Serum anti-Müllerian hormone around the time of ovulation simulated by exogenous hormones in clouded leopards (Neofelis nebulosa).

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 (r2 = .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 (rs = -.692, p = .013). However, serum AMH concentrations were not correlated with the average number of ovarian structures (rs = -.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.

A fluorescence-based sweat test sensor in a proof-of-concept clinical study for COVID-19 screening diagnosis.

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.

Equex STM paste improves post-thawed epididymal sperm motility and acrosome integrity in rhesus monkey (Macaca mulatta).

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.

Identification of feline Kiss1 and distribution of immunoreactive kisspeptin in the hypothalamus of the domestic cat.

In recent years, the Kiss1 gene has been reported in a number of vertebrate species, and a substantial dataset has been acquired to demonstrate the critical role of kisspeptins in the reproductive system; yet limited information is available for carnivores. In the present study, we identified and characterized feline Kiss1 by isolating and cloning its full-length cDNA in the domestic cat hypothalamus and caracal testis, using the method of rapid amplification of cDNA ends. Additionally, we isolated and cloned the 3' end of Kiss1 cDNA, containing kisspeptin-10 (Kp10), from the ovaries of a clouded leopard and Siberian tiger. Nucleotide sequencing revealed that domestic cat Kiss1 cDNA is of 711 base pairs and caracal Kiss1 cDNA is of 792 base pairs, both having an open reading frame of 450 base pairs, encoding a precursor protein Kiss1 of 149 amino acids. The core sequence of the feline kisspeptin Kp10 was found to be identical in all species analyzed here and is highly conserved in other vertebrate species. Using an anti-Kp10 antibody, we found the immunoreactive kisspeptin to be localized in the periventricular and infundibular nuclei of the cat hypothalamus. The results show that kisspeptin is highly conserved among different feline families, and its immunoreactive distribution in the hypothalamus may indicate its physiological function in the domestic cat.

Activation of adenosine monophosphate-activated protein kinase (AMPK) enhances energy metabolism, motility, and fertilizing ability of cryopreserved spermatozoa in domestic cat model.

PURPOSE: Increasing intracellular energy storage by chemically activating adenosine monophosphate-activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5'-aminoimidasole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation. METHODS: In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60 min in the presence of 0 (control), 0.5, 2.0, and 5.0 mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels. RESULTS: AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5 mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0 mM AICAR for 30 min. CONCLUSIONS: Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men.

Gonadotropin-releasing hormone-modified chitosan as a safe and efficient gene delivery vector for spermatogonia cells.

The use of male gonadal tissue as a site for the local delivery of DNA is an interesting concept. Previously, we reported synthesis, physiochemical and biological properties of gonadotropin-releasing hormone (GnRH)-conjugated chitosan as a carrier for DNA delivery to GnRH receptor-overexpressing cells. In this study, the application of modified chitosan as a potential vector for gene delivery to testicular cells was carried out. Transfection efficiency was investigated in mouse-derived spermatogonia cells (GC-1 cells) using green fluorescent protein as a reporter gene. GnRH-conjugated chitosan exhibited higher transfection activity and specificity compared to the unmodified chitosan. Furthermore, the GnRH-modified chitosan showed less cytotoxicity. In conclusion, we have developed and successfully tested the GnRH-modified chitosan for delivery of a transgene of interest to spermatogonia cells in vitro. Such vector could be useful in particular for testis-mediated gene transfer.

SUCCESSFUL LAPAROSCOPIC OVIDUCTAL ARTIFICIAL INSEMINATION IN THE CLOUDED LEOPARD (NEOFELIS NEBULOSA) IN THAILAND.

Captive breeding of clouded leopards (Neofelis nebulosa) is challenging because of mating incompatibility, high incidence of teratospermia in males, and inconsistent ovulation patterns in females. Assisted reproductive techniques, therefore, are necessary to overcome these issues and maintain the genetic diversity in the captive population. The objective was to use laparoscopic oviductal artificial insemination (AI) to breed genetically valuable females (n = 4; aged 4.5-5 yr) that were unsuccessfully paired. Fecal hormone metabolites (estrogen and progesterone) were extracted and measured by enzyme immunoassay for monitoring of ovarian activity 45 days before and 65 days after laparoscopic AI. For timed insemination, females were injected with 200 IU equine chorionic gonadotropin and 1,000 IU porcine luteinizing hormone (pLH) at the 82-hr interval. Ovarian assessment was performed by laparoscopy 44 hr after pLH administration. One nulliparous female out of four presented two ovulation sites on each ovary. The single female that had ovulated was inseminated with chilled semen collected from two males (8 × 106 and 2.7 × 106 motile spermatozoa, respectively, in each oviduct). A significant increase in fecal progesterone concentrations was observed after AI with a concentration peak (500 µg/g dry feces) detected on day 24 after pLH injection, which was then sustained for more than 45 days after the pLH injection. The delivery of two cubs occurred on day 92 after pLH. Microsatellite marker analysis determined that both cubs were sired by the same male. This is the first report of a successful oviductal AI in the clouded leopard.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.

PURPOSE: Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation. METHODS: Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density. RESULTS: Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. CONCLUSIONS: Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors.

This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 µg/mL roscovitine or 0.05 µg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.

Antibody fragments targeting the extracellular domain of follicular stimulating hormone receptor for contraception in male dogs and cats.

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 (KD) 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 KD 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.

Serum anti-Müllerian hormone is an indirect predictor of ovarian reserve in domestic cats.

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.

Development of decellularization protocols for female cat reproductive organs.

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.

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus).

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.

Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species.

This study compared the effects of slow and fast freezing of testicular tissue of wild animals collected at post-mortem on testicular structure and testicular sperm. The testes of seven animals that had died in captivity; three felids (jungle cat, lion and leopard), two cervids (rusa deer and fea's muntjac) and two bovids (Sumatran serows) were cryopreserved using slow- and fast-freezing protocols. There were greater reductions in the integrity of the sperm membrane and DNA in tissues cryopreserved using slow freezing compared to fast freezing (membrane integrity reduced by 21.5 ± 12.4% vs. 13.0 ± 6.9%, P = 0.11 and DNA integrity reduced by 22.7 ± 16.3% vs. 6.6 ± 6.3%, P = 0.13). Histologically, there were similar degrees of detachment and shrinkage of the seminiferous tubules whereas, TUNEL assay revealed a tendency towards more apoptotic changes in the intra-tubular cells of tissues frozen using fast freezing compared to slow freezing (P = 0.09). In conclusion, fast freezing tended to cause less damage to testicular sperm but its protective effect on intra-tubular cells was likely compromised. This is the first report of gamete recovery in the wild and of the comparison in various wildlife species, between testicular tissues cryopreserved using different protocols.

Impaired Testicular Function without Altering Testosterone Concentration Using an Anti-Follicular-Stimulating Hormone Receptor (Anti-FSHr) Single-Chain Variable Fragment (scFv) in Long-Tailed Macaques (Macaca fascicularis).

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 (KD) 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 × 106 cells) to day 56 (1.6 × 106 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.

Seasonal Effects on Body Condition and Characteristics of the Estrous Cycle in Captive Asian Elephants (Elephas maximus) in Thailand: A Retrospective Study.

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.

Gas chromatography-flame ionization detector for sweat based COVID-19 screening.

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.

Long-term preservation of chilled canine semen using vitamin C in combination with green tea polyphenol.

Vitamin C and green tea polyphenol are known to have antioxidant effects. The aim of this study was to evaluate the quality of canine semen after preservation with diluents containing vitamin C and polyphenol at 5 degree C for 4 weeks. In experiment 1, we investigated the effects of vitamin C combined with polyphenol supplementation on chilled semen quality. The addition of vitamin C (0.5 or 1 mM) with 0.75 mg per mL polyphenol to semen extender provided significantly higher percentages of sperm motility and viability during cold storage compared to unsupplemented semen. In experiment 2, we determined the optimal working concentration of vitamin C in the semen extender by comparison of a range of concentrations between 0.1 and 20 mM. Supplementation of 0.5 mM vitamin C plus polyphenol yielded the highest percentages of sperm motility and viability; however, there was no beneficial effect on the plasma membrane and acrosomal integrity of the spermatozoa.

Serum Insulin-like Growth Factor-1 Is a Biomarker of Testosterone Production and Intact Acrosome in Asian Elephants (Elephas maximus).

The objective of this study was to find relationships among serum IGF-1, serum testosterone, seminal plasma IGF-1 concentrations and semen parameters in Asian elephants (Elephas maximus). A total of 17 ejaculates (one to three ejaculates/bull) were collected from seven captive elephant bulls by performing rectal massage. Before each ejaculation, blood samples were obtained for serum IGF-1 and testosterone assays. Subsequently, the semen characteristics of each ejaculate were evaluated. Mean serum IGF-1 concentration of elephant bulls was estimated as 326.3 ± 114.6 ng/mL (median, 286.2 ng/mL; range, 167.4-542.7 ng/mL). An increase in serum IGF-1 concentration was found to correlate with the percentage of spermatozoa with intact acrosomes. In addition, IGF-1 concentration was positively correlated with testosterone level. However, seminal IGF-1 concentrations could not be detected. In conclusion, our findings suggest that serum IGF-1 concentration is likely a biomarker of normal testicular functions, particularly spermatogenesis in elephants. Moreover, this commercial IGF-1 ELISA is eligible for analyzing serum IGF-1 concentration in Asian elephants.

Effects of green tea polyphenols and α-tocopherol on the quality of chilled cat spermatozoa and sperm IZUMO1 protein expression during long-term preservation.

Sperm IZUMO1 protein was recently found to be a crucial mediator in the interaction and fusion with eggs, indicating an important role in assuring the favourable outcome from long-term preservation of chilled semen. The purpose of this study was to investigate whether supplementation of chilled semen extender with green tea polyphenols together with α-tocopherol would provide synergistic effects to prolong sperm survival and maintain IZUMO1 protein stability in cat spermatozoa. Sperm samples were collected from the cat epididymis before being diluted with semen extender containing various concentrations of α-tocopherol (0, 2.5, 5 and 7.5 µg/ml) and 0.75 mg/ml green tea polyphenols and cooled to 4 °C. One sample without antioxidants served as a control. Sperm characteristics and IZUMO1 protein expression were investigated before and after chilling at 3, 6, 9, 12 and 15 days. Using α-tocopherol at 5 µg/ml together with 0.75 mg/ml green tea polyphenols in the semen extender is the most suitable condition to retain the sperm characteristics up to nine days of preservation. Cat IZUMO1 proteins, 17 kDa, were identified at the equatorial segment of acrosome reacted sperm. Without antioxidant, cold storage can gradually degrade the IZUMO1 protein level. Sperm IZUMO1 protein was markedly conserved by supplementation of 5 µg/ml α-tocopherol together with 0.75 mg/ml green tea polyphenols up to 12 days in cold storage. These findings indicate that green tea polyphenols and α-tocopherol have protective effects on the preservation of sperm characteristics and IZUMO1 protein integrity of cat epididymal sperm during long-term chilling.