Young Mares Research Articles

Elemental differentiation and isotopic fractionation during space weathering of Chang’E-5 lunar soil

To investigate the chemical variation during space weathering of young mare basalts, here we report elemental, radiogenic Sr-Nd and stable Fe-Mg-Ca isotopic data of Chang’E-5 sieved soils and breccias. From the coarse fraction to the fine one, the sieved soils display increasing Al2O3 (10.34 wt%–13.36 wt%) and Sr (248 ppm–307 ppm) but decreasing FeO (23.50 wt%–20.22 wt%), MgO (6.88 wt%–5.78 wt%), FeO/Al2O3 (2.27–1.51) and MgO/Al2O3 (0.67–0.43). The contents of rare earth elements (except Eu) and high field strength trace elements do not vary with particle size but correlate with P2O5 contents. Given the limited contribution from contamination by meteorites and exotic materials ejected far away from the landing site, these elemental variations can be explained by differential comminution and distribution behaviors of plagioclase and mesostasis phases. These sieved soils yield a Sm-Nd isochron age (1.84 ± 0.83 Ga) comparable to that of basaltic clasts obtained by U-Pb dating (∼2.0 Ga). However, their Rb-Sr isotopic system is disturbed as indicated by their relatively homogeneous 87Sr/86Sr (0.701425–0.701592) despite variable Rb/Sr (0.017–0.028). These results suggest the Sm-Nd isotopic system is more robust to impact disturbance during space weathering compared to the Rb-Sr isotopic system. Given that the bulk soil still plots on the 2.03 Ga Rb-Sr reference isochron from the pristine plagioclases in CE-5 basalts, this disturbance did not affect the Rb-Sr isotopic system on the bulk scale. The CE-5 bulk soil has higher Mg# (33.6), 87Rb/86Sr (0.06) and present-day 87Sr/86Sr (0.701542) than the mean composition of reported basaltic clasts (Mg#: ∼28; 87Rb/86Sr: ∼0.038; 87Sr/86Sr: ∼0.700941), possibly implying that the bedrocks in CE-5 landing site consist of multiple magma pulses. The δ56Fe (0.122 ± 0.002 ‰ to 0.199 ± 0.008 ‰) and δ26Mg (−0.204 ± 0.016 ‰ to −0.109 ± 0.006 ‰) of sieved CE-5 soils increase with decreasing particle sizes but their δ44/42Ca (0.38 ± 0.04 ‰ to 0.44 ± 0.02 ‰) are relatively homogeneous. Mass balance modelling indicates that differential comminution has limited influence on the Fe-Mg-Ca stable isotopic compositions. We further dismiss the role of solar-wind sputtering, as Ca and Mg are more susceptible to sputtering and thus would be expected to show larger isotope fractionations compared to Fe, which is inconsistent with the observations. Free evaporation may explain the elevated δ56Fe and δ26Mg in fine fractions at given very limited depletion in FeO and MgO. The observed positive correlation between δ56Fe and δ26Mg, however, is much steeper than the slope expected for free evaporation, indicating also other mechanisms (e.g., Fe-Mg inter-diffusion). Since the CE-5 soil has a unique composition compared with Apollo and Luna soils, the chemical differentiation identified in this study provides new insights for establishing a connection between the chemistry and reflectance spectral properties of lunar soil. Our combined Fe-Mg-Ca isotopic study also provides a paradigm to distinguish the role of solar-wind sputtering and impact evaporation, and shows that the inter-particle diffusion process may be an important mechanism for the isotope fractionation among lunar soil components.

Young KREEP-like mare volcanism from Oceanus Procellarum

The Moon’s mare volcanism predominantly occurs within the Procellarum KREEP Terrane (PKT), which is widely thought to be associated with KREEP components within the lunar interior. The Chang’e-5 (CE-5) mission sampled a young (2 Ga) mare basalt Em4/P58 unit of northern Oceanus Procellarum. The geochemistry of the CE-5 mare basalt enables assessment of mantle source compositions which are essential to understand the thermo-chemical mechanism for prolonged volcanism during secular cooling of the Moon. Geochemical compositions of the CE-5 bulk soil, breccias, and basalt clasts from various depths within the drill core consistently display high concentrations of incompatible trace elements (ITE: ∼ 0.3 × high-K KREEP; ∼ 5 μg/g Th) with KREEP-like inter-element ratios, for example for La/Sm, Nb/Ta, and Zr/Y. Exotic impact ejecta, extensive magma differentiation (<70 % fractional crystallization) and significant assimilation of KREEP materials during magma transit and eruption cannot account for the ITE contents and ratios or radiogenic isotope compositions (e.g., εNdinitial of + 8 to + 9 and εHfinitial of + 40 to + 46) of the CE-5 basalts; instead, partial melting of their mantle source played a dominant role. The Chang’e-5 basalt is a chemically evolved low-Ti mare basalt (Mg# of ∼ 34) with enriched KREEP-like ITE compositions but high long-term time-integrated Sm/Nd and Lu/Hf ratios, which represent a hitherto unsampled type of mare basalt. It formed by melting of an augite-rich mantle source (late-stage magma ocean cumulates containing > 30–60 % augite, and little or no ilmenite), with a small amount of late-stage interstitial melt that resembles KREEP (∼1–1.5 modal %, equivalent to 0.2–0.3 μg/g Th in the mantle source). The voluminous mare basalts making up the Em4/P58 unit (>1500 km3) provide compelling evidence for large-scale, ITE enriched young mare magmatism within Oceanus Procellarum. In combination with remote sensing data and with the unique Th-rich Apollo 12 basalt fragment 12032,366–18 (impact ejecta likely from Oceanus Procellarum), this implies that significant portions of the FeO- and Th-rich mare regions of the western PKT may also have formed in a similar way.

Effect of breeding season and age on follicular dynamics and hemodynamics in embryo donor mares subjected to luteolysis after embryo flushing.

Mares are the only companion animals simulating women in the large diameter of their follicles. Horses start reproduction at the age of three years, and some of them live for >30 years, so aging influences their reproductive capacity. Mares are sensitive to summer heat stress as they can sweat like humans. The current work aimed to study the effects of age (young versus senile), season (cold versus hot), and the hormonal treatments during embryo collection on the dominant and subordinate follicular dynamics and hemodynamics and circulating ovarian hormones in embryo donor mares ovulated twice spontaneously before inducing ovulation for flushing embryos. Spontaneous oestrous cycles were studied for young mares (<10 years; N = 6) or senile (>20 years; N = 5) during months of the cold season (November to April) and hot season (May to August). In young embryo donor mares, oestrous cycles after inducing ovulation and luteolysis were studied using Doppler ultrasound. Estradiol (E2), progesterone (P4), nitric oxide (NO), total cholesterol, and lactate dehydrogenase (LDH) were measured in blood serum. A decrease in the dominant follicle antrum diameter (p > 0.05) and LDH (p = 0.016) was observed after inducing luteolysis in young embryo donor mares. Both human chorionic gonadotropin (hCG) and PGF2α treatments increased dominant follicle area (p = 0.0001), antrum area (p = 0.001), perimeter (p = 0.001), granulosa area (p = 0.0001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001). The dominant follicle area, antrum area, perimeter, color area, granulosa area, LDH, cholesterol, NO, and E2 increased (p = 0.0001) during the oestrous cycles of the hot season, but the circulatory % (p = 0.0001) declined. Senile mares had lower dominant follicle area (p = 0.002), antrum area (p = 0.0001), granulosa area (p > 0.05), LDH (p = 0.001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001) but higher circulatory % (p = 0.0001) and color area % (p = 0.023). The dominant follicle possesses the largest diameter, area, perimeter, granulosa area, and color area but the lowest circulatory % during spontaneous oestrous cycles, after inducing ovulation, or luteolysis with significant effects of the day of the spontaneous oestrous cycles on their dynamics and hemodynamics. During hot months, mares treated with hCG ovulated 24 hours later and prostaglandin-induced luteolysis was followed by new ovulation five days later. Follicles ovulated during the hot months were larger than those ovulated during the cold months and both had nearly the same color area %. Senile mares ovulated follicles with a lower area and antrum area but a higher color area %, so senile mares can be used as embryo or oocyte donors during the hot season.

Comprehensive mapping of lunar surface chemistry by adding Chang'e-5 samples with deep learning

Lunar surface chemistry is essential for revealing petrological characteristics to understand the evolution of the Moon. Existing chemistry mapping from Apollo and Luna returned samples could only calibrate chemical features before 3.0 Gyr, missing the critical late period of the Moon. Here we present major oxides chemistry maps by adding distinctive 2.0 Gyr Chang’e-5 lunar soil samples in combination with a deep learning-based inversion model. The inferred chemical contents are more precise than the Lunar Prospector Gamma-Ray Spectrometer (GRS) maps and are closest to returned samples abundances compared to existing literature. The verification of in situ measurement data acquired by Chang'e 3 and Chang'e 4 lunar rover demonstrated that Chang’e-5 samples are indispensable ground truth in mapping lunar surface chemistry. From these maps, young mare basalt units are determined which can be potential sites in future sample return mission to constrain the late lunar magmatic and thermal history.

Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy’s (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy’s solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Assessment of the Virulence of the Burkholderia mallei Strain BAC 86/19 in BALB/c Mice.

Burkholderia mallei is an aerobic, Gram-negative, non-motile bacillus. As an obligate mammalian pathogen, it primarily affects solipeds. Although rarely transmitted to humans, the disease it causes, glanders, is classified as a zoonosis. The bacterium was officially eradicated in Brazil in 1969; however, it reemerged after three decades. This study aims to assess the virulence of a specific B. mallei strain, isolated in Brazil, in BALB/c mice through intranasal infection. The strain, B. mallei BAC 86/19, was obtained from the tracheal secretion of a young mare displaying positive serology but no clinical signs of glanders. Post-mortem examinations revealed macroscopic lesions consistent with the disease, however. In mice, the LD50 was determined to be approximately 1.59 × 105 colony-forming units (CFU)/animal. Mice exposed to either 0.1 × LD50 or 1 × LD50 displayed transient weight loss, which resolved after three or five days, respectively. B. mallei persisted within the liver and lung for five days post-infection and in the spleen for seven days. These findings underscore the detectable virulence of the Brazilian B. mallei BAC 86/19 strain in mice, which are relatively resilient hosts. This research points to the importance of the continued investigation of the virulence mechanisms and potential countermeasures associated with B. mallei infections, including their Brazilian isolates.

Equine maternal aging affects the metabolomic profile of oocytes and follicular cells during different maturation time points.

Introduction: Oocyte quality and fertility decline with advanced maternal age. During maturation within the ovarian follicle, the oocyte relies on the associated somatic cells, specifically cumulus and granulosa cells, to acquire essential components for developmental capacity. Methods: A nontargeted metabolomics approach was used to investigate the effects of mare age on different cell types within the dominant, follicular-phase follicle at three time points during maturation. Metabolomic analyses from single oocytes and associated cumulus and granulosa cells allowed correlations of metabolite abundance among cell types. Results and Discussion: Overall, many of the age-related changes in metabolite abundance point to Impaired mitochondrial metabolic function and oxidative stress in oocytes and follicular cells. Supporting findings include a higher abundance of glutamic acid and triglycerides and lower abundance of ceramides in oocytes and somatic follicular cells from old than young mares. Lower abundance of alanine in all follicular cell types from old mares, suggests limited anaerobic energy metabolism. The results also indicate impaired transfer of carbohydrate and free fatty acid substrates from cumulus cells to the oocytes of old mares, potentially related to disruption of transzonal projections between the cell types. The identification of age-associated alterations in the abundance of specific metabolites and their correlations among cells contribute to our understanding of follicular dysfunction with maternal aging.

Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Reproductive performance of a cohort of Standardbred mares under a commercial breeding system.

Despite being a large commercial breeding industry, there is little published data on the reproductive success of Standardbred mares. To quantify the reproductive performance of Standardbred mares under artificial breeding systems in a commercial setting and determine the incidence of early embryonic and other pre-partum losses. Retrospective cohort study. Data from four commercial farms were collected across four breeding years, and all mares were bred via artificial insemination. A total of 3995 mares contributed 7229 mare years. First-cycle pregnancy rate (FCPR) and end of season pregnancy rate (SPR) were analysed in mixed-effects logistic regression models. Time-to-conception interval was analysed in a Cox regression model. The mean FCPR was 61.4% (confidence interval [CI] 60.3%-62.6%), the mean end of SPR was 84.7% (CI 83.8-85.5%), the mean live foal rate (FR) was 73.1% (CI 72.1%-74.2%). Mares located on-farm were more probable to be pregnant in terms of both FCPR (odds ratio [OR] 1.168, CI 1.018-1.340, p = 0.026) and SPR (OR 2.026, CI 1.673-2.454, p < 0.001), mares inseminated with thawed-frozen semen were less probable to be pregnant in terms of FCPR (OR 0.598, CI 0.457-0.783, p < 0.001) and SPR (OR 0.479, CI 0.354-0.647, p < 0.001) compared with insemination with fresh-extended semen. Older mares (14 years and older) were less probable to be pregnant in terms of FCPR (OR 0.795, CI 0.688-0.919, p = 0.002) and SPR (0.435, CI 0.352-0.538, p < 0.001) compared with young mares (3- to 8-year old). Retrospective data relied on accurate record keeping of stud farms and no mare-treatment or ovulation induction records were available. Live FRs relied mostly on annual foaling returns so fetal/foal deaths may be underrepresented. This study provides substantial baseline data on reproductive performance for Standardbred mares managed under a commercial artificial breeding system.

Adaptive response of estrogen-iron axis in pregnant Purebred Spanish mares of different age.

The maintenance of iron (Fe) homeostasis is vital for the physiological function along life. In sexually mature humans and experimental animals, estrogens downregulate hepcidin (Hpc) expression, in order to improve the intestinal absorption and to mobilize Fe stores for maternal erythropoietic expansion and placental development. However, changes of these mechanisms related to regulation of Hpc on the availability of Fe during gestation with advancing age in mares, remain unknown. The objective of this study was to evaluate the interrelationships between serum Fe, Ferritin (Ferr) and Hpc with estrone (E1) and estradiol-17β (E2) concentrations in pregnant mares of different ages. Blood samples were taken from 40 pregnant Spanish Purebred mares belonging to 4 different age groups, 10 subjects for each group: 4-6 years, 7-9 years, 10-12 years, and > 12 years were used in this study. Fe concentrations of 4-6 and 7-9 years groups were higher (P < .01) than 10-12 and >12 years groups. Ferr concentrations of 4-6 years group were higher (P < .01) than other groups. Hpc concentrations increased and E1 decreased (P < .01) in > 12 years group compared to other age groups. E2 concentrations of 7-9, 10-12 and >12 years groups were higher (P < .01) than those of 4-6 years group; 7-9 years group had higher E2 concentrations (P < .01) than > 12 years group. Fe and Ferr were negatively correlated with Hpc (r = -0.81 and r = -0.67, respectively). E1 and E2 were negatively correlated with Fe (r = -0.23 and r = -0.11, respectively). E2 was positively correlated with Hpc (r = 0.78). In pregnant Spanish Purebred mare, the increase of estrogens, according to the more efficient iron status in response to Hpc inhibition and consequent mobilization of circulating and iron reserve, shows the existence of "estrogen-iron axis" in young mares. Nevertheless, these mechanisms are reversed in old mares, suggesting a less efficient iron metabolism with advancing age. It is hoped that new investigations are needed to understand in depth and clarify further the complex metabolic and hormonal mechanisms involved also in equine species.

Crystallization kinetics of a fastest-cooling young mare basalt of Chang’E-5

Crystallization kinetics of a fastest-cooling young mare basalt of Chang’E-5

Lunar Surface Model Age Derivation: Comparisons Between Automatic and Human Crater Counting Using LRO‐NAC and Kaguya TC Images

AbstractDating young lunar surfaces, such as impact ejecta blankets and terrains associated with recent volcanic activities, provides critical information on the recent events that shaped the surface of the Moon. Model age derivation of young or small areas using a crater chronology is typically achieved through manual counting, which requires a lot of small impact craters to be tediously mapped. In this study, we present the use of a Crater Detection Algorithm (CDA) to extract crater populations on Lunar Reconnaissance Orbiter—Narrow Angle Camera (LRO‐NAC) and Kaguya Terrain Camera images. We applied our algorithm to images covering the ejecta blankets of four Copernican impact craters and across four young mare terrains, where manually derived model ages were already published. Across the eight areas, 10 model ages were derived. We assessed the reproducibility of our model using two populations for each site: (a) an unprocessed population and (b) a population adjusted to remove contaminations of secondary and buried craters. The results showed that unprocessed detections led to overestimating crater densities by 12%–48%, but “adjusted” populations produced consistent results within &lt;20% of published values in 80% of cases. Regarding the discrepancies observed, we found no significant error in our detections that could explain the differences with crater densities manually measured. With careful processing, we conclude that a CDA can be used to determine model ages and crater densities for the Moon. We also emphasize that automated crater datasets need to be processed, interpreted and used carefully, in unity with geologic reasoning. The presented approach can offer a consistent and reproducible way to derive model ages.

Epigenetic regulation of collagen in the mare's placenta: putative relationship with maternal age

Placental plasticity influences fetal development, pregnancy success, and foal growth. Older fertile mares deliver heavier foals and placentas, with increased collagen deposition. In humans, epigenetics regulates placental development and function. Epigenetics, through DNA methylation, also modulates endometrial fibrosis. As such, we hypothesized that as mares get older, collagen deposition in the placenta might be epigenetically modulated. This study assessed if transcription of collagen genes in the equine placenta was epigenetically modulated through DNA methyltransferases (DNMTs), and affected by aging. Immediately after foaling, samples from different chorioallantoic areas (gravid horn, non-gravid horn, and body) were collected from young (n=10; 4-6 years) and older mares (n=10; 12-18 years). Gene transcription of collagen type 1 (COL1A1), collagen type 3 (COL3A1), and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was determined (qRT-PCR). Unpaired T-test and Mann-Whitney test were used with statistical significance set a p <0.05. In older mares, COL1A1 mRNA levels were higher in the gravid than in the non-gravid horn (p<0.05), and also upregulated in comparison to young mares’ placentas (p<0.05). In young mares, these transcripts were increased in the placenta body in comparison to the gravid (p<0.05) and non-gravid horns (p<0.01). In older mares, COL3A1 transcriptionwas higher in gravid and non-gravid horns, with respect to the same placenta regions in young mares (p<0.05). In older mares, DNMT1 mRNA expression in the body was lower than in younger mares (p<0.05). For all placentas, DNMT1 transcripts in the non-gravid horn were lowest, compared to the gravid horn (p<0.01) and body (p<0.001). In young mares, DNMT1 mRNA expression in the body was also higher than in the gravid horn (p<0.05). With mares aging, there was a downregulation of DNMT3A transcripts in gravid and non-gravid placenta horns, compared to young mares’ placentas (p< 0.05). In older mares, its levels were lowest in the gravid horn, compared to the non-gravid horn (p<0.05) and body (p<0.01). Similarly, in older mares’ placentas, DNMT3B transcripts were downregulated in the gravid horn (p<0.05) and placenta body (p<0.001), compared to young mares. DNMT3B mRNA levels were lowest in the non-gravid horn, both in young mares, in comparison to placenta body (p<0.01) and in older mares, with respect to the gravid horn and body (p<0.01). Considering mares aging, hypomethylation indicated by decreased DNMTs transcripts, in parallel with upregulation of collagen gene expression in gravid and non-gravid horns, may suggest epigenetic involvement on placental plasticity. However, further research is needed to unravel the intricacies of epigenetic modulation of collagen in equine placentas.

Placental gene expression reveals reduced angiogenesis in primiparous mares

Primiparous mares produce smaller and lighter foals than in subsequent pregnancies. In most studies, their placenta is also smaller, more efficient and presents structural alterations, but maternal age is a confounding factor. This study aimed to characterize the effects of maternal parity using the mare's first two pregnancies or by comparing young mares with different parities over the same gestation year. The same young mares (4–6-year-old over the study) foaled for the first time in 2018 (primiparous in 2018, P18, n=10) and for the second time in 2020 (multiparous in 2020, M20, n=10). In 2020, another group of young mares foaled for the first time (primiparous in 2020, P20, n=18). All mares were inseminated with the semen of the same stallion and were fed identically over the study. To assess the effect of primiparity, M20 were compared to P18 (effect on the same mare), or P20 (effect in the same year). Placentas were recovered at foaling, measured, weighed and sampled close to the umbilical cord. Stereology was performed using MercartorPro software on HES-stained paraffin-embedded samples and analyzed using a linear model for paired samples or not and permutations with R software. Paired-end RNA-sequencing was performed (Illumina, NextSeq500) on 6 randomly chosen snap-frozen placentas/group. Differential expression was analyzed (DESeq2) using a false discovery rate (FDR) <0.05 cutoff. Gene Set Enrichment Analysis was performed on differential expressed genes using PANTHER overrepresentation test with GOBP database. Placental weight and surface were reduced and the efficiency (foal weight/placental surface, in kg/m2) was increased in primiparous mares in both comparisons (p<0.05). Volume was not modified by parity between the consecutives gestations but reduced in P20 vs M20 (p<0.05). Only the volume of allantoic stroma was significantly reduced in P18 vs M20 (p<0.05), whereas hemotrophic trophoblast and microcotyledonary vessels’ volume tended to be reduced in P20 vs M20 placentas (p<0.1). In consecutive gestations, 118 genes were differentially expressed in placenta while in the same year, only 2 genes (IL4R and FFAR, interleukin-4 and free fatty acid receptors) were differentially expressed (FDR <0.05). Among the 118 genes differentially expressed between consecutive pregnancies, apelin signaling, angiogenesis and catalytic activity were overrepresented. All genes involved in these pathways were under-expressed in P18 mares. Both the apelin (normalized enrichment score, NES=-2.16, FDR <0.05) and its receptor (NES=-2.48, FDR <0.05) were less expressed in the placenta of P18 vs M20. This is also observed in intrauterine growth retardation in humans. Although placental morphology was lightly affected in both analyses, the inter-individual variability seemed to hide parity effects for transcriptomic analysis. Consecutive gestations, however, demonstrate that the smaller placental and foal size in primiparous mares seems to originate from reduced vascularization.

Endometrial gene expression of DEFB4B, LYZ, and SLPI in biopsy samples of mares resistant or susceptible to endometritis

Endometritis is one of the main causes of subfertility in mares and therefore one of the most common reasons for economic losses in the horse breeding industry. Multiparous older mares are more susceptible to the development of persistent endometritis after mating, artificial insemination, or foaling since aging is associated with an altered systemic immune response and anatomical defects that interfere with physiological uterine clearance (Katila et.al. Animals. 2022; 12:779). In this context, their inability to recover from endometritis can lead to degenerative changes in the endometrium (e.g., glandular degenerative changes and associated peri-glandular and/or diffuse stromal fibrosis) (Canisso et.al. International Journal of Molecular Sciences 2020; 21:1432). This study aimed to evaluate possible variations in endometrial gene expression of the genes Defensin Beta 4B (DEFB4B), Lysozyme (LYZ), and Secretory Leukocyte Peptidase Inhibitor (SLPI) between resistant and susceptible mares. These three genes have already been identified as involved in the equine innate immune response and in the chronic uterine inflammatory response (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). Endometrial biopsy samples were collected from 19 mares. Mares were divided into two groups concerning their age and reproductive history: the "resistant group" (RES) (N=4) consisted of maiden mares 3.00±0.00 years old with endometrial biopsy grade I according to Kenney and Doig classification (1986); the "susceptible group" (SUS) (N=15)was composed of multiparous mares 17.33±3.28 years old, with endometrial biopsy of grade IIA, IIB or III. Total RNA was extracted from each sample and the corresponding cDNAs were prepared by retro-transcription using dedicated commercial kits. The samples were then amplified by qRT-PCR procedure as described by Marth and colleagues (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). The housekeeping gene GADPH was amplified in each sample as internal control and for data normalization. To minimize possible experimental errors, for each sample three technical replicates were performed and the quality of the extracted RNA was evaluated by spectrophotometry and gel electrophoresis. The results showed a statistically significant difference in the expression of the genes LYZ (p=0.0003) and DEFB4B (p=0.0273) which were upregulated in SUS versus RES mares. This suggests that lower gene expression found in young mares are attributable to a more balanced local immunomodulation, with consequent reduction of the inflammatory pathways and slower progression towards the deposition of fibrotic tissue compared to older mares. No differences in the expression of the SLPI gene were found between the two groups (p=0.8854). In conclusion, despite these promising results, larger studies are needed before LYZ and DEFB4B genes could be used as biomarkers to identify the mares susceptible to endometritis.

Effect of mare's age and embryo quality on pregnancy and foaling rate in a clinical ICSI program

There are few studies on the quality of in vitro produced (IVP) equine blastocysts related to the mare age. A retrospective study on the outcome of a clinical equine IVP program using donor mares of different ages and comparing their embryo quality and pregnancy potential is presented. Pregnancy, foaling, and pregnancy loss rates are reported for the transfer of fresh ICSI embryos derived from oocytes collected from mares aged 2 - 26 years old. ICSI blastocysts were classified as: Grade A – D6, 7, or 8, good quality; Grade B – D6, 7, or 8, moderate quality and D9 or D10, good quality; Grade C – D9 or D10, moderate quality (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351). Donor mares were divided on the basis of age: young (2-10y), mid-age (11-17y), and old (18-26y). Differences concerning groups were analyzed by Fisher's Exact Test (significance level of P<0.05). Results are presented in Table 1. For grade A embryos, pregnancy and foaling rates were similar among ages, but old mares showed higher pregnancy loss than young mares. For grade B embryos, old mares showed lower pregnancy rates than other groups. For grade C embryos, no difference in pregnancy, foaling, or pregnancy loss was seeing between ages. Embryo grades in each age interval were also analyzed. For young mares, pregnancy rate for different embryo grades was similar. However, foaling rate of grade A embryos was higher, and pregnancy loss of grade A embryos was lower than grade B. In mid-age mares,grade C embryos showed a lower pregnancy rate than grade A and B (tendency, P=0.09). Similarly, grade A embryos showed a higher foaling rate than the others. In old mares, grade A embryos showed higher pregnancy and foaling rates than grade B embryos. Thus, ICSI program outcomes for pregnancy, foaling, and pregnancy loss are related to the mare age. Special attention must be given to the quality of the embryo since pregnancy and foaling rates may vary within the same embryo grade, due to the mare's age.

Effect of fixative solutions in equine ovarian tissue

Histological processing is a classical methodology used for an efficient assessment of ovarian tissue quality. Although it is still controversial which is the most appropriate fixative solution for equine ovarian tissue, the choice of an appropriate fixative is critical to evaluate the improvement of assisted reproductive techniques for fertility preservation. This study aimed to evaluate immediate histological processing, based on an ethanol 70% fixative solution for a short-time exposure (ST-EtOH, 45 min), compared with two classically used fixative solutions [Carnoy (CAR) and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h) on equine ovarian tissue. The end points evaluated were morphology (normal and abnormal) and classes (primordial and developing) of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters. Ovaries (n = 6) from ovariectomized young mares (2–8 years) were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1,444 histological slides. High heterogeneity in the number of follicles per individual mare was observed. Equine ovarian fragments fixed in both CAR treatments had a greater (P = 0.014) percentage of morphologically normal primordial and total (primordial and developing classes combined) follicles compared toST-EtOH and both PFA treatments. Although the follicular diameter, regardless of follicular class, was smaller (P = 0.001) in both CAR treatments compared to the other treatments, the nuclear diameter and the nucleus-follicle ratio were greater (P = 0.001) in both CAR treatments compared to the other treatments. A lower (P < 0.001) percentage of morphologically normal developing follicles was observed in ST-EtOH compared to all other treatments. Both CAR treatments had greater (P < 0.001) stromal cell densities than the ST-EtOH and PFA 6h fixatives. In conclusion, Carnoy's solution is the recommended fixative, rather than ST-EtOH and PFA fixatives, for studies requiring more reliable outcomes concerning cellular morphology of equine ovarian tissue. The ST-EtOH fixative proved to be an acceptable alternative to fix equine ovarian tissue when a quick histological evaluation is required for small samples prior to more complicated, time-consuming, and expensive techniques. PFA can be a reasonable fixative for morphological evaluation of equine ovarian tissue, offering the advantage of being suitable for immunohistochemistry techniques since Carnoy's solution is not recommended for this purpose. More studies are required to further understand the effects of different fixative solutions on the morphological ultrastructure and morphometry parameters in different cellular populations of equine ovarian tissue.

Correlation between age and levels of heavy metals in mares mammary gland secretions

The content of heavy metals in the secretions of the mare's mammary gland should be tested not only because of the possibility of human consumption of mare's milk but also because of the influence of these elements on the development of suckling in the neonatal period and later in adolescence. The aim of the research was to assess the dependence of heavy metals concentration in the secretions of the mammary gland on the mare. Studies were carried out on 16 English Thoroughbred mares. Mares aged 4 to 19 years were divided into 2 research groups: Group Y - young mares 10 years of age (n = 8) Group O - mares older then10 years of age (n = 8) The experimental material, colostrum were collected before 12 hours after delivery and milk were collected on the 3rd day after foaling. The concentration of metals (Zn, Cu, Ni, Mn, Fe, Pb, Cd) was determined by the atomic absorption spectrometry (AAS apparatus Unicam 929 spectrometer). All elements (Ni, Zn, Cu, Mn, Fe, Cd) analysed in colostrum showed higher concentration in young mares. For Ni and Zn differences were significantly higher in young mares while for Mn and Cd those differences were highly significant. In analysed samples of milk Ni, Zn, Mn, Fe and Cd content were higher in young mares, while Cu content were higher in older mares. We reported highly statistical differences for Ni, Mn, and Fe.

Zoonymic laudative nomination of a woman from the aspect of age in the speech of Pirot

When naming a woman from the aspect of her age, the zoonyms in the speech of Pirot positively refer only to a young person (a young woman and a girl). She is also understood as a person of beautiful appearance, which is why all her names (vaklusana, zvoncarica/svoncarica and zdrebica) also belong to the group of names for a (female) person with certain physical characteristics. The terms that represent the original domain of metaphorization in naming a woman of a certain age are a young mare, goat and sheep, with a specific appearance and function in the herd. The semes that participate in the transfer of nomination from the animals to persons are: ?young?, ?beautiful? and ?fertile?. All three zoonymic names are characterized by an average degree of expressiveness, because they express sympathy, affection towards the quality that characterizes the named persons. Only one name (zdrebica) represents a standard lexeme, while the other two have the status of lexical provincialisms. In addition, the standard name fully reflects the conceptualization of a YOUNG WOMAN as a YOUNG MARE characteristic of a modern Serbian speaker, while the other two deviate from the conceptualization of a WOMAN as a SHEEP/GOAT in contemporary Serbian linguistic culture. From the point of view of the value system of female beauty and female attractiveness, their standard is folklore, embodied in black eyes, black eyebrows and a fair complexion. Physically attractive is also a woman of strong build, capable of giving birth and doing physical labour, which is considered as socially desirable behaviour.

Mature lunar soils from Fe-rich and young mare basalts in the Chang’e-5 regolith samples

Space weathering on airless bodies produces metallic iron (Fe0) particles in the rims of mineral grains, which affect visible and near-infrared spectra and complicate the identification of surface materials. The Chang’e-5 mission provides an opportunity to couple information gained from its returned samples with in situ observations and orbital monitoring to gain insight on the details of space weathering on extremely Fe-rich basalts. By putting together all these data, we could extract a soil maturity index (Is/FeO) at the Chang’e-5 landing site of ~66 ± 3.2, indicative of a formation age for the Xu Guangqi crater, whose ejecta dominate the site, of 240–300 Myr ago. In addition, abundant large Fe0 particles were found in the sample, indicating that both the inherited Fe0 particles from late-stage mare basalts and the dense clustering of oversaturated Fe0 in extremely FeO-rich (>17 wt%) basalts contribute to observed Fe0 abundances. We suggest that space weathering of Fe-richer basalt generates Fe0 particles with a larger grain size and faster production rate.