as the levels of estrogen, progesterone and estrogen receptor in rats with stimulated ovulation. However, the mechanism is not clear. This study was designed to investigate the effect of Shoutai pills on the levels of Th1 and Th2 cytokines in rats with stimulated ovulation and the mechanism. The rat model of stimulated ovulation was established by combined injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG). Then the rats were randomly divided into model group (M), Shoutai pills group (S), progesterone group (P) and normal group (N). All the pregnant rats were treated from the first day. The S and P groups were administrated with gavage of Shoutai pills and injection of progesterone respectively, and N and M groups were given the same volume of normal saline and distilled water respectively. After treatment for 7 days, the animals were executed for serum and uterine tissues. The ELISA method was adopted to detect the contents of Th1 cytokines [interferon-γ (INF-γ), interleukin-2 (IL-2)] and Th2 cytokines (IL-4, IL-6, IL-10). The expression of leukemia inhibitory factor (LIF) and leukemia inhibitory factor receptor (LIFR) was detected by Western blotting and real-time PCR. As compared with N group, the expression levels of IFN-γ and IL-2 in M group were significantly increased, and those of IL-4, IL-6, IL-10, LIF and LIFR were significantly decreased (P<0.05). As compared with M group, the levels of IL-4, IL-6, IL-10, LIF and LIFR in S group were significantly increased (P<0.05), and those of IFN-γ and IL-2 were significantly decreased (P<0.05). It was suggested that Shoutai pills can increase the levels of IL-4, IL-6, IL-10, LIF and LIFR as well as reduce the levels of INF-γ and IL-2 in rats with stimulated ovulation. The Shoutai pills may improve endometrial receptivity and promote embryo implantation by maintaining the balance of Th1/Th2 cytokines.

The kisspeptin analog C6 is a possible alternative to PMSG (pregnant mare serum gonadotropin) for triggering synchronized and fertile ovulations in the Alpine goat.

In temperate regions goat’s reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG.

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.

Choice of protocol for the in vivo bioassay of equine Chorionic Gonadotropin (eCG / PMSG) in immature female rats

Equine Chorionic Gonadotropin (eCG) previously known as Pregnant Mare Serum Gonadotropin (PMSG) has been used for decades in regulating reproduction in various domestic animal species. Its use necessitates a good measurement of its bioactivity in commercial preparations. The EUROPEAN PHARMACOPEIA (EP 7.0) recommends 5–6 subcutaneous injections in immature female rats for the in vivo bioassay for eCG as in the case for measurement of FSH bioactivity in the Steelman & Pohley assay (1953). This recommendation is in marked contrast with the classical and long-time used PMSG assay of Cole & Erway (1941) that includes only one subcutaneous injection, 3 days before measurement of ovarian weight. As this difference introduces much confusion in the determination of eCG/PMSG bioactivity in commercial sources, we have performed parallel assays of several PMSG preparations, with both protocols. The single-injection protocol takes into account the long half-life of eCG in bloodstream and provokes an ovarian stimulation at lower concentrations than the multiple-injection protocol. As the single-injection protocol also mimicks the protocol used in cattle, it is preferable to the multiple-injection protocol of the current EP.

Ovarian Stimulation Increases the Risk of Fetal Cardiac Defects of Pups Exposed to Severe Maternal Hyperglycemia

To study the incidence of congenital heart defects (CHD) in offspring born to hyperglycemic mothers with and without ovarian stimulation. Reproductive biology. Mouse model. N/A. Hyperglycemia was induced in CD-1 wild type female mice using a single intraperitoneal dose of 150 mg/kg of streptozotocin. Stimulated dams (SD); (n=3) were injected with pregnant mare serum and human chorionic gonadotropin 48 hours apart. Non-stimulated dams (NSD); (n=4) were not injected. Both groups were mated with normal male CD-1 mice for timed pregnancies. Fetal hearts were extracted on embryonic day 16.5 and histological analyses was performed. Student's t-tests were employed to compare the incidence of cardiac defects in the SD and NSD groups. P ≤ 0.05 was significant. The incidence of CHD in progeny of diabetic dams with and without ovarian hyperstimulation. The average litter size was higher in SD compared to NSD. The average blood glucose for the SD and NSD was similar. Overall, the incidence of cardiac malformations did not differ between the two groups. However, in severe maternal hyperglycemia (>400 mg/dL), there was a higher incidence of fetal cardiac malformations in the pups born to SD vs NSD.

Gonadotrophin stimulation of ewes that are not pregnant following multiple matings during the season

In this study, the aim was to obtain pregnancy following synchronization and to increase profitability in ewes that did not become pregnant during the breeding season after at least 3 matings. Nonpregnant Middle Anatolian Merino ewes (n = 117) were given a progesterone sponge for 9 days. At the time of sponge removal, the ewes received 250 IU pregnant mare serum gonadotropin (PMSG) + 125 µg prostaglandin F2α (PGF2α). Two days later, they were injected with either human chorionic gonadotropin (hCG) (Group I, n = 48) or gonadotropin-releasing hormone (GnRH) (Group II, n = 43), or remained as a control (Group III, n = 26). The ewes were allowed to mate at estrus following the PGF2α injection. Pregnancy diagnosis was performed on day 30 after mating by ultrasonography. Group ratios for estrus (58.3%, 55.8%, 53.8%), conception (78.6%, 87.5%, 14.3%), lambing (90.9%, 90.5%, 50.0%), multiple births (15.0%, 31.6%, 0.0%), and average lamb number for birth (1.15%, 1.31%, 1.0%) were reported. In conclusion, the gonadotropin-supplemented protocols used here were found to be useful in ewes that were not pregnant after multiple matings during the breeding season in increasing the profitability of flocks with this problem.

Progesterone and pregnant mare serum gonadotropin improve the reproductive capacity of long-term non-oestrus replacement gilts

Context It is a common problem that replacement gilts exhibit delayed oestrus and non-oestrus in pig production. Aims This study explored the use of progesterone and pregnant mare serum gonadotropin (PMSG) to promote oestrus and to restore the reproductive capacity of sows. Methods A total of 90 long-term non-oestrus replacement gilts were randomly divided into three groups. Group A consisted of 30 sows that were normally fed for 20 days, followed by injections of 1000 IU of PMSG on the morning of the 21st day and 100 μg of gonadotropin-releasing hormone on the 24th day. Group B consisted of 30 sows that were continuously fed with altrenogest (20 mg/head.day) for 18 days, followed by injections of 1000 IU of PMSG on the morning of the 21st day and 100 μg of gonadotropin-releasing hormone on the 24th day. Group C (control group) consisted of 30 sows that were normally fed for 20 days, followed by injections of 3 mL of physiological saline on the morning of the 21st day and 3 mL of saline on the 24th day. Oestrus identification was performed in all three groups, and sows underwent artificial insemination after the injection of gonadotropin-releasing hormone or saline. Key results We found that the follicles of long-term non-oestrus replacement gilts were not developed, and follicle diameters were &amp;lt;4 mm. The oestrus rate and pregnancy rate of the sows in Group B were significantly lower than those in Group A (30% vs 66.7% and 66.7% vs 90%) respectively. There was no difference in the litter size between Group A and Group B (11.2 vs 11.5). The sows in Group C exhibited no oestrus and no pregnancy. After treatment with progesterone and PMSG, the follicle diameters of sows in oestrus were significantly greater than those of sows in non-oestrus, and the levels of oestradiol, luteinising hormone and follicle-stimulating hormone were significantly higher than those of sows in non-oestrus. Conclusions This study showed that progesterone and PMSG treatment can alter the reproductive hormone levels and follicle diameters in long-term non-oestrus replacement gilts, promote oestrus and restore reproductive capacity in sows. Implications This study provides a method for the use of hormone-treated gilts to maximise the reproductive potential of gilts.

Effects of Vitamin D in Alveolar Bone Remodeling on Osteoblast Numbers and Bone Alkaline Phosphatase Expression in Pregnant Rats During Orthodontic Tooth Movement

Introduction: The vitamin D effect on orthodontic tooth movement in pregnant women remains unknown. The aim of this study was to investigate the post administration of vitamin D effect on osteoblast numbers and bone alkaline phosphatase (BALP) expression in the tension side in pregnant rats during orthodontic tooth movement. Materials and Methods: This study was an in vivo animal experiment; 28 healthy female Wistar rats (Rattus norvegicus) (16–20 weeks’ old) were divided into four groups, with or without intramuscular administration of vitamin D, which were to be observed after 7 and 14 days. Pregnancy was induced with pregnant mare serum gonadotropin and human chorionic gonadotropin. Nickel–titanium coil springs with 30 g/mm2 of force were connected between the right maxillary incisors and the right maxillary first molar. After 7 and 14 days, the animals were sacrificed. Statistical Analysis Used: Analysis of variance with post hoc test (P 0.05). Results: The highest number of osteoblasts occurred in the C-7 group with mean ± standard deviation of 20.54 ± 8.4. Statistically significant differences were seen in decreased osteoblast number between groups (P = 0.001, P 0.05) in the expression of BALP. Conclusion: The post administration of vitamin D during orthodontic tooth movement in pregnant rats produced no significant enhancement on BALP expression and osteoblast number.

Аналіз ефективності застосування гормонального препарату «Фоллімаг» для стимуляції відтворної здатності кролиць

In commercial rabbit meat production, the combination of artificial insemination&nbsp; and management in a batch system is widely used and has replaced natural mating. However, in such systems the success of AI depends largely on the synchronization of oestrus. The irregularity of the oestrus cycle, especially during the lactation period, necessitates a method to synchronize sexual receptivity of the does on the intended AI day. The objective of our experiment was to compare the efficacy of ovulation stimulation in artificial insemination of female rabbit. The task was to compare the results of artificial insemination by using Pregnant Mare Serum Gonadotropin “Follimag” (Mosagrogen) and non-hormonal treatment of (PMSG). The AI technique in rabbits needs an ovulation stimulator or inducer because female rabbits are induced ovulators. In our experiments, we used Surfagon to inducte ovulation by the methodology proposed by Lisin V. I., Sushko A.B., Institute of animal science of the NAAS. Experimental female rabbits were divided into 3 homogenous groups. All animals were maintained and fed in the same way. Artificial light was on from 09.00 to 20.00 h. Receptivity of all does was judged based on vulva colour and turgency just before gonadotropin treatment and in the day before artificial insemination. In two experimental groups with PMSG treatment was observed more pronounced improvement of vulva colour and turgency the day before artificial insemination. Gonadotropin treatment was applied intramuscularly once three days before artificial insemination. We used the dose of 40IU of PMSG – first group, 25IU of PMSG- second group and control group (no oestrus synchronization). The experimental data have shown that the differences between the results of artificial inseminations of rabbit does with the serial use of “Follimag” (PMSG) resulted in 19,29%&nbsp; and 11,36% improvement as compared to the controls (40 IU of PMSG-75,0%;25 IU of PMSG-82,93%;control group-63,64%). In the future carry out further research to monitor the effects of gonadotropin hormones on fertility, estrus, quality and quantity of litter, the physiological condition of rabbit does.

Cryoprotectant combination ethylene glycol and propanediol on mice blastocyst viability post vitrification

The best method to store embryo is vitrification using cryoprotectant, but so far, the viability of the embryo after warming process is decreasing. This objective of this research was to know the effect combination ethylene glycol and propanediol as intracellular cryoprotectant on mice blastocyst viability. The experimental animals were female mice strain Balb/C which was super ovulated using Pregnant Mare Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (hCG). The oocyte collected from the ampulla of tuba fallopian. In Vitro Fertilization was done by placing the sperm and the oocyte in the same medium then cultured until became blastocyst. The blastocyst was classified into: T1: ethylene glycol 30%, T2: propanediol 30%, T3: ethylene glycol 20% and propanediol 10%, and T3: ethylene glycol 10% and propanediol 20%. Each group was vitrified for a week then warmed immediately. Examination of the viability of blastocyst was done using an inverted microscope after warming process. The statistical analysis showed that there were significant differences between single cryoprotectant and combination cryoprotectant group p<0.05. The best cryoprotectant combination was ethylene glycol 10% and propanediol 20%.

58 Transcervical transfer of blastocysts reveals detrimental effect on implantation rate in di (2-ethylhexyl) phthalate-exposed mice

Transcervical transfer is widely used in assisted reproduction techniques, such as cloning and the production of transgenic animals. Using this technique, the number of transferred blastocysts and implanted embryos may be quantified, representing a useful method for assessing the effect of xenobiotics on quality and functionality of the blastocyst. Di (2-ethylhexyl) phthalate (DEHP) is one of the most widely used plasticizers worldwide that causes several detrimental effects, such as decreased embryo quality and a low implantation rate when laboratory animals are treated with very high doses, ranging from 3000 to 3500 µg·kg−1d−1. The aim of this research was to evaluate the effect of environmentally relevant doses of DEHP on blastocyst functionality and ability to implant in an ex vivo study. The CD-1 mice were exposed subchronically (1 month) to 0, 20, 200, and 2000 µg·kg−1d−1 of DEHP via deposition in the mouth, and then they were subjected to a transcervical transfer procedure. The donor female mice were superovulated with pregnant mare serum gonadotropin (5IU) IP, followed by hCG (5 UI) IP 48h later. They were then mated with males of proven fertility. The recipient females (unexposed mice) had the same superovulation protocol as donors, but were mated with vasectomized males. Blastocysts were obtained 96h post-hCG by flushing the oviduct with Gardner-blastocyst medium and were classified according to morphological characteristics. Only expanded blastocysts were transferred transcervically under general anaesthesia protocol using isoflurane (induction: 3-5% and maintenance 2-2.5%). Four days after transcervical transfer, recipient females were injected with 200µL of Chicago Blue 1% IV, killed by cervical dislocation 2h later, following which implantation sites were counted and the implantation rate was calculated with the following formula (implanted blastocysts×100/transferred blastocysts). A decreased implantation rate was evident in 2000 µg·kg−1d−1 DEHP-treated females compared with controls (DEHP 2000: 11.67%±9.64v. control: 71.67%±6.0; P&amp;lt;0.05 according to 1-way ANOVA analysis followed by the Bonferroni post-hoc test). Taken together, these data suggest that the dose of 2000 µg·kg−1d−1 DEPH impairs embryo implantation. We confirm that transcervical transfer is a useful technique in toxicological studies to assess detrimental effects on implantation.

94 Impact of number of embryos transferred on the number of offspring produced in a commercial transgenic founder production operation

The production of transgenic founder dairy goats (cross-bred Saanens, Alpines, Toggenburgs, and Nubians) involves the collection, microinjection, and transfer of numerous embryos into suitable recipient goats to ultimately produce a transgenic founder(s). The objective of this study was to determine the most efficient number of microinjection embryos to transfer to suitable recipients for transgenic founder generation. This is critically important in a commercial production program, as it impacts the goal for the number of embryos collected from donors, number of recipients utilised, and, hence, the overall number of surgical procedures being performed. The entire embryo collection, transfer, and founder-generation process is continuously being evaluated for ways to become more efficient in producing transgenic animals. During LFB USA’s commercial founder-production campaigns over the years (1997-2017), pronuclear microinjection was performed and 3, 4, or 5 embryos were transferred to female goat recipients. The recipients were synchronized using a progesterone implant on Day 0, a prostaglandin injection at Day 7, an injection of 300-500IU of pregnant mare serum gonadotropin on Day 13, progesterone implant removal on Day 14, and surgical transfer of pronuclear microinjected 1- or 2-cell embryos into the oviduct on Day 17. The individual totals and calculation for offspring per embryos transferred was compared for 3, 4, and 5 embryos transferred per recipient and was determined to be (1659/8637) 0.19, (912/4548) 0.20, and (112/675) 0.17, respectively. These embryo efficacy ratios were not significantly different (P&amp;gt;0.05) using the Wald Chi-squared test under logistic regression, and suggests that the number of offspring born is not impacted by number of embryos transferred. Seasonality was also evaluated in this production environment located in North America, with in-season being considered September to December and out-of-season being January to July. Nulliparous recipients during in-season (September to December) embryo transfer operations produced a significant difference, with totals and calculation for (offspring per embryo transferred) of (470/2346) 0.20, (260/1088) 0.24, and (23/190) 0.12 for 3, 4, and 5 embryo transfers, respectively (Table 1). This data indicates that when using nulliparous recipients during the in-season, transferring 4 embryos is optimal for offspring produced. Table 1.Comparison of the individual totals and the calculation of (offspring/embryo) by parity and season

47 Effect of a long-term, high-fat diet on metabolic health and oocyte quality of an outbred (Swiss) versus inbred (C57BL/6N) mouse strain

Maternal metabolic disorders like obesity and diabetes type II are known to affect reproductive physiology, ultimately leading to poor fertility. The oocyte and embryo are extremely vulnerable during the periconceptional period to metabolic stressors, leading to disappointing fertility results. Most mouse model research regarding obesity and Western type diets has been performed on the inbred C57BL/6 strain. However, inbred strains are often linked with decreased fertility. Relying only on inbred strains might also limit translation to human (outbred) physiology. To further explore this, we compared the inbred C57BL/6N to an outbred Swiss strain. Five-week-old Swiss (N=30) and C57BL/6N (B6) (N=29) mice were fed a control (CTRL) or a high-fat (HF) diet for 13 weeks. Diets differed in percentage of fat (10% v. 60%). Body weight gain, serum profile (nonesterified fatty acids, cholesterol, and triglycerides), and oocyte quality were studied. Mature oocytes were collected after hormonal stimulation (IP injection of 10IU of pregnant mare serum gonadotropin followed by 10IU of hCG 48h later). To study oocyte quality, Bodipy (lipid droplets), JC-1 (mitochondrial membrane potential), and Cell-Rox Deep Red stainings were performed, as well as transmission electron microscopy to examine mitochondrial structures. All data were analysed using the t-test. In comparison with the CTRL group, the HF diet increased body weight by 18.09 and 27.87% in Swiss and B6, respectively. The HF significantly increased blood cholesterol levels (103.5v. 143.1 mg/dL in Swiss mice, 141.8v. 185.4 mg/dL in B6 mice) in both strains, and tended to increase blood nonesterified fatty acids (P=0.053) and triglycerides (P=0.075) only in Swiss but not in B6 mice. Oocytes collected from the HF diet group contained a larger total volume of lipid droplets (P&amp;lt;0.05) in both strains compared with controls. The mitochondrial membrane potential and Cell-Rox Deep Red were significantly increased (P&amp;lt;0.05) in oocytes of Swiss mice, but not B6 mice, fed a HF diet. Transmission electron microscopy images from HF oocytes showed mitochondria with abnormal morphology, low electron density, and rose petal appearance, resulting in significantly increased mitochondrial abnormalities in Swiss mice on the HF diet (P&amp;lt;0.05). In B6 mice, both CTRL and HF oocytes contained high proportions of abnormal mitochondria compared with the CTRL group of the Swiss mice, explaining the lack of HF diet effects in B6 oocyte ultrastructure. We conclude that a HF diet has a significant effect on both metabolic health and oocyte quality. However, the Swiss model seems more sensitive to a Western type diet insult, making it more suitable for research focusing on metabolic health and oocyte quality than the B6 strain. The HF diet-exposed Swiss mice showed differences (compared with CTRL) in their serum profile. Alterations in mitochondrial activity, structures, and oxidative stress were induced by HF diet in the Swiss mice and not the B6, although B6 oocytes also showed higher lipid droplet accumulation. Furthermore, even the B6 mice that were fed a normal control diet showed deviant oocyte quality, clearly shown by morphological signs of lower quality and mitochondrial abnormalities.

NRDR inhibits estradiol synthesis and is associated with changes in reproductive traits in pigs.

Cumulus cells secreting steroid hormones have important functions in oocyte development. Several members of the short‐chain dehydrogenase/reductase (SDR) family are critical to the biosynthesis of steroid hormones. NADPH‐dependent retinol dehydrogenase/reductase ( NRDR), a member of the SDR superfamily, is overexpressed in pig breeds that also show high levels of androstenone. However, the potential functions and regulatory mechanisms of NRDR in pig ovaries have not been reported to date. The present study demonstrated that NRDR is highly expressed in pig ovaries and is specifically located in cumulus granulosa cells. Functional studies showed that NRDR inhibition increased estradiol synthesis. Both pregnant mare serum gonadotropin and human chorionic gonadotropin downregulated the expression of NRDR in pig cumulus granulosa cells. When the relationship between reproductive traits and single‐nucleotide polymorphisms (SNPs) of the NRDR gene was examined, we found that two SNPs affected reproductive traits. SNP rs701332503 was significantly associated with a decrease in the total number of piglets born during multiparity, and rs326982309 was significantly associated with an increase in the average birth weight during primiparity. Thus, NRDR has an important role in steroid hormone biosynthesis in cumulus granulosa cells, and NRDR SNPs are associated with changes in porcine reproduction traits.

Pregnancy rate in indigenous ewes by direct transfer of vitrified embryos

The effects of PMSG on superovulation, quality of embryos, and pregnancy rate were studied following transfer of vitrified embryos into indigenous ewes. Three donor and nine recipient ewes were synchronized using two intramuscular doses of Cloprostenol (PGF2α) equivalent to 125 μg Prostaglandin F2α (Ovoprost® Bayer, New Zealand) at an interval of nine days. To ensure ovulation donor and recipient ewes were treated with 600 iu and 250 iu Pregnant Mare Serum Gonadotrophin (PMSG; Folligon®, Intervet, Boxmeer, The Netherlands) at the time of 2nd injection of Prostaglandin F2α. All donor ewes were mated by fertile rams. Embryos were collected from donor ewes on day six after mating by inguinal laparotomy. Grade 1 embryos were vitrified using standard procedure. After thawing the embryos were transferred into the exteriorized uterine horn of the recipient by inguinal laparotomy. Pregnancy diagnosis was performed by trans-abdominal ultrasonography on Day 40 of transfer. Oestrus occurred 31.3 ± 8.1 hours after second injection of prostaglandin and 32.6 ± 8.7 hours in recipients. Oestrus lasted 24.7 ± 9.9 hours in donors and 32.8 ± 12.8 hours in recipients. The mean numbers of corpora lutea and embryos of donor ewes were 11.7 ± 4.0 and 8 ± 2.6, respectively. Total embryo recovery rate of donor ewes was 68.6%. The mean values of qualities of embryo yield of donor ewes were 6 ± 1.7 grade 1, 0.3 ± 0.6 grade 2, 1.3 ± 1.2 grade 3, and 0.3 ± 0.6 grade 4. Twenty embryos were vitrified, 16 embryos were transferred and four recipient ewes were found pregnant. The pregnancy rate of recipient ewes was 44.4%.Bangl. vet. 2017. Vol. 34, No. 1, 27-33

ESR2 Is Essential for Gonadotropin-Induced Kiss1 Expression in Granulosa Cells.

Hypothalamic expression of Kiss1 plays an essential role in the onset of puberty, gonadal development, and ovulation. Estrogens regulate the expression of Kiss1 in the hypothalamus through estrogen receptor-α. Kiss1 is also expressed in the ovary, where its expression correlates with the onset of puberty and progression of the estrous cycle. To date, estrogen regulation of Kiss1 expression in the ovary has not been investigated. We recently observed that gonadotropin-induced Kiss1 expression was absent in Esr2-null rat ovaries even though Esr1 was present. Wild-type granulosa cells abundantly expressed Kiss1 and oocytes expressed the Kiss1 receptor. We characterized estrogen receptor-β (ESR2) regulation of Kiss1 expression in granulosa cells by identifying granulosa cell-specific transcript variants and potential regulatory regions. The Kiss1 promoter, an upstream enhancer, and a downstream enhancer all possessed conserved estrogen response elements (EREs) and showed active histone marks in gonadotropin-stimulated granulosa cells. The transcriptionally active Kiss1 promoter, as well as the enhancers, also revealed enrichment for ESR2 binding. Furthermore, activity of a Kiss1 promoter construct was induced after overexpression of ESR2 and was blocked upon mutation of an ERE within the promoter. Finally, pregnant mare serum gonadotropin and human chorionic gonadotropin administration induced phosphorylation of ESR2 and upregulated the AP-1 proteins FOSL2 and JUNB in granulosa cells. Activated MAPK ERK2 was associated with the ESR2 phosphorylation in granulosa cells, and AP-1 factors could synergistically activate the Kiss1 promoter activity. These gonadotropin-induced changes paralleled Kiss1 expression in granulosa cells. We conclude that gonadotropin-stimulated Kiss1 expression in granulosa cells is dependent on both the activation of ESR2 and the upregulation of AP-1.

Mullerian-inhibiting substance augments ovarian response to superovulation in mice with diminished ovarian reserve

To determine whether pretreatment with Mullerian-inhibiting substance (MIS) affects the oocyte yield of superovulation in a mouse model of diminished ovarian reserve (DOR). Preclinical placebo-controlled animal study. The DOR mouse model was created by giving intraperitoneal (IP) injections of 4-vinylcyclohexene diepoxide (VCD) to 6-week old nu/nu mice (n=3) at 160 mg/kg daily for 5 days. Control mice (n=3) were injected IP with vehicle control (corn oil). Mice were euthanized 14 days later. Dissected ovaries were fixed in 4% paraformaldehyde, embedded in paraffin blocks in an automated tissue processor, cut into 8-um sections, and mounted onto glass slides, which were stained with hematoxylin and eosin. Under light microscopy, ovarian follicles were classified into five groups - primordial, primary, secondary, antral, and atretic - and counted. A cohort of DOR mice was subsequently treated with subcutaneous recombinant human MIS protein (750 ug/kg, n=3) or saline (n=4) twice daily for 40 days. After 30 days of observation, superovulation was performed in standard fashion with pregnant mare serum gonadotropin 7.5 IU IP, followed by human chorionic gonadotropin (hCG) 7.5 IU IP 49 hours later to trigger ovulation. 16 hours after hCG, oocytes were dissected from the oviducts of euthanized mice and were counted. Morphologic assessment of collected oocytes was performed under a stereo microscope, and the percentage of degenerated oocytes collected from each mouse was noted. Oocyte yields and percentage of degenerated oocytes of MIS-treated and saline control mice were compared using student’s t-test. A p-value of < 0.05 was considered significant. VCD administration as described above creates a DOR phenotype in mice, by depleting primordial follicle reserve by 73%, while maintaining ovulatory function. DOR mice treated with MIS produced, on average, 29 oocytes through superovulation compared to saline control mice who produced only 11 oocytes (p = 0.038). Furthermore, more oocytes were morphologically abnormal in controls compared to MIS-treated mice (52.2% versus 30.1%, p = 0.026). In the setting of DOR, treatment with MIS protein significantly enhances oocyte yield following superovulation and leads to a lower proportion of degenerated oocytes. These findings suggest a future clinical application of MIS in IVF protocols, particularly for patients with DOR.