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Contents Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization ( IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection ( ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7 days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7 days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2 hr after a jungle cat ( Felis chaus) aged 10 years died during anaesthesia plus another necropsy-to-sperm recovery time of 25 hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation. [ABSTRACT FROM AUTHOR]

Contents The Indochinese leopard ( Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization ( IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5′-triphosphate ( ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 106 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process ( p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ ( p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced ( p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) ( p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased ( p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development). [ABSTRACT FROM AUTHOR]

Contents Clouded leopards ( Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n = 9) were allocated to simple washing (control; resulting in 11.7% ± 1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9% ± 0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution ( HOS, 60 mOsm fructose solution) containing 5 mM dithiothreitol ( DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 ( TX) (a detergent) was added to the HOS/ DTT-treated samples. After HOS/ DTT incubation, the control samples and sperm-selected samples presented 73.4% ± 3.1% and 73.9% ± 2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8% ± 3.8% and 26.5% ± 3.4%; p = .1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6% ± 10.4% and 21.1% ± 7.9%; p = .06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation. [ABSTRACT FROM AUTHOR]

Cervix is an important part of the reproductive tract; in non-pregnant animals it remains closed during anestrus and diestrus and is open only during estrus. In pathological conditions like pyometra, the cervix may be open or closed but the control mechanism is not clearly known. Prostaglandin E2 (PGE2) is considered to be involved in changes of extracellular matrix via coupling to prostaglandin E receptor subtype 4 (EP4). This study investigated the expression of EP2 in the cervices of bitches during different stages of estrous cycle and those with pyometra. After ovariohysterectomy, cervices were collected from anestrus (n=6), estrus (n=12) and diestrus (n=6), open- (n=10) and closed-cervix pyometra (n=10) bitches. Cervical EP4 expression was observed at all the layers and the stages but the differences in EP4 expression either among bitches in different stages of the estrous cycle and between open- and closed-cervix pyometra were limited to only surface epithelium (SE). In cyclic bitches during estrus and in open-cervix pyometra bitches, significantly higher (P<0.05) EP2 expression was found in SE of uterine part than vaginal part. In SE of the uterine part, the expression was higher in the bitches during estrus than in anestrus and diestrus, and in the bitches affected by open-cervix than those with closed-cervix pyometra. The results suggest that regulation of cervical dilation appeared in the uterine part of the cervix. Moreover, EP2 may be involved in stimulating dilation of the cervix in both estrus and open- cervix pyometra bitches. [ABSTRACT FROM AUTHOR]

Contents This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 μ m). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 μ m in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future. [ABSTRACT FROM AUTHOR]

Contents Cryopreservation of testicular tissue has become a part of gamete preservation in wild animal post-mortem. Using domestic cats as a model for wild felids, this study aimed to (i) investigate the effect of temperature for testicular tissue storage on sperm quality; (ii) compare efficiency of freezing protocols; and (iii) evaluate properties of cryoprotective agents to protect testicular sperm quality. A pair of testes from each cat (n = 9) was cut into four pieces. Three randomly selected pieces were allocated to be (i) fresh controls; (ii) stored at 4°C for 24 h; and (iii) stored at room temperature (28°C) for 24 h. After storage, the testicular tissue from each group was cut into 10 small pieces. One piece was assigned to be a control while the others were assigned to three freezing protocols; −80°C (n = 3), vitrification (n = 3) or two-step freezing (kept above liquid nitrogen vapour for 10 min and submerged in liquid nitrogen) (n = 3). Each of three pieces was frozen using dimethyl sulphoxide (DMSO), ethylene glycol (EG) or DMSO combined with EG. Sperm membrane (SYBR-14/EthD-1) and DNA (acridine orange) integrity were evaluated before and after cryopreservation. The storage of testicular tissue at room temperature decreased the percentage of sperm with intact membrane in fresh tissue (59.5 ± 30.5 vs 87.9 ± 7.0%, p < 0.05). DNA integrity was decreased after 24-h storage either at 4°C or room temperature (p < 0.05). The two-step freezing resulted in a higher percentage of sperm with intact plasma membrane than the other techniques. Dimethyl sulphoxide, EG and DMSO combined with EG provided similar protection for the sperm membrane and DNA from cryodamages. In conclusion, storage of testicular tissue at 4°C is necessary to maintain sperm membrane integrity during transportation of tissue for cryopreservation in the freezing laboratory. The results provide information for male gamete rescue in felid particularly when they die unexpectedly in the field where freezing facilities are not well equipped. [ABSTRACT FROM AUTHOR]

Contents Lipid peroxidation is considered to be an important cause of sperm membrane damage, resulting in an apparent reduction of reproductive fecundity. Recently, a new lipophilic fluorescent dye probe (BODIPY581/591 C11; Invitrogen Singapore Pte Ltd, Singapore) has been demonstrated to be a highly sensitive indicator for the physiological oxidation of cell membrane fatty acids. The objectives of this study were: (i) to detect lipid peroxidation in frozen-thawed epididymal cat spermatozoa using the BODIPY581/591 C11 and (ii) to study the effect of semen extender in protecting sperm membrane from lipid peroxidation [100-mm ferrous ion, ferrous sulphate (FeSO4)]. Epididymal cat spermatozoa were collected from eight male cats. Two straws of sperm sample from each cat were cryopreserved. After thawing, the semen extender from the first straw was removed and the sperm pellet was resuspended with Tris buffer (control). The semen sample from the other straw was equally divided: one sample contained semen extender (treatment A) and one contained no extender (treatment B); both were incubated with FeSO4. Semen samples were labelled with the BODIPY581/591 C11 probe and evaluated by flow cytometry at 0 and 6 h after thawing (control), 6 h after the addition of FeSO4 (treatment A), and 30 min and 6 h after the addition of FeSO4 (treatment B), respectively. The percentage of lipid peroxidation was higher after treatment B (51.3 ± 23.9) and 6-h incubation compared with the control and treatment A (p < 0.05). Furthermore, the percentage of lipid peroxidation after treatment B increased during the incubation time (p < 0.05). In conclusion, the high percentage of lipid peroxidation after treatment B indicated that FeSO4 induced membrane damage in cat spermatozoa, which could be detected by BODIPY581/591 C11. This marker is suggested to be a beneficial tool for the evaluation of lipid peroxidation. Furthermore, the use of semen extender seemed to protect cat spermatozoa membranes from lipid peroxidation. [ABSTRACT FROM AUTHOR]

The objectives of the present study were to evaluate sperm characteristic of captive clouded leopards in Thailand and examine the structural and functional properties of sperm after selection with the single-layer centrifugation (SLC) method. Twenty-two ejaculates from 11 captive clouded leopards (four housed with access to a female in estrus, and seven housed singly) were collected and assessed for semen traits during 2013 to 2015. Twelve fresh ejaculates were chosen from seven males, and each was divided between two sperm preparation methods; (1) simple washing and (2) SLC. Cryopreservation was performed after semen preparation. Sperm qualities after selections including motility, progressive motility, sperm motility index, viability, acrosome integrity, DNA integrity, and morphology were evaluated in fresh, chilled, and frozen-thawed samples. In addition, sperm functionality after cryopreservation was tested by heterologous IVF using domestic cat oocytes. Sperm motility in the ejaculates was 52.5% to 91.3% (76.8 ± 2.0%, mean ± standard error). A high proportion of morphologically abnormal sperm (63.9 ± 2.0%) was observed, with the major abnormality being tightly coiled tail (13.5 ± 0.5%). An interesting observation was that males housed together with a female had a significantly higher proportion of sperm with intact acrosome (47.9 ± 3.4% and 38.4 ± 2.8%) and lower proportion of sperm with bent midpiece and droplet (7.1 ± 0.6% and 10.2 ± 0.5%) than the males living singly. The sperm motility index, intact acrosome, and sperm with normal tail in the fresh and chilled semen samples were improved by the SLC. In the postthawed semen, the SLC selected higher numbers of viable sperm (34.1 ± 2.2% and 27.9 ± 1.8%), sperm with intact acrosome (31.2 ± 2.1% and 24.3 ± 2.2%), and sperm with normal tail (34.2 ± 2.7% and 24.3 ± 2.7%) than simple washing. Also, the proportion of sperm with tightly coiled tail was lower in the SLC-processed than the simple washed samples (8.1 ± 3.1% and 13.5 ± 3.4%). The SLC-processed group had significantly higher penetration rate in heterologous IVF (29.4 ± 3.0%) than the simple washing group (15.8 ± 3.2%). In conclusion, ejaculates of clouded leopards living in Thailand demonstrated teratospermic characteristic similar to the previous reports from other continents. Single-layer centrifugation is a promising tool to select morphologically normal sperm of teratospermic donors. The successes of assisted reproductive technology could be enhanced by the improved quality of postthaw sperm in this species. [ABSTRACT FROM AUTHOR]

Anti-Müllerian hormone (AMH) serves as an indirect marker for predicting primordial follicles that are representative of ovarian reserve. In this study the possibility of using AMH and age to predict the ovarian reserve in domestic cats. Ovaries and blood were collected from 30 cats undergoing routine ovariohysterectomy. The animals were divided into three age groups: prepubertal (<4 mo, n = 10), adult (1-5 y, n = 10), and senior (>5 y, n = 10). Blood was collected at surgery for serum AMH measurements using the AMH Gen II ELISA kit. The intra-assay coefficient of variation (CV) and inter-assay CV were 3.56 % and 7.68 %, respectively. One side of the ovary was processed to determine AMH localization using immunohistochemistry and for a histological count of follicles, which is the gold standard. The expression of AMH protein was quantified from the contralateral ovary by Western blot analysis. Primordial follicles exhibited the most pronounced inverse relationship with age (rho = -0.779, P < 0.05), followed by a positive association with serum AMH concentration (rho = 0.490, P < 0.05), indicating that both age and AMH are potential markers indicative of primordial follicles. Furthermore, secondary (rho = 0.651, P < 0.05) and small antral follicles (rho = 0.648, P < 0.05) were identified as the major sources of circulating AMH, as indicated by the stronger correlation with serum AMH concentrations compared with primary follicles. However, there was no significant correlation between the expression of AMH protein and other factors, including age, primordial follicles, primary follicles, secondary follicles, small antral follicles, and serum AMH concentration. A model for predicting primordial follicle number using serum AMH concentration (AIC = 672.66, P < 0.05) and age (AIC = 668.93, P < 0.05) was established. In conclusion, both serum AMH concentration and age may serve as comparable markers of ovarian reserve in domestic cats. Moreover, AMH is particularly useful in situations where age information is not available., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The increased LH levels resulting from the absence of negative feedback after castration has been linked to long-term health issues. A need exists for an alternative contraceptive agent that functions without interfering the LH pathways. This study aimed to develop antibody fragments against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and evaluate its effects on Sertoli cell functions. Phage clones against the extracellular domain of dog and cat FSHr selected from an antibody fragment phagemid library were analyzed for binding kinetics by surface plasmon resonance. Sertoli cells were isolated from testes of adult animals (five dogs and five cats). Efficacy test was performed by treating Sertoli cell cultures (SCCs) with anti-FSHr antibody fragments compared with untreated in triplicates. Expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB) and vascular endothelial growth factor A (VEGFA) mRNA in SCCs were quantified by RT-qPCR. The results demonstrated that the molecular weight of the purified dog and cat anti-FSHr antibody fragment was 25 kDa and 15 kDa, respectively. Based on protein molecular weight, the antibody fragment of dogs and cats was therefore, so-called single-chain variable fragments (scFv) and nanobody (nb), respectively. The binding affinity with dissociation constant (K D ) was 2.32 × 10 -7  M and 2.83 × 10 -9  M for dog and cat anti-FSHr antibody fragments, respectively. The cross-binding kinetic interactions between the dog anti-FSHr scFv and the cat ECD of FSHr could not be fitted to the curves to determine the binding kinetics. However, the cross-binding affinity K D between the cat anti-FSHr nb and the dog ECD FSHr was 1.75 × 10 -4  M. The mRNA expression of ABP, IHBB and VEGFA in SCCs was less (P < 0.05) in both dogs (12.26, 4.07 and 5.11 folds, respectively) and cats (39.53, 14.07 and 20.29 folds, respectively) treated with anti-FSHr antibody fragments, indicating the Sertoli cell functions were suppressed. In conclusion, this study demonstrated the establishment of species-specific antibody fragments against FSHr in SCCs for dogs and cats. The fragment proteins illustrate potential to be developed as non-surgical contraceptive agent targeting FSHr in companion animals., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study established a novel infield sensing approach based on detection of the volatile compound markers in skin secretions. This was based on analysis of volatile compounds in axillary sweat samples collected from RT-PCR-proven Coronavirus disease 2019 (COVID-19) positive and negative populations using gas chromatography-mass spectrometry (GC-MS). The analysis proposed the possible markers of the monoaromatic compounds and ethyl hexyl acrylate. A portable photo ionization detector (PID) incorporated with the selective material towards the marker compounds was then developed with the pressurized injection approach. This provided the accuracy of 100% in the research phase (n = 125). The developed approach was then applied for screening of 2207 COVID-19 related cases covering the periods of the Alpha, Beta, Delta and Omicron variants of SARS-CoV-2 infection in Bangkok, Thailand. This offered the sensitivity, specificity and accuracy ranges of 92-99, 93-98 and 95-97%, respectively., (© 2024. Crown.)

The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis.

Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2-cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns., Competing Interests: Declaration of competing interest None, (Copyright © 2023. Published by Elsevier Ltd.)

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Anti-Müllerian hormone (AMH) is produced by granulosa cells of the antral follicles. It serves as a promising biomarker for ovarian reserve and responsiveness to ovarian stimulation in humans and domestic animals. This study aimed to validate the AMH Gen II enzyme-linked immunosorbent assay (ELISA) and correlate ovarian structures with serum AMH concentrations after stimulation treatment in clouded leopards (Neofelis nebulosa). Serum samples were collected from 12 women (age 6.21 ± 3.56 years), and serum AMH concentrations were analysed using AMH Gen II ELISA. The animals were divided into two groups based on ovarian structures [preovulatory follicles (>2 mm) and/or corpora hemorrhagica] along with the presence of uterine tonicity visualized laparoscopically around the time of ovulation. Animals that exhibited these reproductive features were identified as the responder group (n = 9, aged 7.59 ± 2.96 years), whereas those lacking the corresponding features were assigned to the nonresponder group (n = 3, aged 2.06 ± 0.53 years). The intra-assay coefficient of variation (CV) and interassay CV was 3.56% and 7.75%, respectively. The linearity of AMH dilution was confirmed (r 2  = .998), and the percentage of recovery ranged from 93% to 115%. The results demonstrated that overall serum AMH concentrations around the time of ovulation were negatively correlated with age (r s  = -.692, p = .013). However, serum AMH concentrations were not correlated with the average number of ovarian structures (r s  = -.535, p = .074). Thus, AMH Gen II ELISA was validated in clouded leopards. Around the time of ovulation, serum AMH decreased with advancing age and ovarian responsiveness cannot be evaluated using serum AMH., (© 2024 Wiley-VCH GmbH.)

Simple approach for rapid screening of corona virus disease 2019 (COVID-19) has been developed. This applied gas chromatography-flame ionization detector (GC-FID) analyzing the potential compound marker in sweat samples obtained from COVID-19 positive and negative volunteers in Bangkok, Thailand. The samples were collected by using cotton rods for 15 min, heated at 90 °C for 5 min, and the volatile compounds in the headspace (HS) were injected (5.00 mL) at 150 °C and separated within 13.7 min. The marker peak was tentatively identified as p-cymene by the authentic standard injection and comparison with the GC-mass spectrometry (GC-MS) and comprehensive two-dimensional GC (GC × GC)-MS analysis. Possible mechanisms for the presence of p-cymene were proposed. The marker peak area thresholds were then varied and optimized via construction of the receiver operating characteristic (ROC) curve. With the optimum threshold, the established method offered the accuracy, sensitivity and specificity of 96 %. This method was insignificantly affected (p-value >0.05) by genders, body mass indices, ages, and use of deodorants as well as the p-cymene containing food. However, the performance could be affected by the population with personal hygiene or experiencing the microbiomes producing p-cymene., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Chadin Kulsing reports equipment, or supplies was provided by Second Century Fund (C2F), Chulalongkorn University. The 90th anniversary of Chulalongkorn University fund (Ratchadaphiseksomphot Endowment Fund) and Researcher assistant fellowship of Chulalongkorn University. Chadin Kulsing reports a relationship with Second Century Fund (C2F), Chulalongkorn University. That includes: funding grants., (Copyright © 2023 Elsevier B.V. All rights reserved.)

FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques ( M. fascicularis ) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein ( ABP ), inhibin subunit beta B ( IHBB ), and vascular endothelial growth factor A ( VEGFA ). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (K D ) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 ( p < 0.05). Total sperm number was reduced from day 0 (36.4 × 10 6 cells) to day 56 (1.6 × 10 6 cells) ( p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) ( p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively ( p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls ( p < 0.05). On day 56, the expression of IHBB was less ( p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period ( p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques.

During the corona virus disease 2019 (COVID-19) pandemic period, rapid screening of covid-19 patients has been of great interest by developing a fluorescent sensor for complexation with nonanal, which is a marker for Covid-19 detection in sweat. Solid phase micro-extraction gas chromatography-mass spectrometry (SPME GC-MS) was initially used to quantify nonanal in armpit sweat samples based on an external calibration curve. A sample containing a nonanal content above the threshold of 1.04 μL is expected to be COVID-19 positive with a sensitivity and specificity of 87% and 89%, respectively, validated by comparison with RT-PCR results. For more practical applications, helicene dye-encapsulated ethyl cellulose, namely EC@dyeNH, was applied to screen 140 sweat samples collected from the foreheads of volunteers. The mixed sensor and sweat solution droplets were then visualized and imaged under blacklight. The COVID-19 positive droplets exhibited yellow fluorescence emission, the brightness of which could be measured by using ImageJ in the grey scale. With the optimum color intensity of >73 for positive results, the screening performance was observed with a sensitivity and specificity of 96% and 93%, respectively. The overall test time of this method is approximately less than 15 min. This alternative method offers a promising practical screening approach for the diagnosis of COVID-19 in sweat.

The aim of this study was to investigate the effects of season on the body condition score (BCS), the characteristics of the estrous cycle (luteal phase [LPL], follicular phase [FPL], estrous cycle [ECL] lengths, and the start of the luteal phase [SLP] and follicular phase [SFP]), and progesterone levels (baseline and peak) of eight captive Asian elephants ( Elephas maximus ) in Thailand. From 2014 to 2019, blood samples were collected weekly for serum progesterone enzyme immunoassays (EIAs). Estrous cycles ( n = 70), including the luteal and follicular phases, and BCS ( n = 70) were recorded. Based on the BCS, the LPL, FPL, and ECL were assigned to the following two groups: normal (BCS = 3.0-4.0, n = 38) and overweight (BCS = 4.5-5.0, n = 32). The findings demonstrated that there was no difference in LPL between the groups. However, in the normal group, the ECL was one week longer (14.9 ± 1.7 vs. 13.9 ± 1.7 weeks; p < 0.05), and the FPL also tended to be one week longer (7.2 ± 1.7 vs. 6.4 ± 1.5 weeks; p = 0.06) than in the overweight group. The mean progesterone level during the rainy, hot, and cool seasons was not statistically different. Based on the yearly averaged BCS from three seasons, the baseline and peak levels of progesterone were classified into the normal ( n = 16) and overweight ( n = 12) groups. Females with a normal BCS tended to exhibit higher progesterone peak levels ( p = 0.08). The majority of peaks appeared during the rainy season (53.57%). The BCS was highest during the hot (4.47) and rainy (4.38) seasons, but not during the cool (4.12) season. The LPL, FPL, and ECL were not affected by the season in which the luteal phase occurred. On the other hand, the rainy season had a significant effect on the SFP, resulting in a longer LPL ( p < 0.05) and ECL ( p = 0.01); both were the longest during the rainy season. In conclusion, the effects of season on BCS may be related to characteristics of the estrous cycle and peak progesterone levels. Ultimately, these findings provide ground knowledge to assist elephant managers and owners in planning breeding activities using seasonal effects and BCS measurements in tropical climates.

Equex STM paste, a water-soluble detergent, exerts the protective effect of egg-yolk during sperm cryopreservation. This study aims to evaluate the post-thaw quality of rhesus monkeys' epididymal spermatozoa in the Tris-citric-glucose egg-yolk extender, supplemented with or without Equex STM paste (0.5%, v/v) (n = 6). Sperm motility, progressive motility, motion characteristics, viability, acrosome integrity and mitochondrial activity were compared immediately post-thaw. Equex STM paste supplementation significantly improved sperm motility (35.0 ± 4.5 vs. 23.7 ± 5.0%), progressive motility (15.4 ± 2.1 vs. 9.8 ± 2.7%) and percentage of sperm with intact acrosome (30.4 ± 4.5 vs. 26.3 ± 4.6%) compared to the controls, respectively. This is the first report applying Equex STM paste for monkey epididymal sperm cryopreservation and is expected to be beneficial as a model for endangered non-human primates., (© 2022 Wiley-VCH GmbH.)