Effect of Garlic Straw with Silage Corn Stalks on Hu Sheep Rumen Fermentation and Microbial Community In Vitro.

Garlic, an important economic crop, provides nutrient-rich straw. When appropriately balanced with silage corn stalks, it is a high-quality forage resource. However, studies on the impact of garlic straw with silage corn stalks on Hu sheep's digestive metabolism and rumen microbiota are scarce. In this study, different addition ratios of garlic straw and silage corn stalks were utilized for in vitro experiments. We designed six experimental groups (CON, G0, G20, G40, G60, G80, and G100) based on varying ratios of garlic straw to silage corn stalks. Rumen microbiota was analyzed through 16S rRNA sequencing. Nutrient composition analysis indicated that garlic straw's relative feeding value (RFV) closely resembled that of silage corn stalks. After 24 h of fermentation, dry matter digestibility and in vitro gas production significantly increased, reaching peak values at a 60% addition ratio. Furthermore, volatile fatty acids (VFAs) such as acetic, propionic, and butyric acid exhibited elevated contents, with the highest yields observed at 60% inclusion. At the genus level, Prevotella, Rikenellaceae RC9 gut group, and Succiniclasticum were identified as the dominant bacterial groups. The gas production test showed a significant decrease in the G80 group compared to others. Microbial analysis revealed a higher abundance of Prevotella in G80 compared to G20, offering valuable insights for reducing greenhouse gas emissions from ruminant animals. Finally, this study predicted the impact of garlic straw with silage corn stalks' addition on Hu sheep's metabolic pathways and biological functions of the rumen microbiota. This research highlights the potential for effectively utilizing garlic straw as a feed resource for Hu sheep and proposes a rational proportion for combining garlic straw with silage corn stalks.

Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR.

Hu sheep, an indigenous breed in China known for its high fecundity, are being studied to improve their growth and carcass traits. MSTN is a negative regulator of muscle development, and its inactivation results in muscularity. The C-CRISPR system, utilizing multiple neighboring sgRNAs targeting a key exon, has been successfully used to generate genes for complete knockout (KO) monkeys and mice in one step. In this study, the C-CRISPR system was used to generate MSTN-edited Hu sheep; 70 embryos injected with Cas9 mRNA and four sgRNAs targeting exon 3 of sheep MSTN were transferred to 13 recipients. Out of 10 lambs born from five recipients after full-term pregnancies, nine had complete MSTN KO with various mutations. No off-target effects were found. These MSTN-KO Hu sheep showed a double-muscled (DM) phenotype, characterized by a higher body weight at 3 and 4 months old, prominent muscular protrusion, clearly visible intermuscular groves, and muscle hypertrophy. The molecular analysis indicated enhanced AKT and suppressed ERK1/2 signaling in the gluteus muscle of the edited Hu sheep. In conclusion, MSTN complete KO Hu sheep with a DM phenotype were efficiently and specifically generated using C-CRISPR, and the C-CRISPR method is a promising tool for farm animal breeding.

PLAG1 g.8795C>T Mutation Regulates Early Body Weight in Hu Sheep by Weakening miR-139 Binding.

Sheep birth and weaning weights indicate their growth and survival. Thus, identifying molecular genetic markers for early body weight is important in sheep breeding. Pleomorphic adenoma gene 1 (PLAG1) is important for regulating birth weight and body length in mammals; however, its relationship with sheep body weight remains unknown. Here, the 3'-untranslated region (3'-UTR) of the Hu sheep PLAG1 gene was cloned, single nucleotide polymorphisms (SNPs) were screened, genotype-early body weight relationships were analyzed, and the possible molecular mechanism was explored. PLAG1 3'-UTR sequences with five forms of base sequences plus poly(A) tails were detected in Hu sheep and the g.8795C>T mutation was identified. Luciferase reporter assay indicated that the g.8795C>T mutation influenced PLAG1 post-transcriptional activity. miRBase prediction showed that the g.8795C>T mutation was located in the miR-139 seed sequence binding region, and miR-139 overexpression significantly decreased both PLAG1-CC and PLAG1-TT activities. Moreover, the luciferase activity of PLAG1-CC was significantly lower than that of the PLAG1-TT, but miR-139 inhibition substantially increased both PLAG1-CC and PLAG1-TT luciferase activities, suggesting that PLAG1 is the target gene of miR-139. Thus, the g.8795C>T mutation upregulates PLAG1 expression by weakening its binding with miR-139, promoting PLAG1 expression, and increasing Hu sheep birth and weaning weights.

miR-27a-3p targets NR5A2 to regulate CYP19A1 expression and 17-ß estradiol synthesis in ovine granulosa cells.

Although 17-ß estradiol (E2) synthesis is important in regulating female fertility, we know little regarding the molecular mechanism of miRNA-regulated ovine E2 synthesis. Here, our experiments with granulosa cells (GCs) from Hu sheep revealed miR-27a-3p involvement in E2 synthesis and its association with ovine litter size. First, we showed that miR-27a-3p of sheep and other mammals share a high nucleotide identity. Next, gain- and loss-of-function assays indicated that miR-27a-3p inhibits CYP19A1 expression and E2 synthesis in GCs. Moreover, we demonstrated that NR5A2 is a direct target of miR-27a-3p. Ovine miR-27a-3p suppresses E2 synthesis via the NR5A2 and CYP19A1 axes. We also identified four single nucleotide polymorphisms in the ovine miR-27a gene, and g.-13 G>A and g 0.24 T > G were significantly associated with the first and the second parity litter size, respectively (P < 0.05). In summary, our findings reveal that miR-27a-3p is a novel regulator of E2 synthesis and may predict litter size of Hu sheep, providing insight into mechanisms underlying granulosa cell function and female fertility.

Comparison of MicroRNA Profiles in Extracellular Vesicles from Small and Large Goat Follicular Fluid.

Extracellular vesicles (EVs), which exist in the follicular fluid of ruminant ovaries, are considered as cargo carriers for the transfer of biomolecules to recipient cells. However, the functions and changes in EVs in antral follicles remain ambiguous. In the present study, we isolated and characterized EVs from goat follicular fluid by means of differential ultracentrifugation and Western blotting of marker proteins. Bioinformatics tools were used to detect miRNA expression levels in EVs. Different miRNA expression patterns of EVs exist in small to large follicles. Thirteen differentially expressed miRNAs (seven upregulated and six downregulated) were identified and used for analysis. A total of 1948 predicted target genes of 13 miRNAs were mapped to signaling pathways, and three significantly enriched pathways (FoxO, MAPK, and PI3K-AKT signaling pathways) were involved in follicular development, as revealed by KEGG enrichment analysis. Our findings suggest that EVs in follicular fluid play biofunctional roles during follicular development in goats.

Analysis of salivary proteomic biomarkers for the surveillance of changes in high-risk status of early childhood caries.

BACKGROUND: Early childhood caries is an urgent public health concern. The aim of this study was to investigate salivary proteomic biomarkers for the surveillance of changes in the high-risk status of early childhood caries. The process involves the screening of specific salivary peptides that were differentially expressed only under dynamic changes in individual caries status. METHODS: Stimulated whole saliva samples were collected from 28 kindergarten children aged 3-4 years in Beijing at baseline and 3 months and 6 months after baseline. A total of 68 samples were collected. In terms of their caries status and progress during the observation period, participants were divided into 3 groups; 7 in the non-caries recurrence group, 6 in the caries recurrence group, and 15 in the healthy control group. Salivary peptides that exhibited no significant differences in cross-sectional comparisons between different groups of caries status but only expressed differentially along with dynamic changes of individual caries were screened using the technique of magnetic beads combined with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The technique of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was employed to identify the proteins from which these peptides were derived. RESULTS: We found two salivary peptides differentially expressed only under dynamic changes in individual caries status in the above comparisons; mass-to-charge ratio (m/z) values of the two peptides were 1045.9 and 2517.6, respectively (P < 0.05). Principal component analysis (PCA) and the decision tree model based on these two peptides showed an acceptable distinguishing ability for changes in the high-risk status of early childhood caries. The source proteins of the two peptides with m/z values of 1045.9 and 2517.6 were identified as submandibular gland androgen regulatory protein 3B (SMR-3B) and mucin-7, respectively. CONCLUSIONS: Two proteins in children's saliva, namely SMR-3B and mucin-7, have the potentiality to serve as candidate biomarkers for dynamic surveillance of changes in high-risk status of early childhood caries.

The involvement of translationally controlled tumor protein during lamb rumen epithelium development.

Early weaning is usually applied to improve the reproductive efficiency of sheep in mutton production, while the development of rumen is of vital importance for sheep weaning age. Translationally controlled tumor protein (TCTP) is a highly conserved protein which participates in multiple tissue and organ development. Thus, we hypothesized that TCTP was involved in sheep rumen development. Histological analyses of sheep rumen epithelium showed that the epithelium formed tough shaped papillae without growing from birth to day 15 of age, after which it rapidly developed to functional epithelia on day 45 of age. We then found TCTP expressed in stratum basale, stratum spinosum and stratum granulosum of rumen epithelium. TCTP protein expression remained at a relative low level from day 0 to day 15 of age, it then significantly increased on day 30 (p < 0.05) and gradually decreased until day 60. Furthermore, to explore the role of TCTP in sheep rumen and its regulation, we found the ratio of Ki67 positive cell in stratum basale cells followed the similar pattern as the expression of TCTP. We also found the ratio of acetate:propionate in rumen fluid decreased from day 30 to day 60 of age (p < 0.05). To conclude, our data indicated that TCTP participated in rumen papillae growth by promoting rumen stratum basale cell proliferation.

Three Complete Linkage SNPs of GDF9 Gene Affect the Litter Size Probably Mediated by OCT1 in Hu Sheep.

Growth differentiation factor (GDF) 9 gene is involved in regulating reproductive traits in animals, but little is known about the promoter, single-nucleotide polymorphisms (SNPs), transcription factor binding sites, and regulating mechanism of GDF9 gene. In this study, the SNPs in the GDF9 promoter region were explored and their transcription mechanisms in regulating GDF9 expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. GDF9 promoter region was amplified by PCRs, and identified SNPs genotyped by sequencing. SPSS16.0 software was used to analyze the association between genotypes and litter sizes. Flow cytometry assay was used to detect cell apoptosis, and dual-luciferase reporter assay was used to discover the promoter activity. A length of 1789 bp promoter region of GDF9 in Hu sheep was obtained by PCR amplification, and luciferase activity assay showed that there was a negative regulatory element in the region within -725 to -309 bp and a positive regulatory element in the region within -309 to +43 bp. Three complete linkage SNPs at -534A/G, -407T/G, and -332C/T were detected, resulting in three genotypes (namely, AA, AB, and BB). The association analysis indicated that the AA genotype ewes had larger litter size at average parity than those with the BB genotype. The -534A/G mutation created a novel binding site for the octamer transcription factor 1 (OCT1), and the Annexin V FITC/PI flow cytometry assay showed OCT1 promoted cell apoptosis in sheep ovarian granulosa cells. Overexpression of OCT1 considerably inhibited the luciferase activity of both genotypes and the inhibition effect of pGL3-BB was higher than that of pGL3-AA. Three complete linkage SNPs of the GDF9 gene regulate the litter size in Hu sheep probably via inhibition of the promoter activity by binding with OCT1 at -534 GG genotype and forming a complex between OCT1 and CCAAT/enhancer-binding protein (C/EBP).

A T > G Mutation in the NR5A2 Gene Is Associated With Litter Size in Hu Sheep Through Upregulation of Promoter Activity by Transcription Factor MTF-1.

Nuclear receptor subfamily 5 group A member 2 (NR5A2), also referred to as LRH-1 or FTF, is an orphan nuclear hormone receptor that is involved in regulating embryonic development, ovarian granulosa cell differentiation, gonadal sex differentiation, and steroidogenesis in mammals. However, little is known about how NR5A2 regulates reproduction in sheep. In this study, we amplified the promoter sequence of NR5A2 and determined that its core promoter region ranged from -721 nt to -281 nt. A T > G polymorphism at -700 nt was detected in the core promoter region. Association analysis found that the litter sizes of Hu ewes at their second and average parities with genotype GG (2.20 ± 0.20 and 1.97 ± 0.06, respectively) were significantly higher than those of ewes with genotype TG (1.68 ± 0.10 and 1.74 ± 0.05, respectively) (p < 0.05) and TT (1.67 ± 0.10 and 1.62 ± 0.06, respectively) (p < 0.05). The litter size of Hu ewes at their third parity with genotype GG (2.10 ± 0.10) was significantly higher than that of ewes with genotype TT (1.56 ± 0.12) (p < 0.05). A luciferase assay showed that the -700G allele increased the luciferase activity relative to the -700T allele. Furthermore, the -700T > G polymorphism created a novel binding site for metal-regulatory transcription factor 1 (MTF-1). A competitive electrophoretic mobility shift assay confirmed that MTF-1 specifically bound with the G-type promoter of NR5A2. An overexpression experiment demonstrated that MTF-1 was involved in the alteration of NR5A2 transcription activity and further increased NR5A2 gene mRNA expression. Our findings revealed that the -700T > G polymorphism promoted NR5A2 expression due to the positive effects on NR5A2 gene transcription activity by MTF-1 and thereby increased fecundity in Hu sheep.

Mutation -388 C>G of NR5A1 gene affects litter size and promoter activity in sheep.

The Nuclear receptor superfamily 5, group A, member 1 (NR5A1) gene encodes a nuclear receptor that regulates the transcription of genes involved in steroidogenesis, follicular development and female fertility. Little, however, is known about the relationship of this gene with reproductive performance in sheep. In this study, the transcription initiation site of Hu sheep NR5A1 gene was located 193 nucleotides (i.e., at -193 nt) before the translational start site (ATG). The core promoter region of the NR5A1 gene ranged from -696 nt to -298 nt, and a C>G mutation at -388 nt was detected in this region. Association analysis indicated ewes with the GG genotype had greater litter size at the second and third parity than those with the CC genotype (P < 0.05). The results from the luciferase assay provided evidence that the -388 G allele increased luciferase activity compared with that of the -388 C allele. Furthermore, the -388 C>G mutation lost a CpG site and gained a novel binding site for the transcription factor, SP1, and results from an overexpression experiment and methylation analysis indicated transcription factor SP1 and methylation of the -388 C>G mutation were both involved in alteration of NR5A1 transcription activity. Results of the present study revealed that the -388 C>G mutation lost a CpG site and promoted NR5A1 gene expression, which completely superimposed positive effects on NR5A1 gene transcription activity by transcription factor SP1, resulting in a fecundity increase in Hu sheep.

Different susceptibility to porcine reproductive and respiratory syndrome virus infection among Chinese native pig breeds.

China is rich in native pig breeds, yet information regarding the susceptibility/resistance of local breeds to porcine reproductive and respiratory syndrome virus (PRRSV) infection is lacking. In the present study, an in vitro method based on assessing PRRSV replication in porcine alveolar macrophages (PAMs) was established to evaluate PRRSV susceptibility/resistance in a commercial pig breed (Landrace) and five native pig breeds from Jiangsu and Anhui provinces in China. Expression levels of cytokines (IL-8, IL-10, TNF-α and IFN-γ), Toll-like receptor 3 (TLR3), CD163 (PRRSV receptor), and sialoadhesin (Sn, PRRSV receptor) in infected pigs were determined using real-time PCR, and the association between PRRSV susceptibility/resistance and the abundance of the cytokines and receptors was investigated. The viral replication rate and titer at 0, 6, 12 18, 24 and 36 hours postinfection (hpi) were determined to assess the proliferation dynamics of PRRSV NJGC in PAMs. Based on the PRRSV proliferation dynamics, the results indicated that Dingyuan pigs were the most susceptible to PRRSV infection, whereas Jiangquhai pigs were the least susceptible to PRRSV infection among the six pig breeds tested, as indicated by measuring PRRSV replication and the viral load in PAMs. The different levels of susceptibility to PRRSV infection in PAMs may be associated with differences in the abundance of CD163 (PRRSV receptor), cytokines IL-8, IFN-γ, and TNF-α in Jiangquhai and Dingyuan pig breeds after viral inoculation.

MC1568 Enhances Histone Acetylation During Oocyte Meiosis and Improves Development of Somatic Cell Nuclear Transfer Embryos in Pig.

An increasing number of studies have revealed that histone deacetylase (HDAC) mediated histone deacetylation is important for mammalian oocyte development. However, nonselective HDAC inhibitors (HDACi) were applied in most studies; the precise functions of specific HDAC classes during meiosis are poorly defined. In this study, the class IIa-specific HDACi MC1568 was used to reveal a crucial role of class IIa HDACs in the regulation of histone deacetylation during porcine oocyte meiosis. Besides, the functions of HDACs and histone acetyltransferases in regulating the balance of histone acetylation/deacetylation were also confirmed during oocyte maturation. After the validation of nontoxicity of MC1568 in maturation rate, spindle morphology, and chromosome alignment, effects of MC1568 on developmental competence of porcine somatic cell nuclear transfer (SCNT) embryos were evaluated, and data indicated that treatment with 10 µM MC1568 for 12 hours following electrical activation significantly enhanced the blastocyst rate and cell numbers. Moreover, results showed that optimal MC1568 treatment increased the H4K12 acetylation level in SCNT one cells and two cells. In addition, MC1568 treatment stimulated expression of the development-related genes OCT4, CDX2, SOX2, and NANOG in SCNT blastocysts. Collectively, our investigation uncovered a critical role of class IIa HDACs in the regulation of histone deacetylation during oocyte meiosis. Furthermore, for the first time, we showed that MC1568 can improve the in vitro development of porcine SCNT embryos. These findings provide an alternative HDACi for improving animal cloning efficiency and may shed more light on nuclear reprogramming.

Thyroid hormones alter estrous cyclicity and antioxidative status in the ovaries of rats.

To expand our understanding of the roles of thyroid hormones on female reproduction, we induced hypo- and hyper-T rat models to investigate the roles of thyroid hormones on estrous cyclicity, as well as the antioxidative status in the ovaries of rats. In the current study, our data show that hypothyroidism (hypo-T) and hyperthyroidism (hyper-T) led to significantly reduced body weights and ovarain weights and delayed vaginal opening day. For hyper-T, thyroxine (T4), tri-iodothyronine (T3), progesterone (P4) and follicle-stimulating hormone (FSH) were significantly increased, while estradiol (E2) and luteinizing hormone (LH) were significantly decreased. For hypo-T rats, serum levels of total T4 and T3, E2, P4, FSH and LH were significantly increased, while concentrations of E2 and LH were significantly decreased. For ovary morphology, the numbers of secondary and antral follicles were significantly decreased with more atretic antral follicles and less corpora lutea in both hyper- and hypo-T groups. Both hyper-T and hypo-T treatment significantly decreased the expressions of thyroid hormone receptor α1 in the ovary. Hypo-T significantly reduced nitric oxide (NO), total NO synthase (tNOS), inducible NOS and constitutive NOS activities, but hyper-T increased them. For antioxidative parameters, hypo-T and hyper-T treatment significantly increased malondialdehyde (MDA) contents. The activities of both glutathione peroxidase (GSH-Px) and catalase (CAT) significantly decreased in the hypo-T group but increased in the hyper-T group. Total superoxide dismutase (T-SOD) activity was significantly increased in the hyper-T group. In summary, thyroid hormones alter estrous cyclicity and antioxidative status in the ovary of the rat may act through the NOS signaling pathway.

Expression and Purification of a Functional Porcine Soluble Triggering Receptor Expressed on Myeloid Cells 1.

Triggering receptor expressed on myeloid cells 1 (TREM-1) plays a vital role in the pathogen-triggered amplification loop required for proinflammatory responses. Blockade of TREM-1 signaling may inhibit expansion of sepsis and prolong survival of animals. In the present study, the gene of porcine soluble TREM-1 was cloned and expressed in E. coli. After purification, the bioactivity of recombinant porcine soluble TREM-1 was tested in vitro on porcine alveolar macrophages. The results showed that supplementation with the recombinant porcine sTREM-1 protein rapidly and dose-dependently attenuated the upregulation of cytokines (IL-1ß, IL-2, IL-4, IL-8, IL-10, IL-12, IL-16, IL-18, and TNF-α) caused by LPS stimulation in the cultured porcine alveolar macrophages. These results indicate that the recombinant porcine sTREM-1 protein can prevent TREM-1-mediated hyperinflammatory responses after exposure to LPS.

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat ß-lactoglobulin (ßLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

[Single nuclear polymorphisms in exon 3 of POMC gene and the association with growth traits in Hu sheep and East Friesian x Hu crossbred sheep].

Pro-opiomelancortin (POMC) plays important roles in the regulation of food intake and energy expenditure. The sheep exon 3 of gene POMC was amplified and sequenced by screening the DNA pools to select single nuclear polymorphisms and analyze the association with the growth traits. Two silent SNP mutations (g.273 T/C and g.456 G/A) in Hu sheep were identified. PCR-restriction fragment length polymorphism (RFLP) was used to test the g.273 T/C and the association between the g.273 T/C polymorphism and some growth traits was analyzed in Hu sheep (n = 162) and East Friesian x Hu crossbred sheep (n=130). The results showed that three genotypes, TT, TC and CC, were detected in Hu sheep with the frequencies of 0.469, 0.438 and 0.093, respectively. Two genotypes, TT and TC, were detected in East Friesian x Hu crossbred sheep with the frequencies of 0.754 and 0.246, respectively. The association analysis showed that in Hu sheep the two-month weaning weight, four-month rump height of genotype CC and the four-month body length, cannon circumference of genotype TC were significantly higher than those of genotype TT (P < 0.05); the four- and six-month weight of genotype CC were significantly higher than those of genotypes TT and TC (P < 0.01); the four-month body height and body length of genotype CC were significantly higher than those of genotypes TT (P < 0.01) and TC (P < 0.05); the four-month cannon circumference of CC genotype was significantly higher than that of TT genotype (P < 0.01). In East Friesian x Hu crossbred sheep the two-month weaning weight, four-month weight, body height, body length, chest depth and cannon circumference of genotype TC were significantly higher than those of genotype TT (P < 0.05); the six-month weight of genotype TC was significantly higher than that of genotype CC (P < 0.01). In conclusion, the exon 3 of gene POMC was associated with growth traits, and C allele was beneficial to the increase of body weight and body size traits of sheep, which potentially afford a good foundation for further study on POMC gene as aided breeding markers for growth traits in sheep.

Cellular Localization of Inhibin alpha-subunit, PKB/Akt and FoxO3a proteins in the ovaries of minipigs.

Experiments were conducted to examine the cellular localization of inhibin alpha-subunit, protein kinase B (PKB/Akt), and FoxO3a proteins in the ovaries of minipigs, Chinese Xiang pigs, by immunohistochemistry. The results indicated that inhibin alpha-subunits were localized in the granulosa cells of follicles at all stages but were not localized in corpora lutea. PKB was localized in the granulosa cells of primordial follicles and in the basal layers of the granulosa cells of preantral and antral follicles, but were not localized in atretic follicles and corpora lutea. FoxO3a was localized in the granulosa cells of follicles at all stages and was extensively localized in the cytoplasma of the luteinized granulosa cells of corpora lutea. Together, the stage- and cell-specific expression patterns of inhibin alpha-subunit, FoxO3a, and PKB suggest that these proteins might play potential roles in follicular development, atresia, and luteinization in the minipig.