Luteal Research Articles

Mechanisms and effects of AdipoRon, an adiponectin receptor agonist, on ovarian granulosa cells-a systematic review.

Granulosa cells, crucial components of ovarian follicles, play a fundamental role in follicle development, hormone production, and overall reproductive health. These cells are integral to steroidogenesis, including the synthesis and secretion of key hormones such as estrogen and progesterone. Dysregulation of granulosa cells can lead to reproductive disorders, including polycystic ovary syndrome and infertility. This systematic review provides a comprehensive evaluation of AdipoRon, a synthetic agonist of adiponectin receptors AdipoR1 and AdipoR2, and its effects on ovarian function, with a particular focus on granulosa cells. Due to the absence of clinical trials, the review centers on preclinical studies to explore AdipoRon's potential therapeutic benefits and to suggest future research directions. A detailed literature search across databases such as PubMed, Scopus, Web of Science, Embase, and Google Scholar was conducted using terms related to AdipoRon and ovarian function. The review encompasses four preclinical studies involving various models: primary granulosa cells from rats, laying hens' granulosa cells, human luteinized granulosa cells, and chicken ovary follicles. Findings indicate that AdipoRon enhances glucose absorption in rat granulosa cells by stimulating glucose transporter 1 expression, modulates steroid hormone secretion in laying hens' granulosa cells, and affects cell proliferation and steroidogenesis in human luteinized granulosa cells. Additionally, AdipoRon, in conjunction with recombinant chicken adiponectin, influences ovarian follicular cell proliferation and steroidogenesis in chicken ovary follicles. This review highlights the need for further investigation into AdipoRon's long-term effects and its potential applications in reproductive health and therapy.

Molecular phylogenetics uncovers two new species in the genus Phyllobates (Anura, Dendrobatidae): the terrible frog gets two new sisters.

True poison-dart frogs (Phyllobates, Dendrobatidae) evolved the ability to secrete batrachotoxins, the most powerful alkaloids known to date. The genus comprises five species whose systematics, at first glance, appeared clear. The most derived clade would include two Colombian species (P.terribilis and P.bicolor) with the highest toxicity, the largest body size, and predominantly yellow body colouration. The other three species (P.aurotaenia, P.vittatus, and P.lugubris) are less toxic on average, have smaller size, and are predominantly black with bright dorsolateral stripes. Recent research has revealed the existence of two major lineages among the three Colombian species. The northern lineage appears to result from a complex evolutionary history, including perhaps introgression among yellow and black taxa. The southern lineage instead revealed the existence of new clades closely related to P.terribilis, black and yellow, that arguably deserve their recognition as new species. Here, available evidence is combined to support the erection of southern populations of P.aurotaenia as a new highly toxic species, sister to P.terribilis, and much closer to it than to any other yellow or black-bodied species, Phyllobatessamperi sp. nov. Their common ancestor is sister to an additional yellow species, which we also describe here as Phyllobatesbezosi sp. nov. Both new species can be externally diagnosed using colouration. Our previous and current analyses also suggest the existence of additional taxa and corroborate multiple transitions in colouration across these hypertoxic taxa.

Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.

The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here, we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cellstreated with a pro-fibrotic or a pro-inflammatory stimulus. Whereas Etanercept did not block Tnf expression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1 expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5months and a downregulation of inflammatory genes at 12months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

PSIII-10 Flaxseed oil-based supplement alters water consumption and plasma fatty acid profile of beef heifers

Abstract The objective of this study was to determine the effects of a flaxseed oil-based supplement (FlaxLic, New Generations, SD) on reproductive parameters, feeding behavior, and lipid profile in beef heifers. Crossbred Angus heifers [n = 60 body weight (BW) = 397kg ± 5.2 kg) were blocked by BW and antral follicle count and randomly assigned to two supplementation treatments: free-choice commercial lose mineral supplement (CON; n = 30) or free-choice flaxseed oil-based supplement (FLAX; n = 30) for 7 wk. Heifers were individually fed via the Insentec feeding system (Insentec RIC, Hokofarm, Marknesse, Netherlands), where basal diet (TMR) and supplements were provided in separate feeders to measure supplement intake and feeding behavior. Further, water intake and behavior were measured via Insentec water system. Individual intake was compiled over 24 h for each animal. Body weight, transrectal ultrasonography, and blood samples were collected weekly. On wk 5, heifers were enrolled in a fixed-time artificial insemination (FTAI) commercial protocol. Largest follicle diameter (LF) and corpus luteum (CL) presence were recorded weekly and on d of the FTAI protocol (d 0, d 7, and d 0). Pregnancy diagnosis was performed 30 d and 60 d following AI. Blood samples were analyzed for progesterone (P4) by radioimmunoassay (RIA) to determine puberty attainment before wk 5, and fatty acid profile was determined via gas chromatography-flame ionization detection. Heifers with a CL or P4 ≤ 1.0 ng/mL were considered pubertal. Data were analyzed using SAS 9.4. ADG was similar between groups (P = 0.90) but was affected by week (P < 0.01). TMR intake was similar in duration and quantity (P > 0.18) between groups. Supplement intake was greater (P < 0.01) for FLAX (0.73 ± 0.03 kg/d) compared with CON (0.18 ± 0.02 kg/d) and from wk 2 to wk 8 (P = 0.01). Time spent consuming the supplements was greater for FLAX compared with CON heifers (62.65 ± 2.11 vs 0.10 ± 0.11 min/d, respectively) over the entire trial (P < 0.01). Further, FLAX heifers consumed less water than CON heifers (32.2 ± 0.39 vs 33.93 ± 0.35 kg/d, respectively; P = 0.03) and from wk 2 to wk 7 (P = 0.03). LF was not different between groups on any of the days of the protocol (d 0, d 7, d 0; P > 0.20). Additionally, P4 (P ≤ 0.20), puberty attainment by wk 5 (P = 0.24), and pregnancy rates (P > 0.48) were similar between groups. Concentrations of arachidonic acid (3.00 ± 0.24 vs 2.32 ± 0.19 mg/mL; P = 0.04), linoleic (60.11 ± 4.34 vs 40.35 ± 4.04 mg/mL; P = 0.002), αLinolenic (16.73 ± 0.26 vs 15.86 ± 0.23 mg/mL; P = 0.02), and total polyunsaturated fatty acids (PUFA; 130.52 ± 5.97 vs 111.19 ± 5.56 mg/mL; P = 0.02) were greater on wk 8 for FLAX compared with CON heifers. Collectively, these findings did not show evidence that the flaxseed oil-based supplement affects variables that impact reproductive performance. However, it altered the plasma lipid profile, increasing omega-3, arachidonic, and linoleic fatty acids, as well as total PUFA.

100 Impact of luteal blood perfusion and estrus expression on pregnancy rates of beef cows exposed to embryo transfer

Abstract This study aimed to evaluate the impact of luteal blood perfusion and estrus expression on pregnancy rates of beef cows exposed to fixed-timed embryo transfer (FTET). Postpartum beef cows (n = 763) from 22 locations were exposed to a 7-d Select Synch + controlled internal drug releasing (CIDR) and breeding indicator patches were affixed upon CIDR removal. Estrus expression was assessed twice daily for 72 h and a 100-µg injection of gonadotropin-releasing hormone was administered to cows that failed to express estrus 72 h after CIDR removal. Brightness mode ultrasonography of the corpus luteum (CL) was conducted to select recipients for FTET 9 d after CIDR removal. Suitable recipients were assigned to receive a single fresh or frozen embryo derived from either in vitro (IVF) or in vivo (IVD) method. After embryo recipient selection, CL morphometries and blood perfusion (BP) were then assessed at the time of embryo transfer via transrectal ultrasonography. Embryo recipients were categorized according to CL area [small (< 3 cm²), medium (3 to 4 cm²), or large (> 4 cm²)] and based on the percentage of luteal area with blood perfusion signals [low (< 25%), medium (25 to 45%), or high (> 45%)]. Pregnancy diagnoses were performed between d 60 and 90 of gestation. Cows that failed to express estrus prior FTET had decreased (P ≤ 0.02) luteal area, luteal diameter, and pregnancy rates compared with cows that expressed estrus. Luteal area category had no effect (P = 0.61) on pregnancy rates. Embryo recipients that expressed estrus had similar (P = 0.12) luteal blood perfusion compared with recipients that failed to express estrus. Embryo recipients with high (P = 0.02) and medium (P = 0.05) BP had greater pregnancy rates compared with embryo recipients with low BP. There was no interaction between estrus expression and luteal BP (P = 0.84). In summary, estrus expression and luteal BP positively impacted pregnancy rates to embryo transfer and may be used to facilitate embryo recipient selection.

387 Impacts of rumen protected omega-6 fatty acid supplementation on cow reproduction and calf growth

Abstract Maternal nutrition during pregnancy is crucial for proper fetal development. Rumen protected omega-6 (ω-6) fatty acids (FA) may improve health and reproductive success in beef cattle. Previous research studied the effects of supplementing ω-6 on cow production either during late gestation or post-breeding but not during both stages. Our objective was to determine the effects of ω-6 supplementation during late gestation and post-breeding on cow reproduction and calf pre-weaning growth. Eighty cows were assigned to either a saturated FA control (C) or rumen protected ω-6 supplements for 40 d before parturition (PRE) and/or 35 d after artificial insemination (AI, POST) using a 2 × 2 factorial: PRE C-POST C(n = 19); PRE C-POST ω-6(n = 19); PRE ω-6-POST ω-6(n = 17); PRE ω-6-POST C(n = 18). Cows were fed ad libitum hay and water when on/off supplementation periods. Supplements were provided in low moisture tubs. Individual animal intake of supplement was measured by a GrowSafe system. Cow body weights and plasma FA concentrations were measured prior to and after supplementation during each period. Calves were weighed at birth, branding, 150 d old and weaning. Reproductive measurements included postpartum estrus activity, estrus response at fixed-time AI (FTAI), and pregnancy rates. Blood plasma for progesterone analysis was collected on d 7 post-AI on all cows. Corpus luteum (CL) area was measured on d 7 via transrectal ultrasonography on a subset of cows during the POST period (PRE ω-6-POST ω-6 n = 8; PRE C-POST C n = 8; PRE C-POST ω-6 n = 8; PRE ω-6-POST C n = 8). The relationship between supplement intake and plasma FA concentration was analyzed via linear regression, and all other variables were analyzed via ANOVA. Supplement intakes within treatments and between the PRE and POST supplemental periods were highly variable. Body weights remained similar for cows (P = 0.52), but POST ω-6 calves had larger weaning weights than POST C calves (P = 0.02). There was a positive linear relationship (r2 = 0.14, P < 0.03) between supplement intake and plasma FA for PRE ω-6 and POST ω-6. Positive linear relationships between intake and plasma ω-6 FA concentration were observed for PRE C (r2 = 0.11, P < 0.05), PRE ω-6 (r2 = 0.2, P < 0.007) and POST ω-6 (r2 = 0.31, P = 0.0005). Post-partum estrus activity was similar (P = 0.81) between PRE C and PRE ω-6 cows, and estrus response at FTAI was similar (P = 0.13) between POST C and POST ω-6 cows. POST ω-6 cows had larger CL areas than POST C cows (P = 0.03); however, blood progesterone concentrations at d 7 did not differ between treatments (P = 0.65). There was no difference in conception to FTAI between POST C and POST ω-6 (P = 0.08). These results indicate that free choice ω-6 FA supplementation during late gestation and post-AI result in large variation in intake between cows that influence plasma concentrations, but did not markedly alter reproductive measures in mature beef cows.

386 Pregnancy supportive metabolites are enhanced in Brahman cows on day 15 of the estrous cycle

Abstract Bos indicus-influenced cattle are more resilient to pregnancy loss while receiving a restricted nutrient diet compared with Bos taurus cattle, but the mechanism behind this resilience is still unknown. To undercover this mechanism, the present study was designed with Angus (Bos taurus), Brangus (Bos indicus-influenced), and Brahman (Bos indicus) cows randomly assigned (d -30) to either a 100% maintenance diet (n = 22), or a 70% maintenance diet (n = 23) for 45 d. All cows were submitted to a 5-d CO-Synch + CIDR synchronization protocol on d -7 with ovulation induction representing d 0. A cervically navigated foley catheter was placed in the uterine horn ipsilateral to the corpus luteum on d 15 of the estrous cycle. Approximately 50 mL of phosphate buffered saline was infused into the uterus then recovered for uterine flush analysis. Recovered uterine fluid was subjected to metabolomic analysis by ultra-high performance liquid chromatography - high resolution mass spectrometry. A significant breed by metabolite interaction occurred (P < 0.001). A treatment by metabolite interaction was also observed (P = 0.015) in which maintenance and restricted cows clustered within their respective treatment when plotted in an orthogonal PCA graph. Of the 101 metabolites analyzed, 7 metabolites (aspartate, glutamate, hydroxyphenylacetate, taurine, uric acid, uridine, and xanthine) differed (P < 0.05) among breeds. Brahman cows had the greatest value of all 7 metabolites (P < 0.05) except hydroxyphenylacetate (P > 0.10). Angus and Brangus metabolomic values were similar for aspartate, taurine, and uridine (P > 0.10) but differed for glutamate, hydroxyphenylacetate, uric acid, and xanthine (P < 0.05). Two metabolites that were greatest in Brahman cows (aspartate and glutamate) contribute significantly to the Kreb Cycle, indicating that Brahman cows have the ability to maintain proper amounts of substrates for energy production compared with Angus cows. Both glutamate and aspartate also aid in the removal of toxic ammonium as they are substrates for a-ketoglutarate and oxaloacetate, respectively, which both consume ammonium in their respective reactions. Furthermore, uric acid (which has a precursor of xanthine) was also greatest in Brahman cows and is a potent antioxidant with strong capabilities of scattering reactive oxygen species. Finally, aspartate also aids in the detoxification of ammonium within the urea cycle. Taken together, these four metabolites (aspartate, glutamate, uric acid, and xanthine) are working together but separately with the overall goal to provide more functional energy, while removing unnecessary toxins which may be two mechanisms in how Bos indicus-influenced cows are better able to sustain optimal reproductive efficiency than Angus cows.

231 Ovarian follicle diameter and corpus luteum volume of beef cows enrolled in fixed-time artificial insemination while receiving chromium propionate supplementation

Abstract We investigated the effects of chromium propionate (CrP) supplementation on reproductive performance of beef cows during the pre-partum and breeding periods. Angus-based commercial suckled beef cows were stratified by predicted calving date, body weight (BW) and randomly assigned to one of two treatments: 1) Control, (n = 30) supplementation of a corn gluten, soy hull pellet feed (50:50) with a mineral pack at 1 kg ּ cows-1 d-1; or 2) CrP, (n = 32) supplementation at 1 kgּ cow-1ּּ d-1 of a corn gluten, soy hull pellet feed (50:50) with a mineral pack containing 1.4 g of CrP (KemTRACE chromium 0.4%, Kemin Industries, Inc., Des Moines, IA). Cows remained on a single pasture equipped with feed bunks capable of monitoring individual supplement intake (SmartFeed, C-lock Inc., Rapid City, SD). Treatments were initiated at 92 d prepartum and ended on d 35 post fixed-time artificial insemination (TAI), for a total of 127 d. Cows were enrolled in the 7-d CO-Synch+CIDR TAI protocol. Ovarian ultrasonography was performed to determine diameter of the largest follicle, and the volume of a corpus luteum (CL) on d -10, -3, 0 (TAI day), and 7. Pregnancy diagnosis was performed by ultrasonography on d 35. Cow BW was recorded on d -92, 0, and 35. Data were analyzed using the GLIMMIX and MIXED procedures of SAS with cow as the experimental and fixed effect of treatment. Age, days postpartum, and initial BW were similar (P > 0.05) between treatments (7.4 and 7.2 ± 0.61 yr, 81.3 and 80.8 ± 4.61 d, 526.2 and 528.6 ± 16.21 kg for Control and CrP, respectively). Supplement intake was similar (P = 0.534) between treatments (1.22 and 1.03 gּ cow-1ּּ d-1, for Control and CrP, respectively). Cow BW was similar (P > 0.05) between treatments on d 0 and 35 (548.5 and 551.4 ± 13.50 kg, 562.8 and 574.6 ± 11.41 kg, for Control and CrP, respectively). Furthermore, estrus expression and TAI pregnancy rate was similar (P > 0.05) between treatments (62.1 and 61.1 ± 1.2% estrus, 55.1 and 81.2 ± 20.1% TAI pregnancy rate, for Control and CrP, respectively). However, CrP cows had a larger follicle (P = 0.028) on d 0 (13.79 and 15.56 ± 0.58 mm, for Control and CrP, respectively). Additionally, on d 7, CrP cows had an increased (P = 0.038) CL volume (15.24 and 17.55 ± 0.68 mm3, for Control and CrP, respectively). Supplementation of CrP to beef cows from the pre-partum until breeding did not change cow BW, but increased follicle diameter at TAI and subsequent CL volume.

389 Impact of a Controlled Internal Drug Release device on circulating and local concentrations of progesterone

Abstract Controlled Internal Drug Release (CIDR) devices have become an important component of reproductive management programs in many beef herds. When a CIDR is inserted into a cow, circulating concentrations of progesterone mimic a functional corpus luteum. Some data, however, suggest that concentration of progesterone following insertion of a CIDR might have negative consequences on fertility in Bos indicus animals. Therefore, the objective of this study was to determine the impact of a CIDR device on circulating and uterine tissue concentrations of progesterone. A blood sample and a uterine biopsy were collected from non-suckled postpartum cycling Brahman cows (n = 10; Cycling) during the midluteal stage. A blood sample and a uterine biopsy were also collected from non-suckled postpartum cycling Brahman cows (n = 9; CIDR) after a CIDR had been in place for 5 d. To collect uterine biopsies from CIDR cows, the uterine biopsy tool was placed inside a plastic covering, both were passed into the cervix of the cow before the tool was extended through the covering and into the uterus. After the biopsy was collected, the tool was carefully removed to prevent sample contamination. Blood samples were centrifuged, and serum was collected to determine concentrations of progesterone. Uterine biopsies were snap frozen until extraction. Biopsies were weighted, thawed, and homogenized in assay buffer. Samples were then extracted with methyl tert-butyl ether and resuspended in assay buffer. Serum concentrations are reported as ng/mL and uterine concentrations are reported as ng/g. Statistical analysis was performed using the GLM procedure in SAS with treatment (CIDR vs Cycling), tissue type (uterine vs serum) and their interaction included in the model. There was a significant effect of treatment (P = 0.0062), tissue (P = 0.0063) and a treatment by tissue interaction (P = 0.0068). Animals with a CIDR had greater concentrations of progesterone than Cycling animals (274.77 vs 4.37 ng, respectively). Uterine samples had greater concentrations of progesterone than serum samples (274.40 vs 4.74 ng, respectively). These increased concentrations of progesterone were mainly from CIDR uterine samples (543.24 ng/g) which had greater (P < 0.001) concentrations of progesterone compared with all other samples, which did not differ from each other (P > 0.98; CIDR Serum 6.29 ng/mL, Cycling Serum 3.2 ng/mL, and Cycling Uterus 5.54 ng/g). In summary, CIDRs increased circulating concentrations of progesterone sufficient to keep animals from exhibiting estrus, and were not different from cows in the midluteal stage of their cycle. Local concentrations in uterine tissue; however, were significantly greater when a CIDR was in place compared with uterine tissue collected during the midluteal phase. The implications of these significantly greater concentrations of progesterone in uterine tissue warrant further investigation.

102 Combination and individual vaccines for bovine viral diarrhea virus and infectious bovine rhinotracheitis effects on reproductive cyclicity and immune response

Abstract Vaccines are known to impact both the immune and reproductive systems. The objective of this research was to identify immunological and cyclic differences following vaccination for Bovine Viral Diarrhea Virus (BVDV) and/or Infectious Bovine Rhinotracheitis (IBR). Brahman cows (n = 15) were administered PGF2α to regress corpus lutea on d -3. Animals (d 0) were immunized (2mL, intramuscular injection) with one of three treatments: 1) Modified Live Vaccine (MLV) for BVDV and IBR (BVD+IBR; n = 5), 2) MLV for BVDV only (BVD; n = 5), or 3) sterile saline (CON; n = 5). Blood samples (30 mL/cow) were collected (d -3, 0, 2, 4, 6, 8, 10, and 14) and peripheral blood mononuclear cells (PBMC) were isolated. PBMC were incubated with propidium iodide and antibodies for specific surface cell markers (CD4, CD8, CD25, CD14, CD86, and CD335). An Amnis FlowSight flow cytometer determined cell type percentage. BVDV and IBR antibody concentrations were analyzed in serum samples (d -3, 0, and 14). Plasma samples (d 0, 2, 4, 6, 8, 10, and 14) were evaluated for interferon (IFN)-γ, interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-36RA, interferon-gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) α, and vascular endothelial growth factor (VEGF) A concentrations by a MagPix multiplex machine using a MILLIPLEX Bovine Cytokine/Chemokine Magnetic Bead Panel. Progesterone (d 0, 2, 4, 6, 8, 10, and 14) and estradiol (d 0) concentrations were determined by RIA. Differences in antibody titers, PBMC populations, and cytokine concentrations were analyzed as repeated measures using PROC MIXED (SASv9.4). Forty percent of BVD+IBR animals experienced abnormal estrus cycles, but no BVD or CON animals did. There was a treatment effect on BVD titer concentration with BVD animals having greater titer concentrations than CON (P = 0.02) but similar concentrations to BVD+IBR (P = 0.18). There was a treatment by time interaction (P < 0.0001) on IBR titers with BVD+IBR animals having greater titer concentrations on d 14 compared with BVD and CON. Treatment had no effect on leukocyte populations (P ≥ 0.1409), but all populations differed over time (P ≤ 0.0045). Antigen presenting cells (CD14+) and natural killer cells (CD335+) were affected by a treatment and time interaction (P ≤ 0.0479). BVD animals had increased CD14+ cells on d 2, 4, 8, and 14 compared with CON, but CON had a greater CD335+ cell population compared with BVD and BVD+IBR on d 10. There tended to be or was a significant treatment by day interaction for IL-1α (P = 0.08), IL-1b (P = 0.03), IL-10 (P = 0.06), IFN-γ (P = 0.05), IP-10 (P = 0.02), MIP-1α (P = 0.02), MIP-1 b (P = 0.01), TNFα (P = 0.01), and VEGFA (P = 0.03). IL-1a, IL-10, IL-1b, and MIP-1 b increased in BVD+IBR from d 0 to 4 or 6 then decreased but not in CON or BVD. Increased VEGFA was observed in CON compared with BVD and BVD+IBR animals. Overall, BVD+IBR resulted in abnormal cycles and changes in cytokine concentrations and leukocyte populations. USDA is an equal opportunity provider and employer.

104 Non-invasive pregnancy diagnosis in Holstein dairy cows using computer vision analysis of thermal images

Abstract Thermal visualization of Holstein dairy cattle skin can be indicative of the pregnancy state (cyclic or pregnant) of the cow by observing the size and shape of skin thermal patterns. However, visual assessment of skin thermal patterns can be subjective and laborious. Further, infrared thermography implementation on-farm requires automated analysis to be feasible for dairy producers. The objectives of this study were to implement a machine learning convolutional neural network (CNN) to detect features in thermal images associated with pregnant (Pregnant) and non-pregnant (Cyclic) and to evaluate the accuracy of pregnancy diagnosis in dairy cows. A total of 938 images from 18 Pregnant cows and 982 images from 18 Cyclic were used to build the CNN model, and 120 images from Pregnant (60 images) and Cyclic (60 images) dairy cows were used to validate the model. Pregnant cows were confirmed to be pregnant 90 d after insemination via transrectal ultrasonography. Cyclic cows were confirmed to be non-pregnant using follicular dynamics and corpora lutea presence via transrectal ultrasonography. Images were recorded using a T620s infrared camera at 640 × 480 (307,200) to train the CNN model and images from an E40 FLIR camera at 320 x 240 (76,800 pixels) were used for the validation test. The CNN model was created using TensorFlow in Google Colab to binary classify images into Pregnant or Cyclic using a single output neuron (1: Pregnant and 0: Cyclic) with different Epochs (10, 25, and 100). The diagnosis accuracy (acc) was 0.72 with a validation accuracy (val_acc) of 0.41 for 10 Epochs, acc 0.76 with val_acc 0.37 for 25 Epochs, and acc 0.80 with val_acc 0.63. The acc and val_acc of the training model and validation test were optimized when using images from the T620s exclusively (Epochs 10: acc = 0.70 and val_acc = 0.64; Epoch 25: acc = 0.74 and val_acc = 0.70; Epoch 100: acc = 0.81 and val_acc = 0.80). Greater resolution of thermal images was proven to increase the accuracy of pregnancy diagnosis (307,200 compared with 76,800 pixels) and the greater number of Epoch used to train the model. Machine learning algorithms can diagnose pregnancy using thermal images with an acceptable evaluation accuracy (> 0.70).

Qualitative and quantitative parameters analyses of buffalo ovaries

Buffalo ovaries were collected from abattoirs in Dhaka city. During collection, left and right ovaries were identified and recorded. After processing, the ovaries were categorized as (i) ovaries without corpus luteum (CL) and (ii) ovaries with corpus luteum (CL). Ovaries were then evaluated based on length (cm), width (cm), weight (g), total number of follicles on the surface of each category of ovaries, number of follicles aspirated, and total number of cumulus-oocyte-complexes (COCs). The experiment's findings indicated variations in the parameters related to the left and right ovarian categories. The CL% (27.12±0.11%) found highest in the right compared to the left (3.24±0.11%). The length (cm), width (cm), and weight (g) (2.75±0.06, 2.17±0.05, 4.74±0.13) were found significantly (p<0.01) higher in the right ovaries compared to left (2.27±0.06, 1.77±0.05, 3.90±0.13). The number of follicles in total and number of follicles aspirated were found significantly (p<0.01) higher (6.35±0.16 & 5.47±0.16) in the left ovaries compared to right (6.16±0.16 & 4.18±0.16). The number of normal COCs and total COCs was significantly (p<0.01) higher in the left ovaries (0.98±0.134 & 1.57±0.17) compared to right (0.84±0.16 & 1.54±0.17). The abnormal COCs were significantly (p<0.01) higher in the right ovaries (0.75±0.107) compared to left (0.57±0.107). Regarding ovarian categories; (76.47±0.01%) showed ovaries without CL, whereas (23.53±0.01%) showed ovaries with CL. The weight (g) was found significantly (p<0.01) higher in ovaries with CL (4.73±0.12) compared to ovaries without CL (4.36±0.12). The number of follicles in total and aspirated were found significantly (p<0.01) higher in ovaries without CL (7.60±0.14 & 5.28 ±0.14) compared to ovaries with CL (4.29±0.14 & 2.29±0.14). Regarding the number of total COCs, ovaries without CL showed a significantly (p<0.01) higher number of total COCs (1.41±0.11) compared to ovaries with CL (1.17±0.11), where the number of normal and abnormal COCs (0.55±0.01 & 0.86±0.03) were found significantly (p<0.01) higher in ovary without CL compared to the ovary with CL (0.5±0.01 & 0.54±0.03). Finally, it can be concluded that the left and ovaries without CL contain more follicles in total and aspirated and normal COCs. Thus, the left and ovaries without CL are best for collecting quality COCs in buffaloes. Res. Agric. Livest. Fish. Vol. 11, No. 2, August 2024: 125-135

Exogenous estrogen partially rescues progesterone deficiency and autophagosome enlargement in Mcoln1 −/− mouse model with lysosomal storage disorder

Objective: Female Mcoln1 −/− mice exhibit progressive progesterone (P4) deficiency, luteal cell degeneration, and premature embryo implantation failure at five months old. We attempted to rescue embryo implantation in non-virgin Mcoln1 −/− mice (5–6 months old) with exogenous P4 treatment on days 1.5 post-coitum (D1.5), D2.5, and D3.5, and observed partially restored luteal cell morphology on D4.5, but unexpectedly found 17β-estradiol (E2) contamination in the P4 working solution. In this study, we aim to investigate exogenous P4 and/or E2 for the partial recovery of luteal cell morphology in infertile Mcoln1 −/− mice. Methods: Control and non-virgin Mcoln1 −/− mice (5–6 months old) were treated with newly ordered vehicle, P4, E2, or P4+ E2 on D1.5 and D2.5 and dissected on D3.5 for P4 and E2 measurements, ovary histology, immunofluorescence, lipid droplet staining, and transmission electron microscopy. Results: E2 treatment significantly increased serum P4 levels in D3.5 Mcoln1 −/− mice. E2 and P4+E2 treatments, but not P4 treatment alone, largely improved the morphology of D3.5 Mcoln1 −/− corpora lutea, indicated by a more contiguous web-like collagen IV expression pattern, increased heat shock protein 60 expression, and reduced accumulation of large lipid droplets. Transmission electron microscopy revealed extremely enlarged autophagosomes and lipid droplets, lysosomes with lamellar structures, and mitochondria with reduced cristae in vehicle-treated D3.5 Mcoln1 −/− luteal cells, while in E2-treated D3.5 Mcoln1 −/− luteal cells, extremely enlarged autophagosomes and lipid droplets were reduced, indicating improved luteal cell ultrastructure. Conclusion: These findings reveal protective effects of high levels of exogenous E2 on P4 production and lysosomal function in Mcoln1 −/− luteal cells.

Recipients' and Environmental Factors Affecting the Pregnancy Rates of a Large, Fresh In Vitro Fertilization-Embryo Transfer Program for Dairy Cows in a Commercial Herd in China.

The main objective of this study was to determine the influence of the recipient dairy cows' breed, lactation number, estrus condition, the type, location and volume of the corpus luteum (CL) and the time of year that the embryo transfer (ET) was performed on the pregnancy rates of a large, fresh in vitro fertilization-embryo transfer program for dairy cows in a commercial herd in China. The recipients were from a herd of dairy cows in Ningxia, a province in northwest China, and we statistically analyzed the data of 495 cows from 2021 to 2023. Cumulus oocyte complexes (COCS) were isolated from follicular fluid obtained through ovum pick-up (OPU) and oocytes were incubated 20-22 h for in vitro maturation (IVM). Embryos were obtained after 10-12 h of in vitro fertilization (IVF) and six days of in vitro culture (IVC). Embryos at the morula or blastocyst stage were transferred to randomly chosen recipients (n = 495). The influence of recipients' breed (Holstein or other), recipients' lactation number (heifers or cows), estrus type (natural or synchronized), CL type (homogeneous, CLhom or cavitary, CLcav), CL side (left or right), volume of the CL and season of transfer (spring, autumn or winter) on pregnancy rates were determined. The pregnancy rates were analyzed by binomial logistic regression with IBM SPSS statistics software, version 26. Pregnancy rates after ET to Holstein cows and other breeds were 43.49% and 42.68%, respectively (p > 0.05). Regarding age, pregnancy rates were 45.56% for heifers and 30.77% for cows (p < 0.05). Pregnancy rates following ET during natural and synchronized estrus were 44.41% and 41.5%, respectively (p > 0.05). Pregnancy rates with a left- or right-side CL were 40.18% and 45.65%, respectively (p > 0.05). The pregnancy rates achieved with a CLhom and CLcav were 44.44% and 39.68%, respectively (p < 0.05). The rates obtained in spring, autumn and winter were 49.26%, 46.02% and 34.64%, respectively (p < 0.05). Moreover, it was found that pregnancy rates were higher in recipients with a CL volume measuring greater than 10 cm3 compared with those with a CL volume measuring less than 10 cm3 (p < 0.05). The comparisons showed that recipients' breed, estrus type or side of the CL had no effect, but the recipients' lactation number, ET season and the type and volume of the CL have significant effects on pregnancy rates during ET.

Stereological and biochemical effects of thymoquinone on ovarian tissue toxicity induced by silver nanoparticles in NMRI mice: An experimental study.

The toxicity of silver nanoparticles (AgNPs) has been proven in the female reproductive system. Thymoquinone (TQ) is a natural antioxidant and bioactive component of Nigella sativa. We evaluated the efficacy of TQ on ovarian tissue following toxicity induced by AgNPs in female mice. 24 female NMRI mice (5-6 wk, an average weight of 33 gr) were randomly divided into 4 groups (n = 6/each): control, AgNPs (500 mg/kg, gavage), TQ (2.5 mg/kg, intraperitoneal injection), and TQ+AgNPs. Mice were treated every day for 35 days. Serum levels of malondialdehyde (MDA), total antioxidant capacity (TAC), luteinizing hormone, and follicle-stimulating hormone were measured. The optical disector and stereological techniques were utilized to estimate the follicular count, their volume at different developmental stages, and the structure of ovarian tissue. In the AgNPs group, the serum concentrations of TAC (p = 0.01), luteinizing hormone (p 0.001), follicle-stimulating hormone, the volume of corpus luteum (p 0.001), and the number of follicles decreased significantly compared to the control group. Nevertheless, AgNPs significantly increased the MDA level. In the TQ+AgNPs group compared to the AgNPs group, a significant decrease in MDA level (p 0.001) and a significant improvement in TAC (p = 0.03), and hormonal levels, the number of primary, preantral, and antral follicles (p = 0.04), and the volume of corpus luteum (p = 0.01) were observed. TQ improved the number of follicles by reducing oxidative stress and lipid peroxidation in AgNPs-damaged ovarian tissue.

Detection of the effect of the KISS1 gene on reproductive parameters in Saanen goats

The KISS1 gene, which activates G protein-coupled receptor-54 (GPR54) and plays a crucial role in the neuroendocrine regulation of GnRH secretion, is known to encode a family of neuropeptides called kisspeptins. Therefore, the KISS1 gene is thought to be associated with sexual maturity, offspring development, and estrus. The g.2124T&amp;gt;A polymorphism of the KISS1 gene, is believed to affect the reproductive system, has been studied in Indonesian goat breeds, Damascus, Zarabi, Baladi, Gaddi, Kaligesing, Guanzong, Xinong Saanen and Boer goat breeds until now. These studies are generally related determining polymorphism frequencies or the effect of genotype on litter size. The study aims to determine the allele and genotype frequencies of the KISS1 gene g.2124T&amp;gt;A SNP by the PCR-RFLP method and define the relationship between the genotypic structures and reproductive parameters such as luteal growth, maximum corpus luteum diameter, and mating rate in Saanen goats (n=30). According to the results, the genotype frequencies in Saanen goats for the KISS1 gene g.2124T&amp;gt;A were 10.35%, 55.17%, and 34.48% for AA, TT and AT, respectively. Moreover, the genotypic structure did not have a statistically significant effect on the investigated fertility characteristics. The average values of expected heterozygosity (He), observed heterozygosity (Ho), the effective number of alleles (Ne), fixation index values (FIS), and the polymorphism information content (PIC) were 0.3996, 0.3448, 1.6655, 0.8629 and 0.855, respectively. The Hardy-Weinberg chi square (χ2) value was found to be 0.5435. In conclusion, it was found necessary to study the KISS1 - g.2124T&amp;gt;A polymorphism in larger herds with different gene pools as a reason for the existence of genotypic variation and the narrow population size.

Local Regulatory Changes of HSD11B1 and NR3C1 in the Follicular and Luteal Tissue During Experimentally Induced Ovulation in the Cow.

The objective of the study was to characterise the expression patterns of the two key components of cortisol action namely HSD11B1 (11-beta-hydroxysteroid dehydrogenase type 1) and NR3C1 (nuclear receptor subfamily 3, group C, member 1, also known as the glucocorticoid receptor) in superovulation induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. Bovine ovaries containing preovulatory follicles or CL were timely defined during induced ovulation as follows: 0 h before GnRH (Gonadotropin-releasing hormone) application, and 4, 10, 20, 25 (follicles) and 60 h (early CL) after GnRH. The low mRNA expression of HSD11B1 and NR3C1 in the follicle group before the GnRH application increased significantly in the follicle group 20 h after GnRH and remained high afterward also in the early CL group. In contrast, the high NR3C1 mRNA decreased in follicles 25 h after GnRH (close to ovulation) and significantly increased again after ovulation (early CL). Our results indicated the involvement of HSD11B1 and NR3C1 as the two key components of cortisol action in the local mechanisms coordinating final follicle maturation, ovulation, follicular-luteal transition and CL development in the cow.

Optimisation of Early Pregnancy Diagnosis andResynchronisation in Beef Cows.

The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler-based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol-progesterone-based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1-12.9 mm (n = 64), 13.0-14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 μg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF-based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.

Testosterone-Induced H3K27 Deacetylation Participates in Granulosa Cell Proliferation Suppression and Pathogenesis of Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is the leading cause of infertility in reproductive-age women. Hyperandrogenism, polycystic ovaries and chronic anovulation are its typical clinical features. However, the correlation between hyperandrogenism and ovarian follicle growth aberrations remains undisclosed. To advance our understanding of the molecular alterations in ovarian granulosa cells (GCs) with excessive androgen, epigenetic changes and affected gene expression in human granulosa-lutein cells (hGCs) and immortalized human GCs (KGN) were evaluated. A PCOS mouse model induced by dihydrotestosterone (DHT) was also established. This study found excessive testosterone significantly decreased the acetylation of lysine 27 on Histone H3 (H3K27ac). H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) data showed down-regulated expression of cell cycle-related genes (CCND1/CCND3/PCNA), which was confirmed by real-time quantitative PCR and western blot. Testosterone application impeding cell proliferation was also proved by Ki-67 immunofluorescence and flow-cytometric analysis. Moreover, testosterone influenced CK2α nuclear translocation, which increased the phosphorylation level of histone deacetylase 2 (HDAC2). Inhibition of CK2α nuclear translocation or silenced HDAC2 expression efficiently retarded H3K27 acetylation. Meanwhile, PCOS mouse model experiments also demonstrated decreased H3K27ac and enhanced HDAC2 phosphorylation in GCs. Cell proliferation-related genes were also downregulated in PCOS mouse GCs. In conclusion, hyperandrogenism in human and mouse GCs caused H3K27Ac aberrations, which are associated with CK2α nuclear translocation and HDAC2 phosphorylation, participating in abnormal follicle development in PCOS patients.

PSIII-23 Identifying genes associated with the retinol biosynthesis pathway in corpora lutea of cyclic and pregnant gilts

PSIII-23 Identifying genes associated with the retinol biosynthesis pathway in corpora lutea of cyclic and pregnant gilts