Association between anti-Müllerian hormone levels and reproductive parameters in Wagyu cattle raised in Brazil.

The production of in vitro embryos has sped up the dissemination of superior genetic material. However, the variation among the cattle response to oocyte and embryo production is a challenging factor. This variation is even higher in the Wagyu cattle as the breed has a small effective population size. The identification of an effective marker related to reproductive efficiency would allow the selection of more responsive females to reproductive protocols. The objective of this study was to evaluate the blood levels of anti-Müllerian hormone and associate it with oocyte recovery and blastocyst rate of embryos produced in vitro in Wagyu cows, as well as observe the hormone circulating levels in males. Serum samples from 29 females with seven follicular aspirations and four bulls were used. AMH measurements were performed using the bovine AMH ELISA kit. A positive correlation was identified between oocyte production and blastocyst rate (r = 0.84, P = 9 × 10-9), and AMH levels with oocyte (r = 0.49, P = 0.006) and embryo (r = 0.39, P = 0.03) production. The mean levels of AMH were different between animals with low (11.06 ± 3.01) and high (20.75 ± 4.46) oocyte production (P = 0.01). Males showed high serological levels of AMH (3829 ± 2328 pg/ml) compared with other breeds. It is possible to use the serological measurement of AMH as a method to select Wagyu females with greater capacity for oocyte and embryo production. Further studies correlating AMH serological levels with Sertoli cell function in bulls are needed.

RNA-Sequencing based analysis of bovine endometrium during the maternal recognition of pregnancy.

BACKGROUND: Maternal recognition is the crucial step for establishing pregnancy in cattle. This study aims to identify endometrial genes and biological pathways involved in the maternal recognition of pregnancy. Caruncular endometrial tissues were collected from Day 15-17 of gestation (pregnant), non-pregnant (absence of conceptus), and cyclic (non-bred) heifers. RESULTS: Total RNAs were isolated from the caruncular endometrial tissues of pregnant, non-pregnant, and cyclic heifers, and were subjected to high-throughput RNA-sequencing. The genes with at least two-fold change and Benjamini and Hochberg p-value ≤ 0.05 were considered differentially expressed genes and further confirmed with quantitative real-time PCR. A total of 107 genes (pregnant vs cyclic) and 98 genes (pregnant vs non-pregnant) were differentially expressed in the pregnant endometrium. The most highly up-regulated genes in the pregnant endometrium were MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, EIF3M, PRSS22, MS4A8, and TINAGL1. Interferon signaling, immune response, nutrient transporter, synthesis, and secretion of proteins are crucial pathways during the maternal recognition of pregnancy. CONCLUSIONS: The study demonstrated that the presence of conceptus at Day 15-17 of gestation affects the endometrial gene expression related to endometrial remodeling, immune response, nutrients and ion transporters, and relevant signaling pathways in the caruncular region of bovine endometrium during the maternal recognition of pregnancy.

Genome-wide association study for carcass weight in pasture-finished beef cattle in Hawai'i.

Introduction: Genome-wide association studies (GWAS) have identified genetic markers for cattle production and reproduction traits. Several publications have reported Single Nucleotide Polymorphisms (SNPs) for carcass-related traits in cattle, but these studies were rarely conducted in pasture-finished beef cattle. Hawai'i, however, has a diverse climate, and 100% of its beef cattle are pasture-fed. Methods: Blood samples were collected from 400 cattle raised in Hawai'i islands at the commercial harvest facility. Genomic DNA was isolated, and 352 high-quality samples were genotyped using the Neogen GGP Bovine 100 K BeadChip. SNPs that did not meet the quality control standards were removed using PLINK 1.9, and 85 k high-quality SNPs from 351 cattle were used for association mapping with carcass weight using GAPIT (Version 3.0) in R 4.2. Four models were used for the GWAS analysis: General Linear Model (GLM), the Mixed Linear Model (MLM), the Fixed and Random Model Circulating Probability Unification (FarmCPU), the Bayesian-Information and Linkage-Disequilibrium Iteratively Nested Keyway (BLINK). Results and Discussion: Our results indicated that the two multi-locus models, FarmCPU and BLINK, outperformed single-locus models, GLM and MLM, in beef herds in this study. Specifically, five significant SNPs were identified using FarmCPU, while BLINK and GLM each identified the other three. Also, three of these eleven SNPs ("BTA-40510-no-rs", "BovineHD1400006853", and "BovineHD2100020346") were shared by multiple models. The significant SNPs were mapped to genes such as EIF5, RGS20, TCEA1, LYPLA1, and MRPL15, which were previously reported to be associated with carcass-related traits, growth, and feed intake in several tropical cattle breeds. This confirms that the genes identified in this study could be candidate genes for carcass weight in pasture-fed beef cattle and can be selected for further breeding programs to improve the carcass yield and productivity of pasture-finished beef cattle in Hawai'i and beyond.

The Outcome and Economic Viability of Embryo Production Using IVF and SOV Techniques in the Wagyu Breed of Cattle.

The Japanese black cattle breed (Wagyu) has an improved metabolism, which allows them to have a higher marbling score when compared with other cattle breeds. However, this may affect other aspects of the animal's physiology, including hormone secretion and their reproductive success, such as their response to synchronization protocols and embryo production. Therefore, the objectives of this study were to test a superovulation protocol (SOV) developed with low doses of FSH and to evaluate the outcome and economic viability of embryo production using the SOV and in vitro fertilization (IVF) approaches in the Wagyu cattle breed. For that, ten Wagyu cows were submitted to five SOVs over a period of 15 months using a standard protocol: CIDR + 3 mg estradiol benzoate (D0), 35 mg FSH (Folltropin®) a.m. and p.m. (D4), 35 mg Folltropin® a.m. and 20 mg p.m. (D5), 20 mg Folltropin® a.m. and 10 mg p.m. (D6), 10 mg Folltropin® and 0.5 mg cloprostenol, both a.m. and p.m., + CIDR removal (D7), 0.05 mg GnRH + insemination 12 and 24 h after (D8) and embryo collection + 0.5 mg of cloprostenol (D16). Thirty days after each SOV, a follicular aspiration was conducted to produce IVF embryos without any pre-synchronization using standard semen in the same group of animals. The average number of embryos produced was 7.63 ± 5.61 (SOV) and 4.52 ± 2.44 (IVF) (p = 0.303). There was no significant correlation between the number of embryos produced by the different techniques (SOV and IVF), indicating that cows that respond well to SOV did not respond well to IVF and vice versa (r = 0.379, p = 0.529). The total cost of each embryo produced by SOV was R$215.00 and R$410.00 for IVF. Therefore, cows that produce less than five embryos by SOV are not economically viable due their lack of response to FSH, and the use of IVF in those animals may be more effective.

Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis.

Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

Free-ranging farm cats: home range size and predation on a livestock unit in Northwest Georgia.

This study's objective was to determine seasonal and diurnal vs. nocturnal home range size, as well as predation for free-ranging farm cats at a livestock unit in Northwest Georgia. Seven adult cats were tracked with attached GPS units for up to two weeks for one spring and two summer seasons from May 2010 through August 2011. Three and five cats were tracked for up to two weeks during the fall and winter seasons, respectively. Feline scat was collected during this entire period. Cats were fed a commercial cat food daily. There was no seasonal effect (P > 0.05) on overall (95% KDE and 90% KDE) or core home range size (50% KDE). Male cats tended (P = 0.08) to have larger diurnal and nocturnal core home ranges (1.09 ha) compared to female cats (0.64 ha). Reproductively intact cats (n = 2) had larger (P < 0.0001) diurnal and nocturnal home ranges as compared to altered cats. Feline scat processing separated scat into prey parts, and of the 210 feline scats collected during the study, 75.24% contained hair. Of these 158 scat samples, 86 contained non-cat hair and 72 contained only cat hair. Other prey components included fragments of bone in 21.43% of scat and teeth in 12.86% of scat. Teeth were used to identify mammalian prey hunted by these cats, of which the Hispid cotton rat (Sigmodon hispidus) was the primary rodent. Other targeted mammals were Peromyscus sp., Sylvilagus sp. and Microtus sp. Invertebrates and birds were less important as prey, but all mammalian prey identified in this study consisted of native animals. While the free-ranging farm cats in this study did not adjust their home range seasonally, sex and reproductive status did increase diurnal and nocturnal home range size. Ultimately, larger home ranges of free-ranging cats could negatively impact native wildlife.

Endocrine regulation of spermatogonial stem cells in the seminiferous epithelium of adult mice.

A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout male life. The seminiferous epithelium is organized into stages of spermatogenesis based on the complement of germ cell types within a tubular section of the testis. The stages exist in close physical proximity and foster diverse phases of germ cell development despite exposure to a similar endocrine milieu that supports coordinated spermatogenesis. The objective of the current study was to identify the population dynamics of SSCs in vivo. We hypothesized that SSC populations and their niches are specifically distributed across the mature seminiferous epithelium in the mouse testis. To test this hypothesis, we conducted stem cell transplantation of germ cells obtained from stage-specific clusters of seminiferous tubules representing areas of high responsiveness to follicle-stimulating hormone (IX-I), androgen (II-IV), and retinoid (V-VIII) signaling. Similarly, we analyzed the expression of genes linked with SSC activity in these groups of stages. No stage-specific differences in the colonization efficiency or the colony number were detected after SSC transplantation, indicating that SSCs are equally distributed across all stages of the seminiferous tubule. In contrast, SSCs obtained from donor stages IX-IV established larger donor-derived colonies due to increased colony expansion. SSCs originating from different stages have varying degrees of stem cell activity in vivo, a notion consistent with Gdnf, Ret, and Bcl6b expression data. These results support the conclusion of a stage-specific, microenvironment-regulating SSC self-renewal and suggest the presence of a transit-amplifying population of undifferentiated spermatogonia in vivo.

Acute ethanol exposure affects spermatogonial stem cell homeostasis in pre-pubertal mice.

Ethanol is a known modulator of neural stem cell development, but the consequences of ethanol toxicity on the cell fate decisions of spermatogonial stem cells (SSCs) is poorly understood. Using an in vivo treatment and stem cell transplantation approach, we investigated the effects of acute ethanol exposure on formation of the growing adult SSC population in neonatal and pre-pubertal mice. Treatment with a single dose of ethanol disrupted SSC homeostasis in vivo evidenced by a significant reduction (7-fold) of stem cell colonization efficiency in the testes of recipient mice following transplantation. Ethanol treatment also increased the rate of apoptosis in adult differentiating germ cells in situ. Gene expression analysis indicates that ethanol exposure has transient and long-term effects on the expression of GDNF and VEGF family molecules and supports the hypothesis that the niche microenvironment for SSCs is sensitive to ethanol toxicity during pre-pubertaland adult life.

VEGFA family isoforms regulate spermatogonial stem cell homeostasis in vivo.

The objective of the present study was to investigate vascular endothelial growth factor A (VEGFA) isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell-specific distribution of VEGF isoforms, receptors, and coreceptors during testis development postnatal d 1-180 suggest a nonvascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that proangiogenic isoforms of VEGF (VEGFA(164)) stimulate SSC self-renewal, whereas antiangiogenic isoforms of VEGF (VEGFA(165)b) induce differentiation of SSC. To test this hypothesis, we used transplantation to assay the stem cell activity of SSC obtained from neonatal mice treated daily from postnatal d 3-5 with 1) vehicle, 2) VEGFA(164), 3) VEGFA(165)b, 4) IgG control, 5) anti-VEGFA(164), and 6) anti-VEGFA(165)b. SSC transplantation analysis demonstrated that VEGFA(164) supports self-renewal, whereas VEGFA(165)b stimulates differentiation of mouse SSC in vivo. Gene expression analysis of SSC-associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSC. These findings indicate a nonvascular role for VEGF in testis development and suggest that a delicate balance between VEGFA(164) and VEGFA(165)b isoforms orchestrates the cell fate decisions of SSC. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis.

Maintaining the male germline: regulation of spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are a self-renewing population of adult stem cells capable of producing progeny cells for sperm production throughout the life of the male. Regulation of the SSC population includes establishment and maintenance of a niche microenvironment in the seminiferous tubules of the testis. Signaling from somatic cells within the niche determines the fate of SSCs by either supporting self-renewal or initiating differentiation leading to meiotic entry and production of spermatozoa. Despite the importance of these processes, little is known about the biochemical and cellular mechanisms that govern SSC fate and identity. This review discusses research findings regarding systemic, endocrine, and local cues that stimulate somatic niche cells to produce factors that contribute to the homeostasis of SSCs in mammals. In addition to their importance for male fertility, SSCs represent a model for the investigation of adult stem cells because they can be maintained in culture, and the presence, proliferation, or loss of SSCs in a cell population can be determined with the use of a transplantation assay. Defining the mechanisms that regulate the self-renewal and differentiation of SSCs will fundamentally improve the understanding of male fertility and provide information about the regulation of adult stem cells in other tissues.