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Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG–hCG, IAS–hCG and eCG–IAS–hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS–hCG and eCG–IAS–hCG methods. However, low fertilisation rates (36.3–38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG–hCG and IAS–hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.

The aim of this study was to investigate the effects of maternal, hormonal, and fetal factors on early fetal volume (FV) measurements in mares obtained by three-dimensional (3D) ultrasound. Furthermore, postpartum parameters were explored in regard to their association with early FV. For this purpose, 149 German warmblood mares that were artificially inseminated and confirmed to be pregnant between days 14–16 of gestation, were examined transrectally at day 45 ± 1 of gestation with the portable 3D ultrasound device Voluson® i (GE, Zipf, Austria). FV was calculated by using the extension software Virtual Organ Computer-aided AnaLysis (VOCAL™). Two different mixed linear models were used to analyze associations between the investigated maternal and fetal factors and the FV. Explanatory variables investigated in the first model were: maternal age, parity, maternal weight, and body condition score, type of pregnancy (recipient or biological mother), barren status (lactating or non-lactating), fetal sex, progesterone (P4) and equine chorionic gonadotropin (eCG) concentrations; and in the second model outcome variables such as gestational length, birth weight, placental weight, fetal sex, and abortion were included in the analysis. The final models revealed a significant relation between FV and eCG (b = 0.011, P = 0.030), as well as with P4 (b = −0.053, P = 0.016), but interestingly P4 was negatively related to FV. Fetal sex showed the most prominent effect on FV (b = −0.256, P = 0.039), with female fetuses being smaller than male fetuses. In the second model none of the investigated parameters were related to early FV except for fetal sex (b = -0.328, P = 0.047), again with female fetuses being smaller. In summary, it was found that FV is related with eCG, P4 and fetal sex, but was no suitable predicting factor for the investigated outcome parameters. Furthermore, the findings suggest that sex specific growth differences exist already in early gestation. The detailed biological mechanism by which P4 and eCG affect fetal size has to be investigated in prospective studies.

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at −80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0–5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

As dogs experience oestrus only once or twice a year, it is necessary to establish an effective method of oestrous induction for efficient breeding. In the present study, we evaluated inhibin antiserum (IAS) on oestrous induction in anoestrous females. Bitches were administered 0.5 ml/kg IAS or a mixture of 50 IU/kg equine chorionic gonadotropin (eCG) and 0.5 ml/kg IAS and 500 IU human chorionic gonadotropin (hCG) administered 7 days after the mixture injection. As a control, bitches received 50 IU/kg eCG, with 500 IU hCG administered 7 days after eCG injection. Blood-tinged vaginal discharge, vulvar swelling, plasma progesterone concentrations and ovarian follicular development were assessed from day 0 to day 14. IAS alone injection did not induce oestrus in bitches at the anoestrous stage. Conversely, vulvar swelling, blood-tinged vaginal discharge and an estimated luteinizing hormone (LH) surge appeared on days 3-7, days 3-6 and days 7-9 after the IAS+eCG mixture injection, respectively, in all five bitches at the anoestrous stage. The average number of developing and ovulated follicles in bitches administered IAS+eCG was 8.8 and 9.6 respectively. A single eCG injection followed by hCG induced oestrous signs, with an average of 8.3 developing follicles and 4.5 ovulated follicles. This study revealed that IAS alone did not induce oestrus, but when IAS was used in combination with eCG, it induced oestrus and promoted a considerable number of ovulations in anoestrous dogs.

The objective was to determine the size of the dominant ovulatory follicle, the ovulation time and the conception rate of cows in anestrus treated with serum equine chorionic gonadotropin (eCG), recombinant chorionic gonadotropin and untreated controls. 57 anestrus primiparous Brangus cows were used with 70 ± 26 days of calving and a body condition of 2.50 ± 0.15. They were synchronized with a FTAI protocol based on estrogen and progesterone devices (DI). Upon removal of DI, 3 groups were made according to the type of eCG applied: serum eCG received 400 IU im of equine Chorionic Gonadotropin, recombinant eCG received 140 IU of recombinant Equine Chorionic Gonadotrophin and Control did without treatment. Follicular size and time of ovulation (normal or abnormal) was determined at DI removal, during FTAI and 7 days later. An ANOVA was performed to determine the effect of the treatment on ovarian dynamics and a chi-square test and correspondence analysis to establish associations. In normal ovulation cows at the FTAI, 94.4% of the serum eCG, 55.5% of the recombinant eCG and 71.4% of the Control cows presented a dominant ovulatory follicle (DOF), which in those treated with serum eCG, it was 1.4 mm greater in relation to the Control (P = 0.0073). The ovulation rate, in normal and abnormal ovulation cows, was 94.4% for serum eCG, and for recombinant eCG and 66.6% for Controls. A significant association was detected between ovulation time and the treated groups (P = 0.0042). Normal ovulation was 38.9% higher in cows with serum eCG in relation to those treated with recombinant eCG and 50.8% in comparison with Control. A significant relationship was observed between the conception rate and the groups treated with the different Gonadotropins (P = 0.0574), being 66.7% in serum eCG, 50% in recombinant eCG and 28.6% in the Control. The use of eCG, either serum or recombinant, in anestrus primiparous cows, stimulates the development of DOF at FTAI, increases ovulation rate and improves conception in relation to untreated cows.

Follicular changes throughout the oestrous phase have been poorly documented in queens because of the location and the small size of ovaries. We investigated follicular development in queens treated with a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) and evaluated the effects of vaginal stimulation by a tomcat on ovulation induction. A hormonal treatment was administered using a simple crossover design. Four queens were administered 150 IU of eCG (day 1) and 250 IU of hCG on day 5 and 6. Half of the queens were mated with a vasectomised tomcat for 3 days after hCG injection. Ultrasound imaging of the ovaries clamped at a subcutaneous site was performed once a day from day 1 to 7, and on day 13, and the serum concentrations of oestradiol and progesterone were examined on day 1, 5, 7 and 13. The mean number of follicles gradually increased with the eCG treatment and decreased after hCG injection. The ovulation rate of follicles was significantly higher in the vaginal stimulation group (70.0%) than in the control group (42.6%). During the hormonal treatments, the serum concentration of oestradiol and progesterone did not differ between the two groups. Ultrasound imaging of the ovaries clamped at a subcutaneous site showed that eCG and hCG treatment promoted the follicular growth and corpus luteum formation, respectively. The combination of hCG injection with vaginal stimulation by a vasectomised tomcat enhanced the ovulation rate of follicles., Ultrasound imaging of the ovaries clamped at a subcutaneous site showed that the combination of hCG injection with vaginal stimulation by a vasectomised male cat enhanced the ovulation rate of follicles.

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

L’étude a eu pour objectif d’évaluer l’efficacité d’un traitement combiné à base de progestérone intravaginale sur l’induction de l’oestrus de femelles croisées au Cameroun. Un total de 116 vaches Goudali (Bos indicus) x Charolaises (Bos taurus) ayant mis bas au moins une fois, sans antécédent pathologique, avec une note d’état corporel supérieure à 2, ont été sélectionnées dans des élevages du département de la Vina (arrondissement de Nyambaka). Elles ont été rassemblées dans un ranch et réparties en lots de 10 animaux. Le protocole d’induction et de synchronisation des chaleurs a consisté en la pose d’un dispositif intravaginal à base de progestérone (Prid Delta) pendant huit à neuf jours. Vingt-quatre heures avant son retrait, une injection de cinq millilitres de prostaglandine F2alpha (Dinoprost, Enzaprost) a été administrée. Au moment du retrait, 500 UI de gonadotropine chorionique équine ont été injectées. Cinquante-six heures après le retrait, l’insémination a été effectuée à heure fixe avec de la semence de taureaux Gyr et Brahman. Les taux d’induction de l’oestrus et de synchronisation, de rétention du dispositif intravaginal, de gravidité en première insémination, de fécondité, et d’avortement ont été respectivement de 100 %, 100 %, 56,0 %, 51,7 %, et 4,3 % (en % des vaches traitées). Le Prid Delta s’est révélé efficace pour l’induction des chaleurs et l’obtention de gravidités chez les vaches Goudali x Charolaises.

The objective of the present study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to different proestrus lengths for Fixed-time AI (FTAI) in beef heifers. In Experiment 1, pre-pubertal heifers (n = 46) received a 6-day estradiol/progesterone-based treatment (J-Synch protocol), and were then allocated into four experimental groups in a 2 × 2 factorial design, to receive or not receive eCG (300 IU) at the time of intravaginal progesterone device removal, and to receive GnRH at 48 h or 72 h after device removal (to induce shortened and prolonged proestrus length, respectively). Endometrial samples were obtained 6 d after ovulation from the cranial portion of the uterine horn. The eCG administration induced greater serum estradiol-17β concentrations before ovulation (P 0.05) and greater proportion of heifers bearing a competent corpus luteum after ovulation (P = 0.054). Delaying GnRH administration from 48 h to 72 h induced a longer interval from device removal to ovulation (i.e., prolonged proestrus; P 0.05), larger diameter of the ovulatory follicle, and greater progesterone concentrations on Day 10-11 after ovulation. Heifers in eCG + GnRH72h group had more uterine receptors in luminal epithelium than those in eCG + GnRH48h group (PR and ERα), and than those in No eCG + GnRH72h group (PR) (P 0.05). No effect of eCG or GnRH treatments was found in endometrial gene expression of progesterone and estrogen receptors. In Experiment 2, a total of 2,598 heifers received the J-Synch protocol associated or not with eCG administration at device removal, followed by FTAI/GnRH at 60 or 72 h after device removal (i.e., prolonged proestrus protocol). Heifers that received eCG had greater P/AI than those not receiving eCG (P 0.05) and there was an interaction between eCG treatment and time of FTAI. The lowest P/AI was found in those heifers that received FTAI/GnRH at 72 h without eCG treatment at device removal (P 0.05), and no differences were found between the other experimental groups. In conclusion, prolonging the length of proestrus in J-Synch protocol improves ovulatory follicular diameter and luteal function; and the administration of eCG at device removal improves preovulatory estradiol concentrations and luteal function. Finally, P/AI was enhanced by eCG treatment and the improvement was more evident when FTAI/GnRH was performed at 72 h after device removal.

Severe immunodeficient mice are an essential tool for the examination of the efficacy and safety of new therapeutic technologies as a humanized model. Previously, non-obese diabetic (NOD)/Shi-scid IL2rγnull (NOG) mice were established as immunodeficient mice by combining interleukin-2 receptor-γ chain-knockout mice and NOD/Shi-scid mice. The NOG mice are used frequently in the research of therapeutic monoclonal antibodies and regenerative medicine for human diseases. Establishment of an efficient production system of NOG mice, using optimized reproductive techniques, is required to accelerate research. In this study, we investigated the efficacy of the superovulation technique using equine chorionic gonadotropin (eCG) and inhibin antiserum (IAS) in NOG mice of various ages (4, 8, 12, 24, or 54 weeks). Additionally, we examined the fertilizing and developmental ability of the oocytes through in-vitro fertilization using frozen–thawed sperm, embryo culture and embryo transfer. The results showed that NOG mice produced the highest number of oocytes at 12 weeks old following the co-administration of eCG and IAS (collectively IASe) (70 oocytes/female). IASe was more effective in increasing the number of oocytes v. eCG at all ages. The IASe-derived oocytes demonstrated the ability to fertilize and develop into blastocysts and pups. Finally, we demonstrated that three strains of genetically modified NOG mice were efficiently produced through the optimized reproductive techniques. In summary, we developed an efficient system for the production of immunodeficient mice using 12-week-old, IASe-treated female NOG mice.

A sincronização do estro é uma ferramenta útil que fornece ao criador de ovinos um planejamento estratégico, concentrando e regularizando os nascimentos em diferentes épocas do ano, o que permite suprir a demanda do mercado. Diferentes protocolos hormonais estão disponíveis no mercado, porém, seus efeitos e resultados ainda não são totalmente claros. Objetivou-se avaliar protocolos de sincronização de estro de curta duração com o uso de hormônio folículo estimulante (FSH) e gonadotrofina coriônica equina (eCG) como indutores de ovulação em borregas mestiças Dorper na contra-estação. As borregas receberam um dispositivo intravaginal contendo 0,36g de progesterona (D0) e após seis dias, o dispositivo foi removido e assim efetuou-se os seguintes tratamentos: T-FSH recebeu aplicação intramuscular de 10mg de dinoprost e 10 mg de FSH (D6); T-eCG recebeu aplicação intramuscular de 10mg de dinoprost e 300UI de eCG (D6). As ovelhas foram submetidas à monta natural. A detecção de estro se deu através da marcação de fêmeas em cio com a deposição da tinta no peitoral do reprodutor. O diagnóstico de gestação foi realizado trinta dias após o último dia de exposição aos reprodutores com ultrassom. O do uso de dispositivo de progesterona por curto período de tempo foi eficiente. Não houve diferença para taxa de estro (100% T-eCG vs. 100% T-FSH). Um maior número de animais do T-eCG obteve manifestação estral mais precocemente (57,6 ± 16,33 para T-eCG vs. 61,2 ± 16,47 para T-FSH), embora não apresentem diferença estatística (P-valor 0,8723). A taxa de prenhez foi similar em ambos os grupos, sendo 60% (12/20) no T-FSH e 45% (9/20) no T-eCG, não havendo diferença estatística (P-valor=0.35). Conclui-se que tanto a aplicação de FSH, quanto de eCG foram efetivos em protocolo de curta duração para sincronização de estro ovino. Palavras-chave: Gonadotrofina. Borregas. Primer Ovino. Ovulação. Biotecnologia Reprodutiva. Abstract The estrus synchronization is a useful tool that provides a strategic planning to the sheep breeder, concentrating and regularizing births at different times of the year, which allows to supply the market demand. Different hormonal protocols are available on the market; however, their effects and results are still not entirely clear. The objective was to evaluate short-term protocols for estrus synchronization using follicle stimulating hormone (FSH) and equine chorionic gonadotropin (eCG) as ovulation inducers in cross-breed Dorper lambs in the counter-season. The lambs received an intravaginal device containing 0.36 g of progesterone (D0) and after six days, the device was removed and the following treatments were performed: T-FSH received intramuscular application of 10 mg of dinoprost and 10 mg of FSH (D6); T-eCG received intramuscular application of 10mg of dinoprost and 300UI of eCG (D6). The sheep were submitted to natural breeding. The detection of estrus occurred through the marking of females in heat with the deposition of ink on the pectoral of the hams. The diagnosis of pregnancy was made thirty days after the last day of exposure to hams with ultrasound. The use of a progesterone device for a short period of time was efficient. There was no difference in estrus rate (100% T-eCG vs. 100% T-FSH). A higher number of animals from T-eCG had earlier estrous manifestation (57.6 ± 16.33 for T-eCG vs. 61.2 ± 16.47 for T-FSH), although without statistical difference (P-valor 0,8723). The pregnancy rate was similar in both groups, with 60% (12/20) in T-FSH and 45% (9/20) in T-eCG, with no statistical difference (P-value = 0.35). It is concluded that both FSH and eCG applications were effective in a short duration protocol for synchronization of ovine estrus. Keywords: Gonadotropin. Lambs. Ovine Primer. Ovulation. Reproductive Biotechnology.

Aim: The aim of this study was to evaluate the effects of the administration of recombinant bovine somatotropin (rbST) at the moment of implementation of the timed artificial insemination protocol, on follicular dynamics and pregnancy rate in crossbred cows. Materials and Methods: A total of 346 cows were used in two experiments with a factorial 2×2 design. The cycling cows (Tcycling) and the anestrous cows (Tanestrous) were considered as factor 1 and the administration of rbST (TrbST) or not (Tcontrol) as factor 2. The experimental protocol: (1) Tcontrol – day 0 (D0), insertion of a progesterone-release intravaginal device (PRID) plus 2 mg of estradiol benzoate (EB); D8, PRID removal, plus 0.150 mg of prostaglandin F2α, and 400 IU of equine chorionic gonadotropin; D9, 1 mg of EB; and with artificial insemination at day 10; (2) TrbST – similar to Tcontrol plus 500 mg of rbST on D0. In experiment I, ultrasound examinations were performed in all treatments. In experiment II, the cows' pregnancy rate was evaluated. Data were analyzed with 5% probability. Results: There was no effect of the protocols on cows cyclicity or follicular growth rate (p>0.05). There was no interaction of the effects, administration of rbST, and the cyclicity of cows on the pregnancy rate. The total pregnancy rate observed was 49.0%. The pregnancy rate in cows receiving rbST was lower for anestrous compared with cycling cows (p

Pregnancy loss during the normal lifespan of endometrial cups (∼37-120-150 days of gestation) may affect a mare’s ability to conceive again in the same breeding season, as equine chorionic gonadotropin (eCG) secretion by retained endometrial cups can lead to abnormal ovulations and follicular growth. While intrauterine kerosene infusion has anecdotally been proposed as a treatment for endometrial cup retention, there are no controlled studies evaluating kerosene’s ability to enhance endometrial cup regression following abortion. The objectives of this study were to assess uterine response, systemic side effects, and efficacy of intrauterine kerosene infusions after abortion. We hypothesized that kerosene infusions would hasten regression of endometrial cups without detrimental effects on the endometrium and the mare’s general health. Twelve light-breed mares were enrolled in the study after an experimentally induced abortion with cloprostenol (n = 12) by 60 ± 2 days of gestation. Mares were randomly allocated to receive an intrauterine infusion with 500 mL of kerosene (n = 6) or 500 mL saline (n = 6) on days 21 and 35 after pregnancy termination. Uterine biopsies were collected at days 7, 21, 35, and 49 post-abortion to evaluate the degree of endometrial fibrosis with Picrosirius Red Stain and to be graded according to the Kenney & Doig 1986 classification. Furthermore, histomorphometry analysis of the endometrium lining, glandular epithelium and glandular density was performed. Endometrial lymphocyte B CD20+, lymphocyte T CD3+, and macrophage IBA-1+ cell populations were characterized by immunohistochemistry. Physical examinations, blood cell counts, and serum biochemistry were performed before, and for 2 days after each uterine infusion. Serum samples were collected for assessment of eCG concentrations. Continuous data were analyzed with MIXED procedure with repeated measures in SAS, categorical data with LOGISTIC procedure of SAS. Significance was set at p

Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterize the levels of NOD-like receptor protein 3 (NLRP3) inflammasome activation in ovaries and liver of mice during obesity progression. Furthermore, we tested the putative role of leptin on NLRP3 regulation in those organs. C57BL/6J female mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for estrous cycle synchronization and ovary collection. In diet-induced obesity (DIO) protocol, mice were fed chow diet (CD) or high-fat diet (HFD) for 4 or 16 weeks, whereas in the hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16 L) or saline (16 C). Finally, the genetic obese leptin-deficientob/ob(+/? and −/−) mice were fed CD for 4 week. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. The estrus cycle synchronization protocol showed increased protein levels of NLRP3 and interleukin (IL)-18 in diestrus, with this stage used for further sample collections. In DIO, protein expression of NLRP3 inflammasome components was increased in 4 week HFD, but decreased in 16 week HFD. Moreover, NLRP3 and IL-1β were upregulated in 16 L and downregulated inob/ob.Transcriptome analysis of CC showed common genes between LEPT and 4 week HFD modulating NLRP3 inflammasome. Liver analysis showed NLRP3 protein upregulation after 16 week HFD in DIO, but also its downregulation inob/ob−/−. We showed the link between leptin signaling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity.

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

Introducción: los objetivos fueron evaluar el estro y la gestación en vacas Bos Taurus, tratadas con un protocolo para la inseminación artificial a tiempo fijo (IATF) con y sin gonadotropina coriónica equina (eCG). Se evaluó también la gestación acumulada con la resincronización de la ovulación 30 días después de la primera IATF. Método: las vacas se asignaron al azar a dos tratamientos: Tratamiento 1 (n = 60), se sincronizó con 2 mg de benzoato de estradiol y un dispositivo intravaginal (DIV) de progesterona (1.3 g) el día 0; el DIV se retiró el día 8 y se aplicaron 25 mg de prostaglandina F2-alfa, 1 mg de cipionato de estradiol y 300 UI de eCG; 48-52 horas después se realizó la IATF. El Tratamiento 2 (n = 50) consistió en el mismo protocolo, sin incluir eCG. Se resincronizó la ovulación en las vacas que resultaron vacías en el diagnóstico de gestación, realizado 30 días después de la primera IATF. En todas las vacas se usó el mismo protocolo aplicado en el Tratamiento 1 para la primera IATF. La segunda IATF se realizó a los 40 días de haber iniciado el programa. Se clasificó la CC en baja (< 2.5), moderada (2.5 a 3.5) y alta (> 3.5). El día de la IATF, el diámetro del folículo preovulatorio (DFP) se dividió en tres categorías (DFP1: < 11 mm; DFP2: 11 a 14 mm; DFP3: > 14 mm). Resultados: el porcentaje de gestación resultó diferente entre tratamientos (p = 0.04; 73 % Tratamiento 1 y 53 % Tratamiento 2). La proporción de vacas en estro antes de la IATF fue de 75 % en el grupo con eCG y 57 % en el testigo (p = 0.04). Las vacas con DFP2 y DFP3 registraron mayor tasa de gestación (p = 0.03), que DFP1. El porcentaje de gestación fue mayor (p = 0.04) en vacas con CC moderada y alta. La gestación general en la primera IATF fue de 63 %. El 51.61 % resultó gestante con la segunda IATF, acumulando 82.1 % en 40 días. Conclusión: la eCG incrementó la proporción de vacas en estro y la gestación; y la resincronización permite acumular un porcentaje adicional de vacas gestantes.

The objective of the current review was to summarize the protocols used for estrous synchronization in ewes during the last two decades. Progesterone (P4) is a major hormone used in most protocols. P4 in the form of a controlled internal drug releasing (CIDR) device, medroxyprogesterone acetate (MAP), and fluorogestone acetates (FGA) has been used for estrous synchronization. Also, gonadotropins such as equine chorionic gonadotropin (eCG) and gonadotropin-releasing hormone (GnRH) are often administered at the end of P4-based protocols to improve fertility. Moreover, the administration of prostaglandins (PG) and ram effects have been used for estrus induction and synchronization of ewes. The findings of previous studies indicate that the outcome of administering various synthetics P4 analogues (CIDR, MAP, and FGA) in ewes is comparable in terms of estrous synchronization/induction. The supplementation of P4-based protocols with eCG, however, improves the estrus response and pregnancy rate during breeding and non-breeding season. On the other hand, PG is effective for successful estrous synchronization during the breeding season only. Often, two injections of PG are administered either 11 or 14 days apart along with P4-based protocols to lyse ovine corpus luteum (CL) when it is receptive to PG i.e., 3 days post-ovulation. Alternatively, the "ram effect" has been shown to improve the efficacy of P4-based protocols and can be used as an alternative to eCG in ewes. The current review describes the methods of synchronization and their outcomes during breeding and a non-breeding season in ewes.

Gonadotropin-releasing hormone agonists (GnRHa) are used as an alternative to human chorionic gonadotropin (hCG) to trigger ovulation and decrease the risk of ovarian hyperstimulation syndrome. GnRHa is less potent at inducing ovarian vascular endothelial growth factor (VEGF), but may also affect endometrial angiogenesis and early placental development. In this study, we explore the effect of superovulation on endometrial angiogenesis during critical periods of gestation in a mouse model. We assigned female mice to three groups: natural mating or mating following injection with equine chorionic gonadotropin and trigger with GnRHa or hCG trigger. Females were killed prior to implantation (E3.5), post-implantation (E7.5), and at midgestation (E10.5), and maternal serum, uterus, and ovaries were collected. During peri-implantation, endometrial Vegfr1 and Vegfr2 mRNA were significantly increased in the GnRHa trigger group (P

The objective was to compare pregnancy per AI (P/AI) between two shortened timed-AI (TAI) protocols in beef cattle. This study also determined whether administration of eCG in heifers and timing of AI in cows would affect P/AI. Cattle were submitted at random to either a modified 5-d Co-synch protocol (Day 0 = progesterone releasing device (CIDR); Day 5 = CIDR removal and 500 μg of cloprostenol (PGF); Day 8 = 100 μg GnRH concurrent with AI) or J-synch protocol (Day 0 = CIDR insertion and 2 mg of estradiol benzoate i.m.; Day 6 = CIDR removal and 500 μg PGF; Day 9 = 100 μg GnRH concurrent with AI). In Experiment 1, 1135 heifers (13-15 mo of age) received an estrus detection patch (Estrotect™) on Day 5 and 579 were selected at random to receive 300 IU of equine chorionic gonadotropin (eCG) at the time of CIDR removal. Patches were scored from 0 to 3 based on color change between initial application and AI; 0 = unchanged, 1 = ≤ 50% change, 2 =50% change, 3 = missing. Estrus was defined to have occurred when the patch was scored 2 or 3. In Experiment 2, 399 cyclic, non-lactating beef cows from 1 location were submitted to either the modified 5-d Co-synch or J-synch protocol and within each protocol cows were TAI at either 66 ± 1 (n = 199) or 72 ± 1 h (n = 200) following CIDR removal. Transrectal ultrasonography was used in both experiments to determine presence of a corpus luteum (CL) on Day 0, and to diagnose pregnancy 35 d after TAI. In Experiment 1, eCG increased estrus rate only in heifers without a CL on day 0 that were submitted to the modified 5-d Co-synch protocol (41.9 vs. 69.6%). Heifers submitted to the J-synch protocol had greater (P = 0.03) P/AI compared with those in the modified 5-d Co-synch (48.7 vs. 41.1%) and heifers that expressed estrus before AI had increased (P 0.0001) P/AI compared to those that did not (53.6 vs. 36.5%). Administration of eCG and presence of a CL tended to affect P/AI (P = 0.13). In Experiment 2, cows submitted to the J-synch protocol tended (P = 0.07) to have greater P/AI compared to those in the modified 5-d Co-synch (74.1 vs. 66.5%). There was no association between P/AI and timing of AI. In summary, the J-synch protocol resulted in greater P/AI than the modified 5-day Co-synch protocol in heifers and cows. Administration of eCG increased estrus rate in heifers without a CL at the start of the protocol and tended to improve P/AI in all heifers. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows subjected to either TAI protocol.

The objective of the present study was to evaluate the administration of Equine Chorionic Gonadotropin (eCG) in a protocol of fixed-time artificial insemination (FTAI) to increase the pregnancy rate in cows subjected to high altitude conditions. These conditions could generate a syndrome of right-sided heart failure and, as consequence, animals trend to have a low body score condition with a decrease in fertility. The research was conducted with 84 lactating cows (Brown Swiss) randomly into two groups with a body condition score between 2.5 and 3.5, allocated at 3100 m above sea level. In the FTAI protocol, 400 IU of eCG was administered (n = 42) or not (n = 42) at progesterone intravaginal device removal. A significant improvement in pregnancy rate was found with eCG treatment in those cows with low body condition score (BCS of 2.5, scale 1–5), while no effect was found in those cows with higher BCS (i.e. 3.0 and 3.5). In conclusion, the addition of 400 IU of eCG in an FTAI in cows under high altitude conditions and low body conditions can improve the fertility.

Purpose This study aimed to examine whether the Tinagl1 might be associated with ovulation in aged females and reproductive age‐associated fibrosis in the stroma of the ovary. Methods To address the ovulatory ability and quality of ovulated oocytes, we induced ovulation by treatment with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) followed by in vitro fertilization. We also performed Picrosirius Red (PSR) staining to evaluate ovarian collagen deposition. Results As compared to ovulation in 8‐ to 9‐month‐old Tinagl1flox/flox mice, the number of ovulated oocytes from Tinagl1flox/flox mice decreased in an age‐dependent manner in mice more than 10‐11 months old, whereas the ovulated oocyte numbers in Tinagl1 −/− mice decreased significantly at 14‐15 months. In vitro fertilization followed by embryo culture demonstrated the normal developmental potential of Tinagl1‐null embryos during the preimplantation period. PSR staining indicated that collagen was found throughout the ovarian stroma in an age‐dependent manner in Tinagl1flox/flox females, whereas those distributions were delayed to 14‐15 months in Tinagl1 −/− females. This timing was consistent with the delayed timing of age‐related decline of ovulation in Tinagl1 −/− females. Conclusions The alleviation of age‐associated depression of ovulation was caused by delayed ovarian collagen deposition in Tinagl1‐null female mice., Effect of female aging on numbers of ovulated oocytes fromTinagl1‐deficient mice.

Many protocols of estrous synchronization in ewes have been developed in the last few decades. The aim of this study was the effect of intravaginal progestogen, medroxyprogesterone acetate (MPA), sponges (a progesterone analogue) with or without equine chorionic gonadotropin (eCG) in out of breeding season of fat-tailed Iranian Shaal breed anestrus ewes to estimate the conception rate in suburb of Tehran-Iran. In total, 50 non-cyclic ewes out of 85 ewes with 7 years old were selected for out of breeding season program in May–June 2018. They were distributed into two groups (control and treatment). The treatment group (no. 25) received 12 days a sponge containing 60 mg MPA and 500 IU eCG on the day of sponge removal. The control group (no. 25) received 12 days of sponge containing MPA only. Rams were introduced to the flock after removal of sponges in treatment group and concomitant in control group. They remained within the flock for 40 days. The ram to ewe ratio was 1 to 6. Transabdominal ultrasonography was performed for pregnancy diagnosis. The obtained data was analyzed using SPSS software version 16. The conception rate in treatment and control groups were differed significantly (P

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild-derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non-reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non-reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2- and/or 4-cell stage using both fresh and frozen-thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non-reproductive seasons. This study is the first to develop IVF-IVC wild large Japanese field mice beyond the 2- and/or 4-cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.

Although thyroid hormone (TH) plays important roles in regulating ovarian development, the mechanism are still unclear. Cytochrome P450 lanosterol 14α-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that involved in folliculogenesis and oocyte maturation, which is regulated by follicle stimulating hormone (FSH). However, the effect of TH on CYP51 expression in ovarian cells is unclear. The objective of this study was to determine the effects of TH on CYP51 in rat ovary. Hypothyroidism rats were induced by 6-propyl-2-thiouracil (PTU), genes expressions in ovary were analyzed by Western blot or qRT-PCR. The data showed that CYP51 was significantly decreased in hypothyroidism, which was accompanied by the down-regulation of mRNA level. Meanwhile, similar tendency was also showed in FSHR expression in hypothyroidism. To evaluate the effect of the gonadotropin on CYP51 and FSHR expression in ovarian cells in vivo, hypo rats were injected subcutaneously with equine chorionic gonadotropin (eCG) respectively. The results showed that eCG reversed CYP51 and FSHR expression in hypo group. Moreover, FSH-induced CYP51 expression was meditated by FSHR. In addition, serum concentration of FSH and E2 were also decreased in hypothyroidism, and E2 was up-regulated by eCG treatment. These results indicate that hypothyroidism changes CYP51 and FSHR expression in ovary, which are regulated by gonadotropin. Moreover, genes changes in ovary are at least partially attributed to steroids biosynthesis.

Follitropin, an important gonadotropin hormone, participates in vitellogenesis and spermatogenesis. Equine chorionic gonadotropin (eCG) can induce gonadotropin hormone activity in non-equid species and exhibits a long biological half-life. Here, we report the production, using silkworm larval and pupal systems, of biologically active recombinant hybrid-type follitropins based on the coding sequence of the eCG C-terminal peptide (CTP) between the mature β- and α-chains of eel. The three constructs, rJeFSH, rJeFSH·eCG, and rJeFSH·2xeCG were produced and verified to be N- or O-glycosylated and secreted mature peptides. Although rJeFSH·eCG contains more elaborate O-linked carbohydrate chains than rJeFSH, it elicited no significant in vitro oocyte maturation, which may be a result of insufficient terminal sialylation of its N-and O-linked carbohydrate chains. Then, a hybrid of rJeFSH·2xeCG extended with two eCG CTP. Furthermore, the receptor binding assay revealed potency of rJeFSH and rJeFSH·2xeCG to be a few folds greater than that of rJeFSH·eCG. The findings of this study will be useful for the development of more efficient GTHs in teleosts, including eels, when various modifications with two or more extended eCG CTP produced by silkworm are included.

Superovulation technique is important to improve the efficiency of oocyte and animal production and reduce the number of oocyte donors. Previously, we have reported that the coadministration of inhibin antiserum (IAS) and equine chorionic gonadotropin (eCG) results in the production of >100 oocytes in a 4-week-old female C57BL/6 mice. It is well established that superovulation depends on the age of the female mice. However, detailed data regarding the ovulation of juvenile, mature, and aged female mice following the administration of IAS and eCG as well as the performance of reproductive technologies using oocytes have not yet been investigated. In the present study, we examined the effect of the age of female mice (3-50 weeks old) on the number of ovulated oocytes via the coadministration of IAS and eCG or eCG alone. Treatment with IAS plus eCG produced the maximum number of oocytes at 4 weeks of age. Moreover, IAS plus eCG produced more oocytes than eCG alone in mice aged between 3 and 5 weeks or 7 and 30 weeks. The fertilization and birth rates were similar between the two treatments at any age. Moreover, after vitrifying and warming the embryos, the survival and birth rates of two-cell embryos were similar between the two treatments. Subsequently, we examined the optimal ages of female mice (between 24 and 34 days) to obtain a high and stable number of oocytes. In mice aged between 24 and 32 days, IAS plus eCG induced the production of more eggs than eCG alone. Notably, the coadministration of IAS and eCG in mice aged between 25 and 31 days resulted in stable ovulation and high number of oocytes. Using the tip of the optimal female aged between 25 and 31 days old, we demonstrated an efficient production of embryos and offspring between homozygous knockout males and few females aged 26-28 days via in-vitro fertilization and embryo transfer. In summary, the coadministration of IAS and eCG resulted in a higher number of oocytes in juvenile, mature, and aged female mice. This treatment may be useful for the efficient production of homozygous mutant mice from a limited number of female mice.

Studies have shown that the use of equine chorionic gonadotropin (eCG), which binds both follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors, could modify the female reproductive tract. We, thus, aimed to quantify the messenger RNA (mRNA) abundance of genes related to cumulus-oocyte complexes (COCs) and embryo quality in Nelore cows (Bos taurus indicus) submitted to ovarian superstimulation using only FSH (FSH group; n = 10) or replacement of the last two doses of FSH by eCG (FSH/eCG group; n = 10). All animals were slaughtered and the ovarian antral follicles from both groups (10-14 mm in diameter) were aspirated for cumulus, oocyte and in vitro embryo production gene expression analysis. The relative mRNA abundance of 96 genes related to COCs development and embryo quality was measured by RT-qPCR. We found that oocytes are more affected by eCG use and that 35 genes involved in lipid metabolism, oxidative stress, transcriptional control, and cellular development were upregulated in the FSH/eCG group. In blastocysts, lipid metabolism seems to be the main pathway regulated by eCG use. We suggest that these multiple effects could be due to the ability of eCG to bind LHR and FSHR, which could activate multiple signal transduction pathways in the superstimulated ovary, further impacting the transcriptional profile of COCs and blastocysts.

Background To directly assess the biological role of oligosaccharides in recombinant equine chorionic gonadotropin (rec-eCG) functioning, cDNA encoding the full-length eCGβ-subunit was fused with the mature protein part of the α-subunit, and we examined the expression levels of deglycosylated eCG mutants, the ovulation rate for deglycosylated mutants in C57BL/6 mice. Results The characterizations of heterodimeric and tethered mutants were studied following their respective secretions in culture medium, molecular weight and ovulation in vivo. Rec-eCG variants containing mutations at glycosylation sites at Asn82 of the α-subunit (eCGβ/αΔ82) and Asn13 of the β-subunit (eCGβΔ13/α) were not efficiently secreted into the culture medium from transfected cells. Western blot analysis revealed that the rec-eCGβ/α proteins have an approximate broad range of molecular weights of 40–46 kDa. Three rec-eCG mutants—a deglycosylated site at Asn56 of the α-subunit (eCGβ/αΔ56), a deletion of the C-terminal region of the β-subunit (eCGβ-D/α), and the double mutant (eCGβ-D/αΔ56)—turned out to have clearly lower (approximately 4–23 kDa) molecular weights. Protein N-glycosydase F (PNGase F) treatment markedly decreased the molecular weight to approximately 2–10 kDa. Normal oocytes were significantly more abundant in the natural eCG–treated group than in mutant rec-eCG–treated groups. In particular, numbers of nonfuntional oocytes were remarkably lower in all rec-eCG groups. Conclusions Our results indicate that the ovulation rates of oocytes are not affected by the deglycosylated rec-eCGβ/α mutant proteins. There are around 20% non-functional oocytes with natural eCG and only 2% with the rec-eCGs tested. These results provide insight into the molecular mechanisms underlying the production of rec-eCG hormones with excellent bioactivity in vivo. Electronic supplementary material The online version of this article (10.1186/s12896-019-0550-6) contains supplementary material, which is available to authorized users.

Spermatogenesis is a complex developmental process that originates from spermatogonia stem cell. This process consists of sequential, highly organized steps of cell proliferation and differentiation resulting in the generation of functional spermatozoa. Many growth factors, hormones, and cell interactions of germ cells with Sertoli cells regulate these processes, and the failure of any of the processes can lead to male infertility The aim of the present study was to determine the effects of equine chorionic gonadotropin on spermatogonial stem cell colony formation and differentiation after in vitro co-culture with Sertoli cells. In this experimental study, Sertoli and spermatogonial stem cells were isolated from 3- to 5-month-old calves. Co-cultured Sertoli and spermatogonial stem cells were treated with equine chorionic gonadotropin in treatment groups before colony assay. The present study showed that equine chorionic gonadotropin increase colony number and decrease colony diameter. Equine chorionic gonadotropin can induce proliferative pathway and inhibit differentiation process in spermatogonia stem cell co-culture with Sertoli cells.

At weaning, 150 primiparous sows were assigned sequentially to receive 400 IU equine chorionic gonadotropin (eCG) with 200 IU human chorionic gonadotropin (hCG) (Gn600), or 400 IU eCG with 400 IU hCG (Gn800), or served as untreated controls. Compared with control and Gn600, the Gn800 had increased estrus response by day 7 and reduced wean-to-estrus intervals (P

This study evaluated the effect of equine chorionic gonadotropin (eCG) on reproductive performance, when incorporated into the first Ovsynch + P4 synchronization following planned start mating (PSM) in pasture-based lactating dairy cows. Two synchrony programs were compared in a randomized controlled trial in Queensland, Australia. Lactating cows from a single dairy herd (n = 782) were randomly allocated to Control and eCG groups. Control cows had their estrous cycles synchronized by treatment with 100 μg gonadotropin releasing hormone (GnRH; im) and insertion of a progesterone (P4) releasing intravaginal device that contained 1.0 g of P4 on Day 0; removal of P4 device and administration of 500 μg of an analogue of PGF2α on Day 7 (cloprostenol; im); 100 μg im of GnRH on Day 9, and fixed-time artificial insemination (FTAI) on Day 10. The eCG group were treated the same as the Control group except for the addition of 400 IU of eCG, im on Day 7 of the first synchronized estrous cycle. Following the first insemination, non-pregnant cows from both groups had their estrous cycles synchronized with the same treatment protocol without using eCG. The effects of eCG on 42d cumulative incidence of pregnancy and pregnancy per AI (P/AI) were determined using logistic regression models. The effect of eCG on time to pregnancy was determined using Kaplan-Meier survival analysis and Cox proportional hazards models. Adjusted 42 d cumulative incidence of pregnancy for eCG and control groups were 47.2 and 39.3% respectively (Odds ratio [OR] = 1.38, 95% CI: 1.01–1.88). Hazard of pregnancy tended to be higher in eCG cows overall (Hazard ratio [HR] = 1.18, 95% CI: 0.99–1.41) and was significantly higher when restricting to the first 42 days after PSM (HR = 1.31, 95% CI: 1.04–1.64). Hazards of pregnancy were not different between groups when restricting to > Day 42 post PSM (HR = 1.00, 95% CI: 0.77–1.31). P/AI tended to be higher in eCG treated cows at the first AI (44.0 vs 37.7%, OR = 1.30, 95% CI: 0.94–1.78). P/AI for second and third AIs were not significantly different between groups. In this herd, a single treatment of eCG at the first synchronized estrus after PSM improved reproductive performance in the short term, but not at subsequent inseminations.

Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment 1, oocytes, corona radiata, cumulus, and granulosa cells were collected from mares classified as young (4-12 years) or old (> 20 years). Transcript abundance was evaluated for connexins -37 (GJA4) and -43 (GJA1); zona pellucida glycoproteins 1, 2, 3, and 4 (ZP1, ZP2, ZP3, ZP4); Tubulin (TUBA1A), and equine chorionic gonadotropin β. For experiment 2, oocytes that failed to cleave following intracytoplasmic sperm injection (ICSI) were stored in salt solution for up to 4 years and used for sperm binding assays. Transcript abundance for GJA1 was decreased in oocytes, corona radiata, and granulosa cells while GJA4 was decreased in cumulus cells from old compared to young mares. Additionally, ZP1 tended to be decreased in corona radiata and cumulus cells from old mares. Oocytes from old mares tended to bind less spermatozoa compared young mares. Oocytes that failed to cleave following ICSI can be used for sperm binding studies for up to 2 years without losses in sperm binding. Our findings suggest that maternal age may contribute to changes in cellular communication and the ZP that could impact sperm binding.

The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation–sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.

Simple Summary Because of their large size, blood, serum, or other substances are often collected from horses for production of biologics and therapeutics used in humans and other animals. There are few international guidelines that provide recommendations for caring for horses kept for these purposes. In this paper, general guidelines are provided to ensure well-being of horses kept for production of biologics. Abstract Various pharmaceutical products have been derived from horse blood and urine for over a century. Production of biologics and therapeutics from these samples is a niche industry and often occurs in regions with little regulation or veterinary oversight. To ensure good welfare of horses maintained for these purposes, guidance has been developed to support the industry.

Timed artificial insemination (TAI) is an efficient reproductive technology in batch farrowing production that aids management in pig farms. However, the effect of TAI on the reproduction performance is still controversial. This study aimed to evaluate the effects of two TAI protocols on the reproductive performance of primiparous sows. A total of 332 weaned sows were randomly allocated into three treatments. Sows assigned to Control (n = 110) were untreated and inseminated on each day in oestrus after weaning. Sows assigned to eG-TAI (n = 112) received equine chorionic gonadotropin (eCG) 24 h after weaning and gonadotropin-releasing hormone (Gonadorelin: GnRH) at oestrus, and were inseminated at 8 and 32 h later if oestrus at 0800, or 16 and 40 h later if oestrus at 1600. Sows assigned to 2e-TAI (n = 110) received eCG and GnRH 24 h and 96 h after weaning, respectively, and were inseminated 16 and 40 h after GnRH administration. Sows showing oestrus at GnRH administration or 64 h after were inseminated immediately, for a total of three inseminations. Ultrasonographic evaluations were performed to determine the follicular diameter and time of ovulation. Most sows in the 2e-TAI and eG-TAI groups ovulated 0–48 h after the GnRH injection. Our results indicated that oestrus rate within seven days after weaning in the experimental groups was higher, and weaning-to-oestrus interval was shorter than in the control group (99.3 h vs 113.5 h, P

Gonadotropins play vital roles in the regulation of female reproductive ability and fertility. Our study aimed to determine the effects of superovulation induced by increasing doses of equine chorionic gonadotropin [eCG; also referred to as pregnant mare serum gonadotropin (PMSG)] on the developmental competence of mouse embryos and on aneuploidy formation during in vitro fertilization (IVF). eCG dose-dependently enhanced the oocyte yield from each mouse. Administration of 15 IU eCG significantly reduced the fertilization rate and the formation of four-cell embryos and blastocysts and increased the risk of chromosome aneuploidy. The IVF-derived blastocysts in the 15 IU eCG treatment group had the fewest total cells, inner cell mass (ICM) cells and trophectoderm (TE) cells. Moreover, more blastocysts and fewer apoptotic cells were observed in the 0, 5, and 10 IU eCG treatment groups than in the 15 IU eCG treatment group. We also investigated reactive oxygen species (ROS) levels and variations in several variables: mitochondrial membrane potential (MMP); active mitochondria; mitochondrial superoxide production; adenosine triphosphate (ATP) content; spindle structures; chromosome karyotypes; microfilament distribution; and the expression of Aurora B [an important component of the chromosomal passenger complex (CPC)], the spindle assembly checkpoint (SAC) protein mitotic arrest deficient 2 like 1 (MAD2L1), and the DNA damage response (DDR) protein γH2AX. Injection of 15 IU eCG increased ROS levels, rapidly reduced MMP, increased active mitochondria numbers and mitochondrial superoxide production, reduced ATP content, increased abnormal spindle formation rates, and induced abnormalities in chromosome number and microfilament distribution, suggesting that a high dose of eCG might alter developmental competence and exert negative effects on IVF-obtained mouse embryos. Additionally, the appearance of γH2AX and the significantly increased expression of Aurora B and MAD2L1 suggested that administration of relatively high doses of eCG caused Aurora B-mediated SAC activation triggered by ROS-induced DNA damage in early mouse IVF-derived embryos for self-correction of aneuploidy formation. These findings improve our understanding of the application of gonadotropins and provide a theoretical basis for gonadotropin treatment.

The objective of the present research was to determine the effect of long synchronization protocols based on reused progesterone devices (controlled internal drug release [CIDR]) associated with different doses of equine chorionic gonadotropin (eCG) on reproductive variables in crossbred sheep (Suffolk × Kathadin × Dorset). The CIDR were used for eleven days in a previous study in sheep from the same herd and were washed and disinfected before reusing. Sixty-four sheep, in the reproductive season, were randomly assigned to four experimental groups (n = 16). Treatments consisted of a group with 10 d CIDR and 300 IU eCG; a group with 10 d CIDR and 400 IU of eCG; a group with 12 d CIDR and 300 IU of eCG; and a group with 12 d CIDR and 400 IU of eCG. A completely randomized design was used. There was an estrous presentation rate of 100% in all treatments. The begining of estrous, gestation rate, fertility rate, type of parturition, and prolificacy index were equal between groups. Progesterone serum concentration was higher in sheep from the 10 d CIDR groups. The CIDR, reused for the second time, associated with 300 or 400 IU of eCG for estrous synchronization in sheep, are effective to obtain good pregnancy rates and ensures higher prolificacy rates.

The aim was to study reproductive outcomes of Nelore (Bos indicus) cows submitted to a 7-d estradiol (E2)/progesterone (P4)-based timed artificial insemination (TAI) protocol, receiving various combinations of doses and hormones. Primiparous (n = 962) and multiparous (n = 1935) cows were submitted to synchronization (n = 2012) and resynchronization (n = 885 non-pregnant cows at pregnancy diagnosis 30 d after TAI) protocols, following a 2 × 2 × 2 factorial arrangement of eight treatments. At the initiation of the TAI protocol (Day -9), all cows received a 1.0 g intravaginal P4 insert, 2.0 mg E2 benzoate and received (PGF1) or not (PGF0) 0.5 mg cloprostenol sodium (PGF). On Day -2, the P4 insert was removed, all cows received 0.5 mg PGF, 300 IU equine chorionic gonadotropin (eCG) and 0.5 (EC0.5) or 1.0 mg estradiol cypionate (EC1.0). On Day 0, cows were treated (G1) with 8.4 μg buserelin acetate (GnRH) or not (G0), concurrently with TAI. The eight treatments were generated: 1) PGF0-EC0.5-G0 (n = 364), 2) PGF0-EC0.5-G1 (n = 363), 3) PGF1-EC0.5-G0 (n = 363), 4) PGF1-EC0.5-G1 (n = 360), 5) PGF0-EC-1.0-G0 (n = 360), 6) PGF0-EC1.0-G1 (n = 363), 7) PGF1-EC1.0-G0 (n = 361), and 8) PGF1-EC1.0-G1 (n = 363). Pregnancy per AI (P/AI) was greater at first AI compared with resynchronization (58.9 [n = 2012] vs. 54.9% [n = 885]). Presence of CL on Day -9 resulted in more cows expressing estrus (81.3 [n = 680] vs. 67.1% [n = 2033]) and greater P/AI (66.0 [n = 692] vs. 54.9% [n = 2106]). There was no difference in P/AI between cows that received or not PGF on Day -9 (58.7 [n = 1447] vs. 56.6% [n = 1450]). In contrast, PGF tended to increase P/AI of cows with CL on Day -9 (with PGF = 69.1 [n = 375] vs. without PGF = 62.5% [n = 317]). Cows that received 1.0 mg EC expressed more estrus than those treated with 0.5 mg (73.8 [n = 1414] vs. 67.9% [n = 1398]) and had greater P/AI (60.2 [n = 1447] vs. 55.1% [n = 1450]). P/AI was greater in cows treated with GnRH at TAI (59.8 [n = 1449] vs. 55.5% [n = 1448]), particularly in cows that did not show estrus (52.7 [n = 393] vs. 38.1% [n = 420]). Moreover, GnRH on Day 0 increased P/AI in cows with BCS3.0 (57.1 [n = 723] vs. 48.6% [n = 698]), in primiparous (50.1 [n = 465] vs. 41.9% [n = 497]) and in cows that received 0.5 mg EC (58.9 [n = 723] vs. 51.3% [n = 727]). In conclusion, 1.0 mg of EC on Day -2 and GnRH at TAI improved P/AI, but the combination of a higher dose of EC and GnRH treatment at AI did not enhance this effect. Furthermore, GnRH improved P/AI especially in Bos indicus cows with lower expression of estrus, such as primiparous, thinner cows, and cows treated with 0.5 mg of EC.

Background : Recent studies have been conducted with the aim of improving the fertility rates in the FTAI programs in beef females. The observation of the estrus expression constitutes an important indicator of fertility in zebu females. Therefore, this work has as an objective to evaluate the impact of the estrus expression on the follicular, luteal and fertility morphofunctional characteristics of Nelore females synchronized for FTAI. Materials, Methods & Results : Sixty five lactating female Nelore ( Bos taurus indicus ) were used. On a random day, denominated day 0 (D0), the 65 Nelore females received a progesterone-releasing device associated to the application of 2.0 mg of estradiol benzoate intramuscularly (IM). On D9, the progesterone-releasing intravaginal devices were removed and was administered 500 μg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 UI of Equine Chorionic Gonadotropin IM. At this point, the animals were marked with a marking stick for the determination of the estrus expression. On D11 of the synchronization protocol, the animals were characterized in two groups: without estrus expression (WO/ ESTRUS) and with estrus expression (W/ ESTRUS). The evaluation of the follicle diameter (FOLD), of the follicle wall area (FOLA), of the follicle wall vascularization (FOLV) and the percentage of vascularization in the area of the preovulatory follicle wall (%FOLV) were conducted on D11 using B-mode ultrasonography and color Doppler and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), the total area of the corpus luteum (CLA), of the area of vascularization of the corpus luteum (CLV), of the percentage of vascularization of the in the area of the corpus luteum (% CLV) and the collection of blood for the evaluation of the serum levels of progesterone were carried out on D24. The analysis of the serum progesterone concentration were performed by the chemiluminescence method using the Access immunoassay systems Progesterone, with a sensitivity of 0.1 ng/mL. The diagnosis of gestation was carried out by transrectal ultrasonography on D45. The animals of the WITH ESTRUS group presented superior FOLD, FOLA, FOV ( P < 0.05) averages to those presented by the cows of the WITHOUT ESTRUS group. In relation to the luteal characteristics, in the WITH ESTRUS group the cows exhibited averages for CLD, CLA, CLV and P4 significantly higher than those of the WITHOUT ESTRUS group. However, the bovines of the WITH ESTRUS group presented a % CLV similar to the presented by the females of the WITHOUT ESTRUS group. As for the fertility rates, there was difference between the experimental groups WITH ESTRUS and WITHOUT ESTRUS. Discussion : The present work demonstrated the impact of the estrus expression on the follicular morphological parameters, therefore in accordance with other studies. These authors show a relation between the estrus expression and levels of estradiol, which is directly related to the sperm transport in the female reproductive tract, oocyte maturation and fertilization directly affecting the fertility of females submitted to the FTAI program. Similarly, there was an interrelation between the estrus expression and the luteal morphological parameters, corroborating with the findings of some researches, where were verified higher luteal morphological parameters associated to higher conception rates in cows that demonstrated estrus behavior in the FTAI. Therefore, the estrus detection can be used as a tool to direct matings in synchronization protocols, promoting the identification of the animals with a higher probability of conception, owing to better follicular and luteal hemodynamic conditions.

Effects were evaluated in Bos indicus cows of eCG and FSH on follicular growth, estrous expression, and pregnancy per artificial insemination (P/AI) as a result of fixed-time artificial insemination (TAI). In Experiment 1, extent of timing-of-ovulation synchronization among cows was evaluated after imposing an estrogen/progesterone-based treatment regimen. At progesterone device removal (D8), cows were administered: eCG, or FSH or served as untreated Controls. In Experiment 2, percentage of cows P/AI was evaluated when the Experiment 1-treatment regimen was imposed. On D10, all cows were artificially inseminated. In Experiment 3, cows were assigned to two treatment groups (Control and eCG) on D8 to evaluate percentage of cows P/AI and estrous expression. In Experiment 1, follicular dynamics were similar among treatment groups. In Experiment 2, follicular growth was greater (P = 0.0001) with the eCG treatment. There was an interaction of treatment × parity (P = 0.007) on percentage of cows P/AI. There was a greater percentage of primiparous cows P/AI in the eCG-treated than Control and FSH-treated cows. There was a greater percentage of eCG-treated multiparous cows pregnant as a result of TAI than Control cows. There was an interaction of treatment × parity (P = 0.005) on P/AI in Experiment 3, in which the eCG effect was more pronounced in primiparous cows. Treatment with FSH, therefore, was not as effective as eCG in stimulation of follicular growth or enhancing percentage of cows pregnant as a result of TAI. Physiological effects of eCG, however, were also more evident in primiparous cows.

Made available in DSpace on 2021-06-25T11:44:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Embrapa This study was conducted in ewes to assess effects of human chorionic gonadotropin (hCG) administration after imposing an estrous induction treatment regimen. Ewes (n = 115) were treated with a 60 mg medroxyprogesterone-intravaginal-sponge for 6 d plus 200 IU of equine chorionic gonadotmpin (eCG) im and 37.5 mu g d-cloprostenol im 36 h before sponge removal (Day 0). After natural mating, ewes having at least one corpus luteum (CL; n = 108) were administered either 1 mL of saline (G-Control; n = 53) or 300 IU of hCG (G-hCG; n = 55) on Day 7.5 after sponge removal (Day 0). Ovarian ultrasonography and blood collection were performed on Days 7.5, 13.5, 17.5, 21.5, and 30.5. Accessory CL (aCL) were observed in 81.5 % (G-hCG) and 0.0 % (G-Control) of ewes (P = 0.0001). Diameter, area, and volume of luteal tissue were greater (P < 0.05) in G-hCG from Day 13.5 to 30.5. Progesterone (P-4) concentrations were greater (P < 0.05) on Days 13.5, 17.5, 21.5 and 30.5 for ewes of the G-hCG group. Pregnancy percentage was similar (P = 0.25) between groups [47.1 % (G-control) compared with 60.0 % (G-hCG)], although total number of lambs produced by estrous synchronized ewes was greater (P = 0.005) in ewes of the G-hCG group (90.9 % compared with 66.0 %). In conclusion, hCG administration 7.5 days after sponge removal from Morada Nova ewes during the non-breeding season is an effective treatment to induce aCL formation, improve luteal tissue biometry and P4 concentrations, and to enhance the total number of lambs born. Sao Paulo State Univ, Sch Agr & Vet Sci, Dept Pathobiol & Vet Theriogenol, Jaboticabal, SP, Brazil Embrapa Caprinos & Ovinos, Rodovia MG 133,Km 42, BR-36155000 Coronel Pacheco, MG, Brazil Univ Fed Vicosa, Dept Vet, Av Peter Henry Rolfs S-N, BR-36570000 Vicosa, MG, Brazil Embrapa Pecuaria Sudeste, Rodovia Washington Luiz,Km 234 S-N, BR-13560970 Sao Carlos, SP, Brazil Univ Fed Fluminense, Fac Vet, Rua Vital Brasil Filho 64, BR-24230340 Niteroi, RJ, Brazil Sao Paulo State Univ, Sch Agr & Vet Sci, Dept Pathobiol & Vet Theriogenol, Jaboticabal, SP, Brazil Embrapa: 22.13.06.026.00.06

Simple Summary Reproductive 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. Abstract 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.

This study was conducted to evaluate the effect of breed types and lactation status on reproductive performance of hormonally-treated Awassi ewes. A total of 282 ewes were treated with intravaginal progestagen sponges and equine chorionic gonadotropin. Ewes were mated with fertile rams following sponge removal. Lambing rate was similar between brown- and black-faced ewes, while the percentage of multiple births, fecundity and prolificacy were greater in black-faced ewes. Lambing rate and the percentage of multiple births were similar between lactating and dry ewes. Fecundity tended to be greater in lactating ewes, while prolificacy was similar regardless of lactation status. Results of the present study indicate that black-faced Awassi ewes respond better to hormonal treatment than brown-faced ewes while the lactation status does not appear to influence the ewes’ response to hormonal treatment.

Approximately 30% of the Association of Zoos and Aquariums cheetah population (~350 total animals) is unlikely to breed naturally due to advanced age, health, or behavioral issues. Aging cheetah females (&ge, 9 y old) are unlikely to become pregnant via natural breeding if they are nulliparous. We previously demonstrated that oocytes recovered from aged females were of similar quality compared with those recovered from younger females (2&ndash, 8 y old). We hypothesize that transfer of 4&ndash, 8 cell embryos produced by in vitro fertilization with oocytes from old donors could result in pregnancy after transfer into younger recipients. Female cheetahs (n = 3 aging donors and n = 3 young recipients) received 300 IU equine Chorionic Gonadotropin (eCG) and 3000 IU Luteinizing Hormone (LH) while fecal metabolites of estrogens and progestogens were closely monitored. At 28 h post-LH injection, oocytes were aspirated laparoscopically from donors and inseminated in vitro with cryopreserved sperm. After 48 h of in vitro culture, resulting embryos (4&ndash, 8 cells) were transferred into the oviducts of recipient females. Pregnancy was confirmed in one recipient via ultrasound 32 days after transfer and by radiograph 62 days after transfer. Two cubs were born naturally after 90 days of gestation, representing the first cheetah births resulting from transfer of embryos produced in vitro.

Made available in DSpace on 2020-12-12T02:43:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 The aim of this research was to evaluate the effects of the temperament of bovine females Bos taurus indicus submitted to a timed artificial insemination (TAI) protocol on the concentrations of cortisol, diameter of the largest follicle, pregnancy by TAI on Days 30 (P/TAI 30) and 60 (P/TAI 60) after TAI. Nellore cows (n = 2702) of 45 ± 7 days post-partum were submitted to the same TAI protocol. On D -10 devices containing 1 g of progesterone were inserted into the vagina and 2 mg of estradiol benzoate were applied intramuscularly. On D -2, the devices were removed and 530 µg of sodium D-cloprostenol, 300 IU of equine chorionic gonadotropin, and 1 mg of Estradiol Cypionate were applied. Forty-eight hours after the final injection, the cows were submitted to TAI (D 0). The temperament of the cows was rated on a numerical scale from 3 to 12 using the composite reactivity score (RS) on D -10. The RS included the evaluation of the movement and breathing while the animal was in the cattle chute and the exit velocity. Cows were classified as Calm (RS ≤ 4; n = 830; Calm group), Reactive (RS = 5 to 8; n = 1313; Reactive group) and Very Reactive (RS ≥ 9; n = 559; Very Reactive group) and 5% of the cows of each group were submitted to blood collection for the subsequent plasmatic cortisol concentration measurement by ELISA. It was observed that Calm group had lower concentration of cortisol than did the Reactive and Very Reactive groups (35.07 ± 1.78, 50.58 ± 2.76, and 54.45 ± 3.39 nmol/L, respectively). The Calm group had diameter of the largest follicle on the day of TAI compared to Reactive and Very Reactive groups (14.4 ± 0.2, 13.6 ± 0.4, and 13.2 ± 0.2 mm, respectively). The Calm and Reactive groups had higher P/TAI compared to the Very Reactive Group on Day 30 (56.4%, 57.0%, 51.0%, respectively) and on Day 60 (50.0%, 47.3%, 42.7%, respectively). In conclusion, temperament influences the reproductive efficiency of bovine females Bos taurus indicus submitted to TAI protocols. University of São Paulo Faculty of Animal Science and Food Engineering, Duque de Caxias Norte Avenue, 255, Campus Fernando Costa, CEP 13.635-900 MF Vetplan, José Ferreira Street, 130, CEP 13.890-000 Boehringer Ingelheim Animal Health, Carlos Grimaldi Avenue, 1701, CEP 13.091-908 Biogénesis-Bagó Animal Health Ltda, Manoel Ribas Avenue, 985, CEP 80.810-000 São Paulo State University (UNESP) FCAT, Comandante João Ribeiro de Barros Road (SP 294), Km 651, CEP 17.900-000 University of São Paulo Faculty of Veterinary Medicine and Animal Science, Duque de Caxias Norte Avenue, 225, Campus Fernando Costa, CEP 13.635-900 University of Florida North Florida Research and Education Center Institute of Food and Agricultural Sciences São Paulo State University (UNESP) FCAT, Comandante João Ribeiro de Barros Road (SP 294), Km 651, CEP 17.900-000

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step.

Several metalloproteinases (MMPs) are present and functional in the chicken ovary and regulate the extracellular matrix (ECM) during follicle development, ovulation, atresia, and regression. The regulation of the abundance of MMPs in avian ovarian follicles, however, is largely unknown. The aim of the present study was to examine effects of equine chorionic gonadotropin (eCG) on abundance of selected MMPs and relevant tissue inhibitors of MMPs (TIMPs) in the hen ovary. The MMP-2 and MMP-9 activity was also determined. Results indicated there were effects of eCG on abundances of MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, TIMP-2, and TIMP-3 mRNA transcript and/or protein relative abundances in white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1) ovarian follicles. The response to eCG depended on the stage of follicle development, layer of follicular wall, and the type of MMPs or TIMPs affected by eCG. Furthermore, there was a pause in egg laying when eCG was administered and there were morphological changes in the ovary following eCG treatment that were associated with alterations in MMP-2 and MMP-9 activity. In general, the results indicate that eCG, which has primarily follicle stimulating hormone (FSH)-like bioactivities, is a negative regulator of MMP abundance and activity in the largest yellow preovulatory follicles. Results from the present study indicate the gonadotropins, especially FSH, by the regulation of transcription, translation, and/or activity of proteins of the MMP system have effects on the mechanisms that underlie ECM remodeling and cell function throughout ovarian follicle development in the chicken ovary.

Reducing the number of sperm needed to produce a litter with artificial insemination (AI) allows greater use of higher genetic merit boars. Induced ovulation with single fixed-time artificial insemination (SFTAI), combined with intrauterine (IUI) or deep uterine insemination (DUI), could improve fertility with low numbers of sperm. The objectives of the study were to determine the fertility effects of sperm numbers and the site of insemination. At weaning (0 h), sows (n = 534) were assigned by parity and estrus induction method (equine chorionic gonadotropin [eCG] or Control) to receive 1,200 × 106 sperm by IUI; 600, 300, or 150 × 106 sperm by IUI or DUI; or 75 × 106 sperm by DUI. At 80 h postweaning, sows received OvuGel and 26 h later a SFTAI using pooled semen. Sows were exposed to boars once daily and ultrasound was performed to determine follicle size and time of ovulation. Following SFTAI, sows were slaughtered 27 d after AI to determine pregnancy and litter traits. Data were analyzed using different models to test for effects of estrus induction, interaction of three levels of sperm (600 to 150) with two levels for site (IUI vs. DUI), and the overall effects of AI method (eight treatments). There was no effect (P > 0.05) of estrus induction on estrus (93%) within 5 d of weaning or on follicle size (6.1 mm) at OvuGel, but wean-to-estrus interval (3.8 vs. 4.0 d) was slightly reduced (P < 0.01) as was AI-to-ovulation interval (15.9 vs. 17.0 h, P = 0.04) for eCG and Control, respectively. There was no effect (P > 0.05) of estrus induction on pregnancy rate (78.6%), number of corpora lutea (CL; 21.7), or number of viable embryos (12.2). There was no effect of number of sperm or site of insemination and no interaction (P > 0.05) on pregnancy rate (range: 80.9% to 70.5%), but AI occurring after ovulation reduced the pregnancy rate (P < 0.02). The total number of embryos (range: 16.5 to 10.3) was not affected by estrus induction, number of sperm, or site of insemination (P > 0.05), but was influenced by AI treatment (P < 0.01). Treatments with a higher number of sperm (1,200 and 600) had more embryos compared with those with a lower number of sperm (300 to 75). The numbers of embryos also increased with the number of CL (P < 0.0001). These results suggest that the lower number of sperm affects litter size more than the pregnancy status. Acceptable fertility can be achieved with low numbers of sperm when using a SFTAI and uterine deposition, but AI-to-ovulation interval and ovulation rate influence final fecundity.

The genus Mus consists of many species with high genetic diversity. However, only one species, Mus musculus (the laboratory mouse), is common in biomedical research. The unavailability of assisted reproductive technologies (ARTs) for other Mus species might be a major reason for their limited use in laboratories. Here, we devised ARTs for Mus spretus (the Algerian mouse), a commonly used wild-derived Mus species. We found that in vitro production of M. spretus embryos was difficult because of low efficacies of superovulation with equine chorionic gonadotropin or anti-inhibin serum (AIS) (5–8 oocytes per female) and a low fertilization rate following in vitro fertilization (IVF; 15.2%). The primary cause of this was the hardening of the zona pellucida but not the sperm’s fertilizing ability, as revealed by reciprocal IVF with laboratory mice. The largest number of embryos (16 per female) were obtained when females were injected with AIS followed by human chorionic gonadotropin and estradiol injections 24 h later, and then by natural mating. These in vivo-derived 2-cell embryos could be vitrified/warmed with a high survival rate (94%) using an ethylene glycol-based solution. Importantly, more than 60% of such embryos developed into healthy offspring following interspecific embryo transfer into (C57BL/6 × C3H) F1 female mice. Thus, we have devised practical ARTs for Mus spretus mice, enabling efficient production of embryos and animals, with safe laboratory preservation of their strains. In addition, we have demonstrated that interspecific embryo transfer is possible in murine rodents.