Improving the meiotic competence of small antral follicle-derived porcine oocytes by using dibutyryl-cAMP and melatonin.

OBJECTIVE: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. METHODS: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. RESULTS: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. CONCLUSION: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

Bone Morphogenetic Protein 15 (BMP-15) Improves In Vitro Mouse Folliculogenesis.

Multilayered secondary follicles were encapsulated in a 0.5% alginate matrix and cultured in a 3D culture system supplemented with bone morphogenetic protein 15 (BMP-15; 15 ng/mL) for 12 days. The in vitro development of ovarian follicles was evaluated. On day 12, the follicle diameter, follicle survival rate, and antrum formation rate were significantly higher for follicles cultured in BMP-15-supplemented medium than those cultured in regular medium. The percentage of ovulated metaphase II oocytes retrieved from follicles cultured in BMP-15-supplemented medium was greater than that of oocytes retrieved from follicles cultured in regular medium. The secretion of P4 was significantly higher on days 6, 8, and 10 in follicles cultured in BMP-15-supplemented medium. The result for E2 tended toward significance on day 12. Intracellular reactive oxygen species levels were higher and glutathione levels were lower in mature oocytes from the in vitro culture than in mature oocytes from an in vivo control. A 3D culture system using an alginate matrix and supplemented with BMP-15 effectively improves the outcomes of in vitro ovarian follicle culture.

Increased luteal tissues after secondary corpus luteum formation leads to enhanced progesterone concentrations and improved fertility in repeat-breeder dairy cows during heat stress condition in tropical climate.

Relatively, little is known about the corpus luteum (CL) function in early pregnancy after the successful treatment of luteal phase deficiency in repeat-breeder dairy cows when exposed to extreme environments under tropical climate. To investigate the influence of increased tissues of corpora lutea (CLs) by inducing secondary CL based on progesterone (P4) concentration and fertility in repeat-breeder dairy cows undergoing the fixed-time artificial insemination (FTAI) protocol, 32 cows were treated with gonadotropin releasing hormone (GnRH) on day 5 post-induction (experiment 1). In experiment 2, 213 cows were bred using the short-term FTAI protocol. On day 5 post-FTAI, cows were divided into two groups: treatment with (GnRH5-treated group) or without (GnRH5-untreated group) GnRH. The temperature-humidity index ranged from 77.3 to 82.8. Cows bearing two CLs had greater P4 concentrations than cows bearing only one CL on their ovaries (P < 0.05). Pregnancy rates were greater in GnRH5-treated group than the GnRH5-untreated group (P < 0.01). Moreover, repeat-breeder cows bearing two CLs had a greater likelihood of pregnancy (odds ratio = 20.86) than cows bearing only one CL on their ovaries (P < 0.01). Under heat stress condition, the results highlighted that increasing luteal tissues by creating secondary CL leads to enhanced peripheral P4 concentrations and improved pregnancy outcomes in repeat-breeder dairy cows.

Ovarian luteal category at the time of exogenous progesterone treatment alters pre-ovulatory follicle size and pregnancy outcome but not initial GnRH treatment in repeat-breeder crossbred dairy heifers submitted to the 7-day fixed-time AI protocol.

Repeat breeding is a substantial problem in heifer and cow breeding leading to greater infertility for female dairy herds. The aim of present study was to investigate the impact of corpus luteum (CL) presence and category and the first gonadotropin-releasing hormone (GnRH) administration concurrent with exogenous progesterone (P4) treatment on the largest follicle (LF) size and pregnancy rate (PR) in repeat-breeder crossbred dairy heifers submitted to the fixed-time artificial insemination (AI) protocol. Heifers (n= 243) were synchronised with (+GnRH) or without (-GnRH) first GnRH in the 7-day P4-GnRH-prostaglandin F2α-based programme. Each GnRH group was divided on presence of CL into two groups (+CL and -CL) in a 2 × 2 factorial arrangement. The PR was similar among -GnRH-CL (20.7%), -GnRH+CL (68.8%), +GnRH-CL (30.4%), and +GnRH+CL (68.3%) groups. However, presence of CL in heifers produced a 43.6% increase in PR compared to PR of heifers without CL (odds ratio = 6.550). Heifers bearing large-sized CL had greater large-sized LF on the day of fixed-time AI and PR. Plasma P4 concentration was positively related with CL diameter (r= 0.845; p < 0.001). The diameter of ovarian LF on the day of fixed-time AI was positively associated with P4 concentrations (r= 0.512; p < 0.001). We highlight that ovarian CL presence and category at the time of exogenous P4 treatment alters pre-ovulatory follicle size and PR but not initial GnRH treatment in repeat-breeder crossbred dairy heifers submitted to service with the 7-day fixed-time AI programme.

Understanding the Ovarian Interrelationship with Low Antral Follicle Counts (AFC) in the In Vivo Bos indicus Cow Model: Unilateral and Bilateral Main AFC as Possible Biomarkers of Ovarian Response to Hormonal Synchronisation.

The antral follicle count (AFC) is a test in which the number of oocyte-containing follicles that are developing in both ovaries are visually counted. The count of these follicles strongly relates to the population of the growing follicle reserve on the ovaries. However, the importance of the main number of antral follicle populations (mAFC) in mono-ovulatory animal species has yet to be completely elucidated. Moreover, the investigation of the ovarian interrelationship with unilateral mAFC (main number of antral follicle populations appearing on only one side of the ovary) and bilateral mAFC (main number of antral follicle populations appearing in equivalent numbers on both sides of the ovary) and how understanding this interrelationship can offer possible indicators of ovarian response to hormonal induction have not yet been investigated in mono-ovulatory Bos indicus beef cows. The aim of this study is to investigate the different ovarian interrelationships of mAFC (unilateral and bilateral mAFC) at the time of exogenous hormonal stimulation on the total number of AFC (left and right ovaries) at the beginning of the hormonal protocol for ovarian stimulation and ovarian response at the completion of exogenous hormonal stimulation as well as their usefulness as possible biomarkers of successful hormonal stimulation in Bos indicus beef cattle. Beef cows (n = 104) with low total numbers of AFC (4.7 ± 2.4 follicles) were stimulated with a gonadotropin-releasing hormone-progesterone-prostaglandin F2α-based protocol. At the beginning of the hormonal protocol, ovarian ultrasound scans were performed to evaluate AFC from both ovaries of cows. Beef cows were divided into two groups, unilateral (n = 74) and bilateral mAFC (n = 30), according to the ovarian interrelationship. At the completion of the hormonal stimulation, ovarian ultrasound scans were performed to evaluate the dominant follicle (DF) and cows with DF > 8.5 mm in diameter emerging on their ovaries were defined as having experienced a response to hormonal stimuli. There was a difference of 19.1% between Bos indicus cows bearing unilateral mAFC that produced an increase in ovarian response (odds ratio = 2.717, p < 0.05) compared to the responsive rate of cows displaying bilateral mAFC (82.4% vs. 63.3%). In unilateral mAFC, cows bearing mAFC ipsilateral to the ovary of dominant follicle (DF) had a higher responsive rate than cows bearing mAFC contralateral to the DF ovary (50.0% vs. 32.4%, p < 0.05). In mAFC ipsilateral to the DF ovary, pregnancy rates were greatest in cows bearing mAFC and DF on the right ovary compared with cows bearing mAFC and DF on the left ovary (25.0% vs. 9.1%, p < 0.05). In primiparous and multiparous cows, unilateral mAFC occurs with a greater (p < 0.05) frequency than bilateral mAFC (69.0% and 72.0% vs. 31.0% and 28.0%, respectively). In unilateral mAFC, primiparous cows bearing mAFC ipsilateral to the DF ovary had a greater responsive rate than primiparous cows bearing mAFC contralateral to the DF ovary (55.0% vs. 20.0%, p < 0.05). In mAFC ipsilateral to the DF ovary, responsive and pregnancy rates were greatest (p < 0.05) in multiparous cows bearing mAFC and DF on the right ovary compared with multiparous cows bearing mAFC and DF on the left ovary (58.1% and 22.6% vs. 25.8% and 3.2%, respectively). Furthermore, there was a positive correlation between the mean diameter of AFC at the time of the exogenous hormonal trigger and the mean diameter of DF at the completion of hormonal synchronisation (p < 0.05). Our findings emphasise that the ovarian interrelationship with unilateral mAFC at the time of the hormonal trigger might be a promising biomarker for predicting success in ovarian response to hormonal stimulation of mono-ovulatory Bos indicus beef cows with low AFCs.

In Vivo Follicular and Uterine Arterial Indices as an Indicator of Successful Hormonal Stimulation for Inactive Ovaries in Repeat-Breeder Crossbred Dairy Cows Using a Short-Term Progesterone-Based Programme.

An investigation of vascularity of ovarian and uterine arteries after hormonal treatment for inactive ovaries using the short-term progesterone-based programme had not yet been explored in repeat-breeder crossbred dairy cows. To investigate the in vivo follicular and uterine arterial indices as an indicator of successful hormonal stimulation for inactive ovaries in repeat-breeder crossbred dairy cattle, 59 cows with inactive ovaries were induced with a 5-day progesterone-based protocol. At the completion of hormonal synchronisation, cows were divided into two groups according to the size of the largest follicle (LF) on their ovary: small (≤10.0 mm) and large (>10.0 mm) LFs. Vascularities of LF and uterine artery (UtA) were evaluated using a colour Doppler tool. Cows that presented with large LF had greater follicular and UtA vascular indices (p < 0.001) and pregnancy rate (p < 0.01) than cows bearing small LF on their ovary. There was a positive correlation (p < 0.001) between follicular size and LF and UtA vascular indices. Our findings highlighted that in vivo LF and UtA vascular indices at the completion of hormonal stimulation might be a promising indicator for predicting success in ovarian response to hormonal stimulation for inactive ovaries of infertile crossbred dairy cows.

Associations among thermal biology, preovulatory follicle diameter, follicular and luteal vascularities, and sex steroid hormone concentrations during preovulatory and postovulatory periods in tropical beef cows.

The objectives were to evaluate effects of tropical seasons on thermal biology, preovulatory follicle (POF) diameter, POF and luteal vascularities, and estradiol (E2) and progesterone (P4) concentrations; and to determine the associations among the values for these variables during preovulatory and postovulatory periods in Thai native cows in tropical climates: cold, hot, and rainy seasons. Development and vascularity of the POF and corpora lutea (CL) were evaluated using color Doppler ultrasonography. The temperature-humidity index (THI) was greater when the preovulatory period occurred during the rainy season when compared with the occurrence during the hot and cold seasons of the year. Furthermore, POF diameter was less when the THI was greater. The THI was greater when the postovulatory period occurred during the rainy season when compared to the occurrence of the postovulatory period during the hot and cold seasons of the year. Furthermore, the CL vascularity and P4 concentration were less when the THI was greater. The THI was inversely correlated with CL vascularity and P4 concentrations. When the THI was greatest during the hot and rainy seasons of the year, there were the greatest negative effects on POF size, POF and CL blood flow, and concentrations of E2 and P4 during the preovulatory and postovulatory periods. While native Bos indicus are capable of adapting to tropical conditions, there are still negative effects, such as impaired POF and CL functions, when the THI induces heat stress.

Uterine artery flow velocity waveform, arterial flow indices, follicular dynamics, and sex hormones during preovulatory period in synchronized ovulatory cycle of Bos indicus beef cows.

A greater understanding of the uterine artery's (UtA) biology is essential to the increase in female reproductive abilities. The UtA flow velocity waveform, blood flow volume (BFV), pulsatility and resistance indices (PI and RI), blood flow velocities, dynamics of the dominant follicle (DF), and estradiol (E2) and progesterone (P4) levels in an induced ovulatory cycle were evaluated in Thai native cattle. Twenty cows were induced with synchronized ovulation through a P4-releasing device, from Day -9 to Day -4, concurrent with the administration of two doses of a gonadotropin-releasing hormone on Day -9 and Day -1, and two doses of prostaglandin F2α on Day -4 and 8 h later. Day 0 was designated as the day of ovulation. The cows underwent Doppler sonographic determination and blood collection from Day -4 to Day 0. The cows were classified in the non-ovulating (n = 5) and ovulating groups (n = 15). The ovulating cows presented higher BFV values, blood flow velocities, DF growth rates, and E2 levels; yet lower PI values and P4 concentrations, than those of the non-ovulating cows. The BFV values and the blood flow velocities were greater, but the RI and PI values were lower in the ovulatory side UtA than in the contraovulatory side UtA. The BFV values were positively correlated with blood flow velocities, DF growth rates and E2 concentrations in the ovulating cows; confirming the importance of UtA blood flow, follicular growth, and E2-vasodilation during preovulatory phase in the induced ovulatory cycle of Bos indicus beef cows.