Injection Of Equine Chorionic Gonadotropin Research Articles

Comparison of efficiency between two artificial insemination methods using fresh semen in domestic cat (Felis Catus)

The objective of this study was to compare pregnancy rates in domestic cats using fresh semen for the bilateral intrauterine insemination (BIUI) method and the novel uterine body insemination (UBI) method. Queens received a single injection of equine chorionic gonadotropin (eCG) (200 IU; IM) to induce ovarian follicular development and, after 83 h, an injection of human chorionic gonadotropin (hCG) (100 IU; IM) for final oocyte maturation and ovulation induction. Thirty-four hours after hCG administration, 3 × 106 fresh spermatozoa were used for insemination by the BIUI (n = 8 queens) or by the UBI (n = 7 queens) techniques respectively. Pregnancy rates were 75.00% (6/8) by BIUI and 42.85% (3/7) by the UBI method. The mean litter size was 3.0 ± 0.86 for the BIUI, and 2.0 ± 1.0 for the UBI method. Spontaneous abortion occurred on day 35 of pregnancy in one queen following the UBI method. Our findings showed that the BIUI of queens with fresh semen resulted in higher pregnancy rates than the novel UBI method; also, acceptable pregnancy rates were achieved following BIUI with fresh semen in the domestic cat.

The Effectiveness of Pharmacological Synchronization of the Estrous Cycle in Hinds (Cervus elaphus L.): A Pilot Field Trial.

Simple SummaryReproductive biotechnics of Cevidae species may increase valuable traits for the agricultural sector, and improve genetic diversity in relatively small farmed populations. The first step of biotechnics in terms of suitability for embryo transfer or insemination is preparation of females for proper phase of the estrous cycle. Based on available synchronization protocols applicated in red deer and domestic ruminant females we estimated the protocol for hinds in view of future routine use. Progestin analogues in the form of an insert (CIDR) or a sponge (Chronogest) and injection of equine chorionic gonadotropin (eCG; Folligon) were used on the appropriate day for inducing the estrus. The effect of synchronization was checked by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA, pregnancy-associated glycoproteins (PAG)s concentration by EIA and insemination effectiveness—pregnancy rate. The better method of the estrous cycle synchronization found down to be progestin sponge and eCG based protocol in hinds considering hormone responsiveness and pregnancy rate.The aim was to estimate the effective pharmacological method of the estrous cycle synchronization by checking the effects of synchronization by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA and artificial insemination. The experiment was performed at the red deer farm in Rudzie (North-East Poland; 3 year’s old). The herd (N = 14) was kept away from bulls and was divided in two groups of seven animals. In the Group I, CIDR insert (0.3 g of P4) was applicated intravaginally for 12 days; a second insert replaced the first one for the next 12 days, and next 200 IU of equine chorionic gonadotropin (eCG) was injected intramuscularly (Folligon). Estrus was expected 48 h after eCG injection. In the Group II, Chronogest sponge (20 mg of flugestone acetate) was applicated intravaginally and after 7 days replaced with second chronogest sponge for 7 days. After removing the sponge, on the same day eCG was injected and estrus was expected after 48 h. Artificial insemination was provided with frozen-thawed semen twice: 12 and 24 h after expected estrus. The peripheral blood from the jugular vein was collected each time when the inserts or sponge were applicated and 40 days after insemination. The concentration of P4 and E2 in plasma was measured by RIA. The effectiveness of insemination was monitored by pregnancy-associated glycoproteins determination and observed by the number of calves born. Two pregnancies were confirmed in Group I and five in Group II based on PAG concentration. One newborn was observed in Group I and five in Group II. Both methods of synchronization are effective in hinds based on the received profile of steroids. Although the sponge shape in case of chronogest is better comparing with CIDR, which was not completely deposited in the vagina of hind, potentially leads to bacteria inflammation, and it disturbs the rightful endocrine regulation. Moreover, pregnancy rate and hormone responsiveness were better in Group II.

Estrous expression during a fixed-time artificial insemination protocol enhances development and interferon-tau messenger RNA expression in conceptuses from Bos indicus beef cows

Expression of estrus (EST) near the time of fixed-time artificial insemination (FTAI) increases pregnancy success in beef females. This outcome has been associated with improved pregnancy establishment and maintenance, although research is still warranted to validate this theory. Hence, this experiment compared ovarian, uterine and conceptus factors associated with pregnancy establishment in Bos indicus beef cows according to estrous expression during a FTAI protocol. One hundred lactating multiparous Nelore cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device on day −11, a 12.5 mg injection of prostaglandin F2α on day −4, P4 device removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of equine chorionic gonadotropin on day −2, and FTAI on day 0. An estrous detection patch was attached to the tailhead of each cow on day −2, and estrous expression was defined as removal of >50% of the rub-off coating from the patch at FTAI. Overall, 39 cows expressed EST, 55 did not express EST (NOEST), and six cows lost their patch and were discarded from the experiment. Ovarian ultrasonography was performed at FTAI, and on days 7 and 15 of the experiment. Blood samples were also collected on days 7 and 15. Only cows without a corpus luteum (CL) on day 0, and with a CL on days 7 and 15 remained in the experiment (EST, n=36; NOEST, n=48). On day 15, cows were randomly selected within each group (EST, n=29; NOEST, n=30) for conceptus collection via transcervical flushing, followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Within cows not assigned to conceptus collection, blood samples were collected for whole blood RNA extraction (day 20) and pregnancy status was verified by transrectal ultrasonography (day 30). Diameter of dominant follicle on day 0 and plasma P4 concentrations on day 7 were greater (P⩽0.02) in EST v. NOEST cows. Conceptus length and messenger RNA (mRNA) expression of prostaglandin E synthase and interferon-tau were greater (P⩽0.04) in EST v. NOEST cows. Moreover, EST cows diagnosed as pregnant on day 30 had greater (P<0.01) blood mRNA expression of myxovirus resistance 2 on day 20 compared with NOEST. In summary, estrous expression near the time of FTAI enhanced pregnancy establishment factors in B. indicus cows, including conceptus development and mRNA expression of interferon-tau.

Effect of lipopolysaccharide on circadian clock genes Per2 and Bmal1 in mouse ovary.

In mammals, circadian rhythms are associated with multiple physiological events. The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on circadian systems in the ovary. Immature female mice were received an intra-peritoneal injection of equine chorionic gonadotropin (eCG) and LPS. Total RNA was collected from the ovary at 6-h intervals throughout a 48h of experimental period. The expression of the circadian genes period 2 (Per2) and brain and muscle ARNT-like 1 (Bmal1) such as circadian genes was measured by quantitative PCR. Although expression of Per2 and Bmal1 in the ovary did not display clear diurnal oscillation, LPS suppressed the amplitude of Per2 expression. Additionally, LPS inhibited the expression of cytochrome P450 aromatase (CYP19) and luteinizing hormone receptor (LHr) genes in the ovary of eCG-treated mice. Our data suggest that Per2 may be associated with the inhibition of CYP19 and LHr expression by LPS in the ovaries of immature mice.

Regulation of interleukin-11 expression in ovulatory follicles of the rat ovary.

The aim of the present study was to examine the regulation of interleukin (IL)-11 expression, as well as the role of IL-11, during ovulation in gonadotropin-primed immature rats. Injection of equine chorionic gonadotropin (eCG), followed by human CG (hCG) to induce superovulation stimulated expression of the Il11 gene in theca cells within 6h, as revealed by northern blot and in situ hybridisation analyses. Real-time reverse transcription-polymerase chain reaction analysis showed that the IL-11 receptor, α subunit gene was expressed in granulosa and theca cells and that injection of hCG had no effect on its expression. IL-11 protein expression was stimulated in theca cells by hCG. LH-stimulated increases in Il11 mRNA levels in cultured preovulatory follicles were inhibited by protein kinase A and mitogen-activated protein kinase kinase inhibitors. Toll-like receptor (TLR) 2 and TLR4 were detected in preovulatory follicles, and the TLR4 ligand lipopolysaccharide, but not the TLR2 ligand Pam3Cys, increased Il11 mRNA levels in theca cells, but not in granulosa cells. Treatment of preovulatory follicles with IL-11 stimulated progesterone production and steroidogenic acute regulatory protein (Star) gene expression. Together, these results indicate that IL-11 in theca cells is stimulated by mitogen-activated protein kinase signalling and TLR4 activation, and increases progesterone production during ovulation.

Stresses on Female Mice Impair Oocyte Developmental Potential: Effects of Stress Severity and Duration on Oocytes at the Growing Follicle Stage.

Although previous studies found that 1-time acute stress applied during follicle maturation impaired oocyte competence, it is unknown whether repeated chronic stress, which is known to cause animal behavioral adaptation, would damage oocytes when applied during follicle growth. In this study, female mice were exposed to repeated restraint stress (RRS) or unpredictable stress (UPS) for different days before equine chorionic gonadotropin injection to initiate oocyte prematuration development and to observe effects of different stressors on oocytes in the growing follicles. The results showed that although oocyte pre- and postimplantation development was unaffected when mice were exposed to RRS or UPS once a day for 4 days, development was impaired when mice were exposed to RRS for 8 or more days or to UPS twice a day for 4 days (4 × 2). The 4 × 2 UPS caused more oxidative stress in oocytes and severer apoptosis in antral follicles than did the 4-day RRS. The RRS mice were stressed consistently from days 1 to 23 of restraint, and the stress that a mouse had 4 × 2 UPS was severer than that from 4-day RRS. The results suggest that (1) the degree that a stress damages oocytes is the product of duration × severity of the stress; (2) RRS impaired oocyte developmental potential through cumulative effects on growing follicles; and (3) preantral follicles were not as sensitive to stress as antral follicles were.

A simple method for inducing estrous cycle stage-specific morphological changes in the vaginal epithelium of immature female mice.

The vaginal epithelium of the adult female laboratory rodent changes from mucous secretion to cornification over the course of the estrous cycle. The morphophysiological changes occur with such regularity, accuracy and precision that the specific stage of the estrous cycle in the rat can be determined by inspection of the vaginal opening and/or exfoliative vaginal cytology. However, in the mouse, post-mortem vaginal histology is often required to determine the estrous cycle stage for ensuring the required level of reliability. Consequently, an excess number of female adult mice are needed to allow for the delivery of sufficient numbers of mice in a desired estrous cycle stage. In this study, we demonstrate that the standard procedure for oocyte superovulation and collection in the laboratory mouse (e.g. injection of equine chorionic gonadotropin followed 48 h later by human chorionic gonadotropin) can also be reliably used to induce changes in the epithelium of 3.5-week-old mouse vaginas in an estrous cycle stage-specific manner (e.g. establishment and replacement of a mucous secreting epithelium with a cornified epithelium; induction of cornification-associated loricrin expression). The superovulation protocol thus allows for the efficient and economic induction of estrous cycle stage-specific characteristics in the Müllerian duct-derived vagina thereby avoiding the necessity of post-mortem identification of the estrous cycle stage. In addition, our study indicates that the laboratory mouse vagina is an excellent organ for studying the sequence of events leading to cornification.

Diagnosis and treatment of a functional follicular cyst in a Persian queen cat: A case report

In the present report, diagnosis and treatment of a case with follicular ovarian cysts in a 5-year-old Persian queen cat is described. In response to palpation of spines, the queen cat presented herself in lordosis and danced up and down with her rear legs. Trans-abdominal ultrasonography examination showed 2 cysts in the left ovary of the queen. Serum estrogen assay indicated elevated level of 17 β-estradiol concentration (105 pg/ml). However, progesterone concentration was normal (0.3 ng/ml). Accordingly, the queen was diagnosed with functional follicular cysts. The queen was treated with an administration of hCG intra-muscularly. Thirty (30) days after the administration of hCG, an injection of equine chorionic gonadotropin (eCG) (50 IU) was given intra-muscularly. Natural mating was done with a fertile Persian tom cat. In conclusion, it seems that treatment of functional follicular cysts can be applied to preserve fertility in cats.

Cumulus cell-expressed type I interferons induce cumulus expansion in mice.

Ovulation resembles the inflammatory response. The purpose of the present study was to examine the expression and role of type I interferons (IFNs) Ifnalpha and Ifnbeta in mouse ovaries during the process of ovulation. An in vivo injection of equine chorionic gonadotropin (CG)-human CG (hCG) stimulated Ifnalpha and Ifnbeta mRNA in cumulus-oocyte complexes (COCs) within 6 h. Type I IFN receptor (Ifnar1 and Ifnar2) genes were also expressed in preovulatory follicles without a change by hCG. Immunofluorescent study revealed the expression of protein signals of Ifnalpha, Ifnbeta, and Ifnar1 in cumulus cells. Treatment of COCs with Ifnalpha or Ifnbeta in vitro induced cumulus expansion that was comparable to that mediated by epiregulin. In cultured COCs, the levels of Ifnalpha and Ifnbeta mRNA increased by epiregulin and follicle-stimulating hormone, but not by prostaglandin E2. Ifnalpha and Ifnbeta activated multiple signaling events (signal transducer and activator of transcription-1/3, Akt, and mitogen-activated protein kinase 1/2) and stimulated the expression of genes known to impact COC expansion (Has2, Ptx3, Tnfaip6, and Ptgs2). Interestingly, treatment of COCs with Toll-like receptor (TLR) 2 and TLR4 ligands (lipopolysaccharides, Pam3Cys, and hyaluronan fragments) increased Ifnalpha and Ifnbeta mRNA, while coculture with anti-TLR2/4 neutralizing antibody abolished these effects. Taken together, these results demonstrate that the type I IFN system is operating in mouse cumulus cells and plays a role in the induction of cumulus expansion during the ovulatory process in mice.

The effects of intramuscular or intravenous injections of gonadotropin releasing hormone at fixed-time artificial insemination on pregnancy rates of Bos indicus beef cows

The effect of an intramuscular versus intravenous administration of gonadotropin releasing hormone (GnRH) at fixed-time AI (FTAI) on the pregnancy rates of crossbred Bos indicus beef cows was evaluated. Pluriparous nursing calv cows (n=120) were synchronized as follows: d 0 cows received a 2.0 mg injection of estradiol benzoate (EB) and insertion of a controlled intravaginal progesterone releasing device containing 0.558 g of progesterone, d 8 removal of the progesterone device , a 0.15 mg injection of prostaglandin F2α (PGF), a 1.0 mg injection of EB, and 400 IU injection of equine chorionic gonadotropin. Fifty-four hr after PGF, all cows were exposed to FTAI and a 0.084 mg injection of GnRH was administered either via Vena caudalis (n=60), or via Longissimus dorsi (n=60). Cows were inseminated with the same sire and by a single AI technician. Pregnancy was determined by the transrectal ultrasonography on d 40 after AI. Cows receiving the intravenous administration of GnRH had higher (P = 0.04) pregnancy rates than the cows receiving the intramuscular injection of GnRH (65 vs 46.6%, respectively). It was concluded that the intravenous administration of GnRH at the time of AI improved the pregnancy rates of crossbred Bos indicus beef cows submitted to FTAI.

211 REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AS A TOOL TO ELUCIDATE THE BIOLOGICAL ACTIVITY OF COMMERCIAL EQUINE CHORIONIC GONADOTROPIN (eCG)

Superovulation in ruminants can be induced with a single injection of equine chorionic gonadotropin (eCG). However, ovarian response is sometimes lower than expected because of, among other factors, the source of eCG used. This study aimed at establishing the physical-chemical profile of commercial eCG, in order to find differences which can be related to their biological activity. Four different commercial eCG products for veterinary use (A, B, C, D) and one eCG chemical reagent from Sigma (St. Louis, MO, USA), here used as reference preparation, were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C4-Grace Vydac 214 TP 54-column (25 cm × 4.6 mm I.D.), with UV detection at 220 nm. All eCG preparations presented at least three peaks with retention times (tR) of ~27(I), 34(II), and 36(III) minutes, with a peak at tR = 27 min common to A, C, D, and Sigma eCG, whereas preparation B did not present this peak. A bioassay test was carried with all of these preparations. Immature 21- to 25-day-old Wistar female rats received the equivalent to 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The average ovarian weight for preparations A, C, D, and Sigma were ~0.0795 ± 0.0107 g, whereas preparation B was 0.035 ± 0.007 g (P &lt; 0.01). Preparation B was not different from saline (0.034 ± 0.002 g). In order to establish which one of these three peaks presented the highest biological activity, a mass equivalent to 10 IU of eCG from peaks I, II, and III of Sigma and of product A were studied. The average ovarian weight of animals injected with material from peak II and III (~0.0285 ± 0.003 g) were similar to that of the control whereas peak I produced ovarian weights of 0.059 ± 0.007 g and 0.075 ± 0.010 g for Sigma and product A, respectively (P &lt; 0.01). These results suggest that the lack of ovarian response to eCG treatments can be related to differences in the physical-chemical profile of commercial eCG products and that RP-HPLC is a fast and reliable tool for detecting these differences. Supported by FAPESP (Grant 11/13096-0).

Antioxidant supplementation overcomes the deleterious effects of maternal restraint stress-induced oxidative stress on mouse oocytes

In this study, using a mouse model, we tested the hypothesis that restraint stress would impair the developmental potential of oocytes by causing oxidative stress and that antioxidant supplementation could overcome the adverse effect of stress-induced oxidative stress. Female mice were subjected to restraint stress for 24 h starting 24 h after equine chorionic gonadotropin injection. At the end of stress exposure, mice were either killed to recover oocytes for in vitro maturation (IVM) or injected with human chorionic gonadotropin and caged with male mice to observe in vivo development. The effect of antioxidants was tested in vitro by adding them to IVM medium or in vivo by maternal injection immediately before restraint stress exposure. Assays carried out to determine total oxidant and antioxidant status, oxidative stress index, and reactive oxygen species (ROS) and glutathione levels indicated that restraint stress increased oxidative stress in mouse serum, ovaries, and oocytes. Whereas the percentage of blastocysts and number of cells per blastocyst decreased significantly in oocytes from restraint-stressed mice, addition of antioxidants to IVM medium significantly improved their blastocyst development. Supplementation of cystine and cysteamine to IVM medium reduced ROS levels and aneuploidy while increasing glutathione synthesis and improving pre- and postimplantation development of oocytes from restraint-stressed mice. Furthermore, injection of the antioxidant epigallocatechin gallate into restraint-stressed mice significantly improved the blastocyst formation and postimplantation development of their oocytes. In conclusion, restraint stress at the oocyte prematuration stage impaired the developmental potential of oocytes by increasing oxidative stress and addition of antioxidants to IVM medium or maternal antioxidant injection overcame the detrimental effect of stress-induced oxidative stress. The data reported herein are helpful when making attempts to increase the chances of a successful outcome in human IVF, because restraint was applied at a stage similar to the FSH stimulation period in a human IVF program.

Psychological Stress on Female Mice Diminishes the Developmental Potential of Oocytes: A Study Using the Predatory Stress Model

Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr2+ activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation.

Distribution of Adenylyl Cyclases in the Rat Ovary by Immunofluorescence Microscopy

In the mammalian ovary both gonadotropins and local cytokines, acting through G-protein coupled receptors, govern the physiology of the ovary in part by regulating the cyclic adenosine 3',5'-monophosphate via adenylyl cyclases. The nine transmembrane adenylyl cyclases and a soluble adenylyl cyclase are regulated by a diversity of ligands. In this study we have examined the rat ovaries, prior to and subsequent to gonadotropin treatment, for the presence of different transmembrane adenylyl cyclases by indirect immunofluorescence microscopy. Adenylyl cyclase I immunoreactivity was observed in the nuclei of oocytes in preantral and antral follicles along with some staining in granulosa cells. Equine chorionic gonadotropin injection increased adenylyl cyclase I staining in granulosa cells. Adenylyl cyclase I staining was also observed in luteal and endothelial cells. Adenylyl cyclase II was observed throughout the ovary, including granulosa cells and the ovarian surface epithelium. Adenylyl cyclase II staining was also found to increase in granulosa cells after equine chorionic gonadotropin injection. Adenylyl cyclase III was distributed primarily in theca and smooth muscle cells of arterioles, with faint staining in the oocytes of equine chorionic gonadotropin-injected ovaries. Adenylyl cyclase IV staining was present throughout the ovary, including the nuclei of oocytes. Adenylyl cyclase VIII staining in granulosa cells increased subsequent to equine chorionic gonadotropin injection and remained in luteal cells. Our study reveals the redundancy of adenylyl cyclases present in the rat ovary and, therefore, implies potential regulation of follicular and corpus luteum physiology by cyclic adenosine 3',5'-monophosphate generated through distinct adenylyl cyclases.

Wild-Derived Inbred Mice No Longer ART-Resistant1

Wild-Derived Inbred Mice No Longer ART-Resistant1

Maternal-Restraint Stress Increases Oocyte Aneuploidy by Impairing Metaphase I Spindle Assembly and Reducing Spindle Assembly Checkpoint Proteins in Mice1

Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.

Use of ultrasound biomicroscopy to evaluate induced ovarian follicular growth and ovulation in mice

Recent advances in image technology, including significant gains in spatial resolution, have made realtime sequential ovarian evaluations possible in small rodents, allowing longitudinal (continued) studies of the ovarian cycle and reducing the required number of experimental animals. The aim of this study was to evaluate exogenous stimulated follicular growth in mice using high-resolution ultrasound technology. Female mice (n = 15) received a 5 IU intraperitoneal injection of equine chorionic gonadotropin (eCG) and 48 h later a 5 IU injection of human chorionic gonadotropin (hCG), and were allowed to mate thereafter. In experiment 1, animals (n = 7) were evaluated every 6 h, from 3 to 51 h after eCG injection, with an ultrasound biomicroscopy (UBM) equipped with a realtime 45 MHz microvisualization probe (RMV 707b). The ovaries were identified and follicular population quantified, and follicles were classified according to the diameter as small (≤449 µm) or large (≥450 µm). A significant change in the distribution of follicle population according to category was observed only 45 h after eCG injection (P < 0.05). In experiment 2, animals (n = 8) were evaluated every 2 h, from 2 h to 10 h after hCG treatment. The largest follicles reached a maximum size (596.7 ± 106.0 µm) 5.8 ± 2.3 h after hCG injection. As expected, the population of large follicles decreased thereafter, indicating the progress of ovulations, but large follicles were still detected late after treatment (10.1 ± 1.1 h). In conclusion, UBM can be used to evaluate follicle dynamics in superstimulated mice (C57BL/6 and BALB/c); significant changes in follicle distribution only occur at later stages after eCG stimulation; and hCG-induced ovulations may not occur synchronously in mice.

Maternal Restraint Stress Diminishes the Developmental Potential of Oocytes

Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.

Selective Modulation of Follicle-Stimulating Hormone Signaling Pathways with Enhancing Equine Chorionic Gonadotropin/Antibody Immune Complexes

The injection of equine chorionic gonadotropin (eCG) in dairy goats induces the production of anti-eCG antibodies (Abs) in some females. We have previously shown that Abs negatively modulate the LH and FSH-like bioactivities of eCG, in most cases, compromising fertility in treated females. Surprisingly, we found out that some anti-eCG Abs improved fertility and prolificity of the treated females, in vivo. These Abs, when complexed with eCG, enhanced LH and FSH ability to induce steroidogenesis on specific target cells, in vitro. In the present study, we analyzed the impact of three eCG/anti-eCG Ab-enhancing complexes on two transduction mechanisms triggered by the FSH receptor: guanine nucleotide-binding protein alphaS-subunit/cAMP/protein kinase A (PKA) and beta-arrestin-dependent pathways, respectively. In all cases, significant enhancing effects were observed on ERK phosphorylation compared with eCG alone. However, cAMP production and PKA activation induced by eCG could be differently modulated by Abs. By using a pharmacological inhibitor of PKA and small interfering RNA-mediated knock-down of endogenous beta-arrestin 1 and 2, we demonstrated that signaling bias was induced and was clearly dependent on the complexed Ab. Together, our data show that eCG/anti-eCG Ab-enhancing complexes can differentially modulate cAMP/PKA and beta-arrestin pathways as a function of the complexed Ab. We hypothesize that enhancing Abs may change the eCG conformation, the immune complex acquiring new "biased" pharmacological properties ultimately leading to the physiological effects observed in vivo. The modulation of ligand pharmacological properties by Abs opens promising research avenues towards the optimization of glycoprotein hormone biological activities and, more generally, the development of new therapeutics.

Estrus Induction and Fertility Rates in Response to Exogenous Hormonal Administration in Postpartum Anestrous and Subestrus Bovines and Buffaloes

A total of 130 animals (82 cattle, 48 buffaloes) with histories of anestrous 60-90 days post-partum and belonging to different agroclimatic zones of Punjab were subjected to rectal palpation and blood samplings at least three times at weekly intervals. The body condition score (BCS) of each animal was also recorded. The animals were divided into two groups; viz., true anestrous (Gp-I) and subestrus (Gp-II) through rectal palpation of ovaries and plasma progesterone (P4) concentrations. Furthermore, the Gp I and II animals were divided into treatment (Gp Ia, 40 cattle and 16 buffaloes; Gp IIa, 12 cattle and 14 buffaloes) and control groups (Gp Ib, 20 cattle and 8 buffaloes; Gp IIb, 10 cattle and 10 buffaloes). True anestrous animals (Gp Ia) were treated with 3 injections of hydroxyprogesterone caproate (750 mg, i.m.) at 72-hr intervals followed by injection of equine chorionic gonadotropin (eCG; 750 I.U., i.m.) 72 hr after the last progesterone injection. The animals were bred at the first estrus after the induced one. The first service conception rate (FSCR), overall conception rate (OCR), services per conception and pregnancy rate of the true anestrous treated cattle (Gp Ia) were 44.4%, 48.0%, 2.08 and 60.0%, respectively. In the true anestrous control cattle (Gp Ib), only five that were observed to be in estrus failed to conceive. In the anestrous treated buffaloes (Gp Ia), the FSCR, OCR, services per conception and pregnancy rate were 50.0%, 62.5%, 1.6 and 62.5%, respectively. No buffalo amongst true anestrous control (Gp Ib) showed estrus. The subestrus animals (Gp IIa) were administered Prostaglandin F(2alpha) (PGF(2alpha); 25 mg Dinoprost, i.m.) and bred at induced estrus. Amongst the Gp IIa animals, all cattle (100%) and twelve buffaloes (85.7%) responded to treatment. Of these animals, the FSCR and pregnancy rate at induced estrus in the cattle were 50.0% each, whereas they were 66.6% and 57.1%, respectively, in the buffaloes. The subestrus control animals (Gp IIb) remained infertile. In summary, the plasma P(4) profile can be used to differentiate true anestrous and subestrus animals and thus to determine a hormonal therapy. Furthermore, fertile estrus can be induced with hormonal therapy in anestrous and subestrus bovines.