Search Results (26)

Seasonal reproduction in sheep reduces reproductive efficiency. Melatonin (MT) plays a crucial role in reproductive processes. The purpose of this study was to assess the effects of a 5-day MT implant pretreatment on estrus synchronization and reproductive performance in sheep during seasonal anestrus. A total of 40 multiparous Mongolian sheep were selected and randomly divided into two groups. In the MT group (n = 20), the ewes received an MT implant for 5 days, and then, they were given a progesterone (P4)-containing vaginal sponge for 14 days with equine chorionic gonadotropin (eCG) administered (330 I.U. per ewe; I.M.) at sponge removal. Control (CON) ewes (n = 20) were similarly treated but did not receive MT implants. The results demonstrated that MT implantation significantly improved serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GSH-Px), increased post-ovulatory luteal diameter and serum P4 levels, and reduced ovarian apoptosis. Compared with the CON group, the MT group showed significantly higher pregnancy (68.23% vs. 50.59%) and lambing rates (63.53% vs. 47.06%; number of lambed ewes/number of total ewes) following cervical-timed artificial insemination. Ovarian transcriptome analysis revealed 522 differentially expressed genes (DEGs) in the MT group compared with the CON group, including 355 upregulated and 167 downregulated DEGs. In addition, MT significantly enhanced proliferation and inhibited apoptosis in cultured granulosa cells (GCs) and luteal cells (LCs) in vitro. Moreover, it enhanced the antioxidant capacity of GCs and LCs probably by activating the NRF2 signaling pathway as well as stimulating steroid hormone synthesis. In conclusion, MT implantation 5 days before applying the conventional P4-eCG protocol enhances ovine reproductive outcomes during seasonal anestrus. MT implantation has a beneficial role on the growth and function of ovarian cells. These findings offer novel evidence supporting the functional role of MT in mammalian reproduction, and would be informative for optimizing estrus synchronization in sheep. Full article

This study examines the reproductive outcomes of Lacaune-crossbred ewes and hoggets in intensive production systems, focusing on vaginal prolapse and hydrometra associated with flaws in estrus synchronization (E.S.) protocols. Data from multiple farms were combined for analysis due to the absence of significant variation at the farm level. The findings revealed a strong association between vaginal prolapse, parity, and litter size, with hoggets carrying multiple fetuses facing the highest risk (p < 0.0001). This highlights the need to reconsider equine chorionic gonadotropin (eCG) administration in hoggets, as it increases the likelihood of multiple pregnancies and, consequently, prolapse. Additionally, a progressive rise in hydrometra prevalence was observed with repeated synchronization cycles in ewes, increasing from 0.51% after the third treatment to 12.33% after the fourth (p < 0.0001). Notably, in this study, the four synchronization cycles were applied over a relatively short period (7.22 ± 1.64 months), further supporting concerns that excessive hormonal treatments within a compressed timeframe exacerbate reproductive dysfunction. The results corroborate previous reports that prolonged progesterone exposure can impair uterine function, leading to fluid retention and hydrometra. To mitigate these risks, estrus synchronization protocols should be critically reassessed—especially by extending the interval between successive treatments—to protect reproductive health and animal welfare. These findings not only underscore the need for more welfare-conscious practices in intensive sheep farming but also encourage further research aimed at refining hormonal management strategies in dairy ewe reproduction. Full article

Goats are primarily raised in marginal, arid, and semi-arid production systems and exhibit a seasonal reproductive pattern. Extending their reproductive season would enhance food security and improve the economic viability of goat production. Therefore, the objective of this study was to assess the effects of SeVE supplementation during the anestrus season on ovarian activity in anestrous goats subjected to an estrus synchronization protocol. The response variables included estrus induction (EI), ovulatory rate (OR), corpus luteum diameter (CLD), left and right ovary diameters (LOD and ROD), embryo implantation (EMBRYO), and pregnancy rates (PREG). Adult, anestrous, crossbred dairy goats (n = 32) from northern semi-arid Mexico (i.e., Comarca Lagunera, 26° N) were randomly assigned to two homogeneous groups based on age, live weight (LW), and body condition score (BCS): (1) the supplemented group (SeVE; n = 16), received 5 mg selenium plus 350 IU vitamin E, and (2) the control group (CONT; n = 16), received physiological saline. Estrus was induced using a combination of progesterone (P4, 20 mg), equine chorionic gonadotropin (eCG, 400 IU), and exposure to the male effect. While no differences (p > 0.05) were observed between the groups for LW and BCS, the variables EI, OR, EMBRYO, and PREG rates were higher (p < 0.05) in the SeVE group than the CONT group. No differences (p > 0.05) were observed between the groups for CLD, LOD, or ROD. Thus, SeVE-targeted supplementation improved reproductive outcomes without affecting ovarian structure size (i.e., a non-dimensional ovarian SeVE action), suggesting that this precision supplementation strategy certainly enhances reproductive efficiency. This was possibly related to selenium-induced increases in P4 synthesis, and the antioxidant effects on follicular development correlate with improved endometrial development, conceptus survival, embryo growth, and pregnancy maintenance. Such improvements could contribute to the sustainability of marginal production systems in the semi-arid regions of northern Mexico, supporting food security and increasing economic returns for goat producers and their families. Full article

Equine chorionic gonadotropin (eCG) has been shown to improve reproductive performance in cattle by stimulating terminal follicular growth. This study was designed to assess the effects of 400 IU of eCG on TFG and its consequences for luteal development in indigenous cows undergoing ovulation synchronization. Specifically, we investigated the follicular and luteal dynamics following eCG treatment using structural markers (follicular and luteal diameters and luteal area) and functional markers (P4 profiles and luteal blood perfusion) to evaluate CL development. A total of twelve cows were assigned into two groups: an untreated control group and an eCG-treated group (n = 6 in each). Ultrasonography was used to scan the ovaries and monitor the development of TFG and ovulation from day 0 to ovulation. The diameter in TFG was measured daily, and the terminal follicle (TF) was identified as the preovulatory follicle throughout the study period. Ovulation was defined by the disappearance of the TF. Following ovulation, the progression of CL development in both groups was observed on days 5–6, and the mid-stage CL was identified between days 9 and 12 post-ovulation. The results indicated a faster increase in the terminal follicular diameter (TFD). From day 4 to ovulation, the eCG-treated group showed a significantly greater terminal follicular growth rate (TFGR) compared to that in the untreated control group. This enhanced follicular growth in the eCG-treated group was associated with more robust CL blood perfusion and maturation. The Doppler imaging assessments revealed a significant increase in the blood perfusion within the CL, which corresponded with the accelerated TFG. Pearson’s correlation analysis showed strong positive relationships between follicular growth, luteal function, and CL blood perfusion, underscoring the interconnectivity of these processes. In conclusion, our results highlighted the key role that eCG administration has in TFG and CL development and function in White Lamphun cattle. Full article

Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter CHO-K1 cells. The expression from one, among nine isolates, peaked at 364–470 IU/mL on days 9 and 11. The molecular weight of rec-eCG β/α ranged from 40 to 47 kDa, with two distinct bands. PNGase F treatment reduced the molecular weight by 8–10 kDa, indicating N-glycosylation. Rec-eCG β/α demonstrated dose-responsive cAMP activity in cells expressing eLH/CGR, with enhanced potency in rLH/CGR and rFSHR. Phospho-ERK1/2 activation peaked at 5 min before declining rapidly. β-arrestin 2 recruitment was receptor-mediated in cells expressing hFSHR and hLH/CGR. This study provides insights into the mechanisms underlying eCG’s FSH- and LH-like activities. Stable CHO DG44 cells can produce large quantities of rec-eCG. eCG activates pERK1/2 signaling via the PKA/cAMP pathway and facilitates β-arrestin 2 recruitment. Full article

Introduction: Equine chorionic girdle cells can give rise to the endometrial cup, a structure that secretes equine chorionic gonadotropin (eCG). To date, the mechanisms underlying the proliferation, differentiation, invasion, and hormone secretion of equine chorionic girdle cells are not fully understood. During human pregnancy, interleukin-6 (IL-6) is maternally expressed to stimulate the invasion and migration of human syncytiotrophoblast cells and influence the synthesis of human chorionic gonadotropin. Although many previous studies reported the same upregulation of IL-6 during equine placentation, its effects on equine chorionic girdle cells have never been tested. Methods: In this study, we tested the effects of different concentrations of IL-6 on the morphology, differentiation, proliferation, hormone production, invasion, migration, and gene expression profiles of equine chorionic girdle cells. Results: We found that, with respect to morphology and proliferation, IL-6 had no significant effect; with respect to eCG production, 30 ng/mL IL-6 significantly increased the expression of genes related to eCG production, whereas treatment with 70 ng/mL IL-6 downregulated these genes. However, no significant increase in intracellular protein levels or eCG secretion was observed following treatment with any concentration of IL-6. Regarding cell migration and invasion, we found that IL-6 treatment had no significant effect on the migration capability of equine chorionic girdle cells but did enhance the invasion capability of equine chorionic girdle cells and upregulated the expression of the invasion-related genes, Mmp2 and Mmp9. Finally, our transcriptomic study revealed that in equine chorionic girdle cells, IL-6 treatment mainly affected the expression of genes related to the NOD-like receptor signaling pathway and the JAK–STAT signaling pathways, which are involved in immune and inflammatory responses. Conclusions: In summary, this study demonstrates the positive effects of IL-6 on the cytokine secretion and invasive ability of equine chorionic girdle cells. Full article

To improve the efficiency of prostaglandin F_2α (PG)-based estrus synchronization in sheep, this study assessed the effect of the gonadotropin-releasing hormone (GnRH) included in the long-interval PG treatment regimen for sheep estrus synchronization during the breeding season. In experiment 1, 30 multiparous Mongolian sheep (3–4.5 years old) were randomly divided into three groups. In the progesterone (P4)–equine chorionic gonadotropin (eCG) group (P4-eCG, n = 10), the ewes were synchronized with intravaginal P4 sponges for 14 days, and received an injection of 330 IU of eCG at sponge withdrawal. In the PG group (n = 10), the ewes received two doses of 0.1 mg PG with a 14 day interval. In the PG-GnRH-PG group (n = 10), the ewes were synchronized by two doses of 0.1 mg PG with a 14 day interval like that in the PG group, but received 50 μg of GnRH 7 days after the first injection of PG. It was found that, at the end of treatment, the number of corpus luteum on the ovaries and the concentration of the serum P4 in the PG-GnRH-PG group were significantly higher than that of the PG and P4-eCG groups. In experiment 2, 59 multiparous Mongolian sheep (3–4.5 years old) were assigned to three groups, like in experiment 1 (n = 20, 20 and 19 for the P4-eCG, PG and PG-GnRH-PG groups, respectively). The estrus of ewes in the PG-GnRH-PG group was more synchronous compared to the PG group. After insemination of the estrus ewes, the pregnancy rate was numerically but not significantly higher in the PG-GnRH-PG group than that in the P4-eCG and the PG groups. In a field test, 285 multiparous Hu sheep (3–4.5 years old) were randomly assigned to a P4-eCG group (n = 142) and PG-GnRH-PG group (n = 143). Timed artificial insemination showed no significant differences in the rates of pregnancy and lambing between the PG-GnRH-PG and P4-eCG groups. We conclude that the addition of GnRH in the long-interval PG protocol may improve the efficiency of PG-based estrus synchronization, and would represent a potential alternative to the conventional P4-eCG based protocol during the breeding season in sheep. Full article

The lutropin/chorionic gonadotropin receptor (LH/CGR) belongs to the G protein-coupled receptor family, characterized by conserved leucine residues in their carboxyl-terminal cytoplasmic tails. This study aimed to investigate the functional significance of the equine LH/CGR (eLH/CGR) trileucine motif in signal transduction. Wild-type eLH/CGR (eLH/CGR-wt) and mutant receptors, in which the trileucine motif was altered to alanine (eLH/CGR-ALL, LAL, LLA, and AAA), were analyzed in transfected cells. The expression levels of mutants ranged from 60% to 78%, with eLH/CGR-AAA showing the lowest level. Although the trileucine motif did not individually affect cAMP responsiveness, the combined mutant (eLH/CGR-AAA) significantly reduced cAMP response, surface receptor levels and enhanced receptor internalization rates. Activation of phospho-ERK1/2 was rapid in all mutants, peaking at 5 min, but eLH/CGR-ALL and LAL mutants exhibited a sharp decline in activity at 15 min. Notably, the eLH/CGR-LLA and AAA mutants showed similar phospho-ERK1/2 activity as the wild type. The eLH/CGR-AAA mutant also displayed a two-fold reduction in PKA signal transduction. These findings suggest that while individual leucine residues of the trileucine motif do not affect cAMP responsiveness, the entire motif plays a crucial role in receptor trafficking and signaling, specifically influencing PKA and phospho-ERK1/2 pathways. Full article

The present study evaluates the effect of including microencapsulated hot chili pepper (MHCP) in the diet of crossbred dairy cows on the volume and quality of milk and on ovarian morphofunctionality. Twenty-four crossbred females in their lactating period were used. The cows were divided into two experimental groups, a control (CT) and an MHCP -supplemented group (CP) given 1 g a day per animal of microencapsulated hot chili in concentrate for 42 days. Over seven weeks of daily milk production was measured, and sample milk was collected weekly for composition analysis. Animals were subject to an ovulation synchronization protocol on day 0 (D0), and an intravaginal progesterone (P4) implant, estradiol benzoate, and prostaglandin (PGF2α) were administered. On D8, the P4 implant was removed and PGF2α, equine chorionic gonadotropin, and estradiol cypionate were administered to the animals. The ovarian dynamics were evaluated in B mode and color Doppler. There were significant differences (p < 0.05) in the group X time interaction, the volume of milk produced, and the amount in kg/day of milk components. There was a higher percentage of vascularization in the preovulatory follicle in the CP group (p ≥ 0.10). The findings show that the inclusion of MHCP in the diet of dairy cows does influence their milk production and reproduction. Full article

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115–149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000–5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34–40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39–46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC₅₀ value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system. Full article

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000–7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32–36 kDa, while that of tethered rec-eel LH-M increased to approximately 38–44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC₅₀ value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system. Full article

An intravaginal sponge impregnated with progesterone is commonly used for estrus induction and synchronization in ewes. Although using an intravaginal sponge containing progesterone positively affects the synchronization rate, varying degrees of vaginitis occur during its application. This study aimed to investigate the impacts of various intravaginal treatment options on the vaginitis severity and pregnancy rate in Merino ewes synchronized with intravaginal sponges impregnated with progesterone. During the breeding period, 589 ewes, aged 2–6, received intravaginal sponges for 14 days. The control group (CON) received no treatment, whereas vaginal sponges absorbed with enrofloxacin (ENR), Lactobacillus plantarum (LAC), or Lactobacillus plantarum supernatant (CFS) were applied in the treatment groups. All groups received 500 IU of equine chorionic gonadotropin intramuscularly. The ENR group showed lower scores in vaginal discharge and sponge odor compared with the CON, LAC, and CFS groups. Although estrus responses did not differ between groups, the pregnancy rate tended to be higher in the ENR group. In conclusion, intravaginal ENR application, but not LAC or CFS, reduced vaginitis severity and tended to increase pregnancy rates in ewes synchronized with intravaginal sponges impregnated with progesterone. Full article

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) β-subunit, was attached between the β- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-wt was 32–34 and 34–36 kDa, respectively, and that of LH-M and FSH-M was 40–43 and 42–45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8–10 kDa. The EC₅₀ value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system. Full article

Fixed-timed artificial insemination (FTAI) protocols for beef cattle in South America are primarily based on estradiol esters and intravaginal progesterone-releasing devices (IVPD). The objective of this study was to determine the optimal gonadotropin-releasing hormone (GnRH)-based protocol as an alternative to the use of estrogen-based protocols in grazing Bos taurus suckling beef cows. All cows received an IVPD on the day of protocol initiation and prostaglandin F_2α (PG) plus equine chorionic gonadotropin (eCG) treatments at the time of IVPD removal. In Experiment 1, cows (n = 235) were randomly assigned to one of four treatments: (i) 7-day estradiol = 2 mg of estradiol benzoate (EB) at IVPD insertion on Day 9 and 1 mg of estradiol cypionate (ECP) at IVPD removal on Day 2; (ii) 7-day GnRH = 10 µg of GnRH at IVPD insertion on Day 10, IVPD removal on Day 3 and GnRH at FTAI; (iii) 7 & 7 estradiol = PG at IVPD insertion on Day 16, EB on Day 9 and ECP at IVPD removal on Day 2; (iv) 7 & 7 GnRH = PG at IVPD insertion on Day 17, GnRH on Day 10, IVPD removal on Day 3 and GnRH at FTAI. In Experiment 2, cows (n = 462) were randomly assigned to one of four treatments: (i) 6-day estradiol = EB at IVPD insertion on Day 9, IVPD removal on Day 3 and GnRH at FTAI; (ii) 7-day estradiol; (iii) 7-day GnRH; (iv) 7 & 7 GnRH. In Experiment 1, plasma progesterone concentrations and percentage of cows with a corpus luteum (CL) at IVPD removal, and pregnancy per AI (P/AI) were greater for cows subjected to GnRH-based protocols compared with cows subjected to estrogen-based protocols (p < 0.01). In Experiment 2, cows subjected to the 7 & 7 GnRH protocol had the greatest P/AI (p < 0.01). In summary, GnRH-based FTAI protocols resulted in similar or greater P/AI compared to estrogen-based FTAI protocols in grazing postpartum Bos taurus suckled beef cows. The greatest P/AI was attained with the 7 & 7 GnRH protocol. Full article

This study analyzed the effects of dietary supplementation with by-pass linseed oil (LO; rich in α-linolenic acid) on maternal antioxidant systems at Days 14 and 16 of pregnancy in Sarda ewes. This trial used sixteen dry ewes. Eight ewes (CT group) were fed with a control diet without LO, and eight ewes (LO group) were fed with a diet supplemented with LO (10.8 g of α-linolenic acid/ewe/day). Both diets had similar crude protein and energy levels. The experiment included 10 days of an adaptation period and 31 days of a supplementation period. This supplementation period was divided into Period −2 (from Day −15 to −8), Period −1 (from Day −7 to −1; before synchronized mating period/Day 0), Period +1 (from Day +1 to + 7 after mating), and Period +2 (from Day +8 to +15 after mating). Estrous synchronization was induced in all the ewes using an intravaginal sponge (45 mg fluorgestone acetate) for 14 days and equine chorionic gonadotropin (350 UI/ewe) at the end of the treatment. On Days 14 (CT, N = 4; LO, N = 4) and 16 (CT, N = 4; LO, N = 4) after mating, the ewes were slaughtered. Samples of plasma, uterine, and luteal tissues were collected. Thiols, total antioxidant activity (TEAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were measured. On Day 16, thiol and TEAC in luteal tissues were higher in the LO group when compared with the control one (p < 0.05). Moreover, TEAC was higher for the LO group in uterine tissues on Days 14 and 16 (p < 0.05). SOD activity was higher in the LO group in luteal and uterine tissues on Day 14 and Day 16, respectively (p < 0.001). On Day 16, uterine MDA content was lower for the LO group (p < 0.001). No differences were found between groups at the plasmatic level. However, the by-pass LO supplementation enhanced the analyzed antioxidant parameters in luteal and uterine tissues. In conclusion, these results demonstrate that by-pass LO supplementation exerted a positive effect on antioxidative defenses on maternal structures during the embryo-maternal recognition period in ewes. Thus, this could contribute to improving the maternal environment during the embryo-maternal recognition period in mammals. Full article