A caffeine pre-treatment and sole effect of bone-marrow mesenchymal stem cells-derived conditioned media on hyperglycemia-suppressed fertilization.

As a common metabolic disorder, hyperglycemia (HG) affects and disrupts the physiology of various systems in the body. Transplantation of mesenchymal stem cells (MSCs) has been used to control the complications of disease. Most of the therapeutic properties of MSCs are attributed to their secretome. This study aimed to investigate the effects of conditioned media extracted from sole or caffeine pre-treated bone-marrow-derived MSCs on hyperglycemia-induced detrimental impact on some aspects of reproduction. The HG was induced by intraperitoneally injection of streptozotocin (65 mg/kg) and nicotinamide (110 mg/kg). Twenty-four male Wistar rats (190 ± 20 g) were divided into control, HG, and the hyperglycemic groups receiving conditioned media of proliferated MSCs solely (CM) or MSCs pre-treated with caffeine (CCM). During the 49-day treatment, body weight and blood glucose were measured weekly. Finally, HbA1c, spermatogenesis development, sperm count, morphology, viability, motility, chromatin condensation, and DNA integrity were examined. Also, testicular total antioxidant capacity (TAC), malondialdehyde, sperm fertilization potential, and pre-implantation embryo development were evaluated. A one-way ANOVA and Tukey's post-hoc tests were used to analyze the quantitative data. The p < 0.05 was considered statistically significant. The CM and with a higher efficiency, the CCM remarkably (p < 0.05) improved body weight and HG-suppressed spermatogenesis, enhanced sperm parameters, chromatin condensation, DNA integrity, and TAC, reduced HbA1c, sperm abnormalities, and malondialdehyde, and significantly improved pre-implantation embryo development versus HG group. The conditioned media of MSCs solely (CM) and more effectively after pre-treatment of MSCs with caffeine (CCM) could improve spermatogenesis development, sperm quality, pre-implantation embryo development, and testicular global antioxidant potential during hyperglycemia.

Response to hormonal treatment and conception rates of Sahiwal cows subjected to fixed time artificial insemination in pastoral dairy systems.

This study aimed at determining factors influencing response of Sahiwal cows/heifers to fixed time artificial insemination protocol in pastoral systems in Kenya. Available cows/heifers were inspected for conformity to Sahiwal breed characteristics, parity, body condition score, and subsequently rectal palpation to determine pregnancy status, ovarian structures, and estimated ovarian diameter. Consequently, these animals were injected with 100 µg of gonadotrophin-releasing hormone. On days 7 and 9, only responsive cows/heifers were injected with 500 µg of cloprostenol and 100 µg of gonadorelin Acetate, respectively. On day 10, animals were inseminated and separated from bulls for 45 days and pregnancy diagnosis done after 90 days. Analysis of variance was performed to determine the effects of production system, parity, and ovarian structures on ovary diameters pre- and post-hormonal treatment. Logistic regression was used fitting a logit function to account for the binomial distribution of conception. Overall, 56.2%, 23.1%, and 20.7% of the animals had follicles (F), corpus luteum (CL), and corpus albicans (CA), respectively, at day 0, and 16.6%, 68.6%, and 14.8%, respectively, at day 7. Human and environmental factors had no influence on conception. Among the animal factors, only the ovarian structures at day 7 had a significant effect on conception. Ovaries with CL at this time were about 6 times significantly more likely to conceive than those with F. For higher conception rates, animals with ovaries with CL should be recruited into the FTAI program as they are significantly more likely to conceive than those with other ovarian structures.

Conservação de peças anatômicas em cloreto de sódio: um estudo com produto de fecundação / Conservation of anatomical parts in sodium chloride: a study with fertilization product / Conservación de partes anatómicas en cloruro sódico: un estudio con producto de fertilización

Introdução: Os materiais de origem humana geralmente são conservados em formaldeído, para possibilitar o estudo da anatomia humana, tal conservante possui baixo custo e boa fixação, contudo é toxico. Diante do exposto é necessário, o estudo de outros métodos de conservação, menos prejudiciais, como a solução de NaCl 30%. Objetivo: Comparar a conservação de peças anatômicas em solução de NaCl à 30% e formaldeído a 10%. Método: Pesquisa experimental, exploratória e descritiva, realizada com dois produtos de abortamento, no laboratório de anatomia de uma universidade pública, no estado do Paraná/BR. Foi realizada fixação em solução de formol 10%, em seguida uma amostra foi lavado em água corrente e armazenado em solução de NaCl à 30%. Após 6 meses da conservação em solução salina, foram coletadas amostras, estas foram submetidas a análise de crescimento bacteriano. Avaliou-se tonalidade e turgor cutâneo, odor e peso, bem como crescimento bacteriano. O estudo seguiu os preceitos éticos (CAAE: 53740121.9.0000.9247). Resultados: Foram realizadas observações após 24h, 7, 30, 60, 90 e 180 dias. O feto em solução de NaCl não possui odor, e diminuição do turgor da pele. Ambas a amostras não apresentaram crescimento bacteriano. Considerações finais: A solução de NaCl a 30% desidrata a pele, mas não altera significativamente a forma e estrutura, ainda não possui odor e nem toxicidade, o que garante benefícios a saúde de quem os manipula, bem como tal concentração de NaCl inibe de forma efetiva o crescimento bacteriano nos tecidos e na própria solução, se demostrando eficaz na conservação.

Introduction: The materials of human origin are usually preserved in formaldehyde, to enable the study of human anatomy, this preservative has low cost and good fixation, however it is toxic. Therefore, it is necessary to study other less harmful preservation methods, such as 30% NaCl solution. Objective: To compare the preservation of anatomical specimens in 30% NaCl solution and 10% formaldehyde solution. Method: Experimental, exploratory and descriptive research, carried out with two abortion products, in the anatomy laboratory of a public university, in the state of Paraná/BR. Fixation in 10% formaldehyde solution was performed, after which a sample was washed in running water and stored in a 30% NaCl solution. After 6 months of preservation in saline solution, samples were collected and submitted to bacterial growth analysis. Skin tone and turgor, odor, weight, and bacterial growth were evaluated. The study followed the ethical precepts (CAAE: 53740121.9.0000.9247). Results: Observations were made after 24h, 7, 30, 60, 90 and 180 days. The fetus in NaCl solution had no odor, and decreased skin turgor. Both samples showed no bacterial growth. Final considerations: The 30% NaCl solution dehydrates the skin, but does not alter significantly the shape and structure, and also has no odor or toxicity, which guarantees health benefits to those who handle them, and such concentration of NaCl inhibits effectively the bacterial growth in the tissues and in the solution itself, proving to be effective in conservation.

Introducción: Los materiales de origen humano suelen conservarse en formol, para posibilitar el estudio de la anatomía humana, este conservante tiene bajo coste y buena fijación, sin embargo es tóxico. Por ello, es necesario estudiar otros métodos de conservación menos nocivos, como la solución de NaCl al 30%. Objetivo: Comparar la conservación de especímenes anatómicos en solución de NaCl al 30% y en solución de formaldehído al 10%. Método: Investigación experimental, exploratoria y descriptiva, realizada con dos abortos, en el laboratorio de anatomía de una universidad pública, en el estado de Paraná/BR. Fue realizada fijación en solución de formaldehído al 10%, después de lo cual la muestra fue lavada en agua corriente y almacenada en solución de NaCl al 30%. Tras 6 meses de conservación en solución salina, se recogieron las muestras y se sometieron a análisis de crecimiento bacteriano. Se evaluaron el tono y la turgencia de la piel, el olor, el peso y el crecimiento bacteriano. El estudio siguió los preceptos éticos (CAAE: 53740121.9.0000.9247). Resultados: Las observaciones se realizaron después de 24h, 7, 30, 60, 90 y 180 días. El feto en solución de NaCl no tenía olor, y la turgencia de la piel disminuyó. Ambas muestras no mostraron crecimiento bacteriano. Consideraciones finales: La solución de NaCl al 30% deshidrata la piel, pero no altera significativamente la forma y estructura, además no tiene olor ni toxicidad, lo que garantiza beneficios para la salud de quienes los manipulan, y dicha concentración de NaCl inhibe eficazmente el crecimiento bacteriano en los tejidos y en la propia solución, demostrando ser eficaz en la conservación.

Modulatory effects of R10 fraction of garlic (Allium sativum L.) on hormonal levels, T cell polarization, and fertility-related genes in mice model of polycystic ovarian syndrome.

Polycystic ovary syndrome (PCOS) is an inflammatory endocrine-metabolic disorder related to reproductive system characterized by polycystic ovarian morphology, androgen excess, and chronic anovulation. Current treatments haven't been very successful in PCOS treatment and the problem still remains as a challenge. Therefore, new approaches should be applied to overcome the disease. Previous studies demonstrated immunomodulatory effects of R10 fraction of garlic in the treatment of inflammatory conditions such as cancer. Considering previous studies suggesting immunomodulatory therapy for PCOS, therapeutic effects of R10 fraction was evaluated in a mouse model of PCOS. To do so, PCOS was developed by intramuscular injection of estradiol valerate. Treatment with R10 fraction, isolated from garlic, was performed and the alterations in hormonal levels (estradiol, progesterone, and testosterone), T cell polarization markers (IFN-γ, IL-4, and IL-17), and expression of fertility-related genes (Gpx3 and Ptx3) were evaluated. The results showed that hormonal levels were elevated in PCOS model comparing to normal animals but were markedly modulated after treatment with R10 fraction. Moreover, a severe disturbance in T cell polarization with a significant reduction of fertility-related genes expression were detected in PCOS-induced ovaries. Treatment with R10 fraction also represented modulatory effects on T cell polarization by increasing IL-4 and decreasing IL-17 and IFN-γ levels. Accordingly, fertility-related genes were also modulated following treatment with R10 fraction in PCOS. Our study elucidated that R10 fraction of garlic possess immunomodulatory effects alleviating PCOS symptoms. This approach could be adjusted to give rise the optimum therapeutic results and considered as a candidate therapeutic approach for PCOS.

Potassium Fertilization Stimulates Sucrose-to-Starch Conversion and Root Formation in Sweet Potato (Ipomoea batatas (L.) Lam.).

A field experiment was established to study sweet potato growth, starch dynamic accumulation, key enzymes and gene transcription in the sucrose-to-starch conversion and their relationships under six K2O rates using Ningzishu 1 (sensitive to low-K) and Xushu 32 (tolerant to low-K). The results indicated that K application significantly improved the biomass accumulation of plant and storage root, although treatments at high levels of K, i.e., 300-375 kg K2O ha-1, significantly decreased plant biomass and storage root yield. Compared with the no-K treatment, K application enhanced the biomass accumulation of plant and storage root by 3-47% and 13-45%, respectively, through promoting the biomass accumulation rate. Additionally, K application also enhanced the photosynthetic capacity of sweet potato. In this study, low stomatal conductance and net photosynthetic rate (Pn) accompanied with decreased intercellular CO2 concentration were observed in the no-K treatment at 35 DAT, indicating that Pn was reduced mainly due to stomatal limitation; at 55 DAT, reduced Pn in the no-K treatment was caused by non-stomatal factors. Compared with the no-K treatment, the content of sucrose, amylose and amylopectin decreased by 9-34%, 9-23% and 6-19%, respectively, but starch accumulation increased by 11-21% under K supply. The activities of sucrose synthetase (SuSy), adenosine-diphosphate-glucose pyrophosphorylase (AGPase), starch synthase (SSS) and the transcription of Susy, AGP, SSS34 and SSS67 were enhanced by K application and had positive relationships with starch accumulation. Therefore, K application promoted starch accumulation and storage root yield through regulating the activities and genes transcription of SuSy, AGPase and SSS in the sucrose-to-starch conversion.

Amelioration of sperm fertilizability, thyroid activity, oxidative stress, and inflammatory cytokines in rabbit bucks treated with phytogenic extracts.

This study investigated the beneficial effect of phytogenic extracts on semen quality, reproductive hormones, thyroid activity, immunity, hepatic antioxidant activity, and fertility in rabbit bucks. We divided 70 bucks into seven groups (10 in each). Group 1 was fed a basal diet (control); groups 2, 3, and 4 were fed the control diet with 30, 60, and 90 mg/kg of turmeric, respectively; and groups 5, 6, and 7 were fed the control diet with 50, 75, and 100 mg/kg of garlic extract, respectively, for 8 weeks. Rectal and skin temperatures decreased, while follicle-stimulating hormone, luteinizing hormone, triiodothyronine, thyroxine, testosterone, immunoglobulin M, tumor necrosis factor-alpha, and interleukin-6 in blood serum and glutathione peroxidase in the liver increased in all groups (p < .05). Garlic extract (100 mg/kg diet) increased adenosine triphosphate and glutathione in the liver tissues. All treatments significantly increased net semen volume, percentages of progressive motility, livability, curled tail, and intact acrosomes of spermatozoa, sperm cell concentration, and outputs of total and motile spermatozoa, while significantly decreased percentage of sperm abnormality. In conclusion, dietary supplementation of turmeric or garlic extract can be used as a suitable tool for enhancing the hepatic antioxidant activity, immunity, and semen quality in rabbit bucks.

Effects of phthalates on the functions and fertility of mouse spermatozoa.

Phthalates are common environmental pollutants that are presumed to negatively impact male fertility including animals and humans. Particularly, these potential xenoestrogens may alter male fertility by binding to specific sperm receptors. Although several studies have characterized the toxic effects of single phthalates, epidemiological studies indicate that humans are typically exposed to phthalate mixtures. Here, we tested an environmental-related phthalate combination composed of 21 % di(2-ethylhexyl) phthalate, 15 % diisononyl phthalate, 8% diisobutyl phthalate, 15 % dibutyl phthalate, 35 % diethyl phthalate, and 5% benzylbutyl phthalate. Specifically, the effects of short-term exposure (90 min) to various concentrations (1, 10, 100, and 500 µg/mL) of this phthalate mixture on several important sperm processes, oocyte fertilization, and embryo production were assessed. All phthalate concentrations significantly decreased sperm motility and hyperactivity by compromising the sperm's ability to generate ATP. Additionally, short-term phthalate exposure (>10 µg/mL) also induced abnormal capacitation and the acrosome reaction by upregulating protein tyrosine phosphorylation via a protein kinase-A-dependent pathway. Furthermore, phthalate exposure (particularly at doses exceeding 10 µg/mL) significantly affected fertilization and early embryonic development. Together, our findings indicate that the studied phthalate mixtures adversely affected sperm motility, capacitation, and acrosome reaction, which resulted in poor fertilization rates and repressed embryonic development. Moreover, the lowest-observed-adverse-effect dose of the phthalate mixture tested can be assumed to be < 1 µg/mL in vitro.

Ocean acidification increases polyspermy of a broadcast spawning bivalve species by hampering membrane depolarization and cortical granule exocytosis.

Ensuring that oocytes are fertilized by a single sperm during broadcast spawning is crucial for the fertilization success of many marine invertebrates. Although the adverse impacts of ocean acidification (OA) on various marine species have been revealed in recent years, its impact on polyspermy and the underlying mechanisms involved remain largely unknown. Therefore, in the present study, the effect of OA on polyspermy risk was assessed in a broadcast spawning bivalve, Tegillarca granosa. In addition, the impacts of OA on the two polyspermy blocking processes, the fast block (membrane depolarization) and the permanent block (cortical reaction), were investigated. The results show that the exposure of oocytes to two future OA scenarios (pH 7.8 and pH 7.4) leads to significant increases in polyspermy risk, about 1.70 and 2.38 times higher than the control, respectively. The maximum change in the membrane potential during oocyte membrane depolarization markedly decreased to 15.79 % (pH 7.8) and 34.06 % (pH 7.4) of the control value. Moreover, the duration of oocyte membrane depolarization was significantly reduced to approximately 63.38 % (pH 7.8) and 21.91 % (pH 7.4) of the control. In addition, cortical granule exocytosis, as well as microfilament migration, were significantly arrested by OA treatment. Exposure to future OA scenarios also led to significant reductions in the ATP and Ca2+ content of the oocytes, which may explain the hampered polyspermy blocking. Overall, the present study suggests that OA may significantly increase polyspermy risk in T. granosa by inhibiting membrane depolarization and arresting cortical granule exocytosis.

Piperonyl butoxide, a synergist of pesticides can elicit male-mediated reproductive toxicity.

A semi-synthetic methylenedioxyphenyl compound piperonyl butoxide (PBO) has been used as a ubiquitous synergist to increase the insecticidal effect of pesticides for agricultural and household use. Despite previously demonstrated effects of PBO, the detailed mechanism of PBO in spermatozoa and reproductive toxic effects on male germ cells have not been fully elucidated. Therefore, this study evaluated the effects of PBO on various sperm functions during capacitation and clarified the mechanisms of reproductive toxic effects on male fertility at different concentrations of PBO (0.1, 1, 10, and 100 µM). Sperm motility and kinematics were assessed using computer-assisted sperm analysis and the status of capacitation was evaluated using combined H33258/chlortetracycline (CTC) staining. Intracellular adenosine triphosphate (ATP) and cell viability levels were also measured. In addition, protein kinase A (PKA) activity and protein tyrosine phosphorylation were evaluated. In addition, in vitro fertilization was performed to determine the effects of PBO on cleavage and blastocyst formation rates. We found that PBO significantly decreased sperm motility, kinematics, and acrosome-reacted and capacitated spermatozoa. In addition, PBO suppressed the intracellular ATP levels and directly affected cell viability. Moreover, PBO detrimentally decreased the activation of PKA and altered the levels of tyrosine-phosphorylated proteins. Consequently, cleavage and blastocyst formation rates were significantly reduced in a dose-dependent manner. In line with our observations, the synergist of pesticides PBO may directly and/or indirectly cause disorder in male fertility. Hence, we suggest that careful attention is made to consider reproductive toxicity when using PBO as a synergist.

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Melatonin regulates ATP content and fertilising capacity of Onychostoma macrolepis spermatozoa by inhibiting ROS accumulation during semen storage in vitro.

Melatonin (MLT) is an efficient antioxidant that protects spermatozoa against damages caused by oxidative stress. In this study, to maintain good function of Onychostoma macrolepis spermatozoa during semen preservation invitro at 4°C, different concentrations of MLT (0.5, 1 and 2µM) were added to the semen. After storage (0, 24, 48 and 72h), 1µM MLT in semen markedly improved sperm quality, as reflected by better plasma membrane integrity, the relative steady level of reactive oxygen species (ROS) and slower rate of decrease in mitochondrial membrane potential. Activated spermatozoa in semen with 1µM MLT had higher kinematic performance (i.e. percentage of motile and progressive spermatozoa and the beat cross frequency; P<0.05) and longer duration of sperm motility (P<0.05) compared with spermatozoa in semen withother MLT concentrations. Furthermore, 1µM MLT maintained higher ATP concentrations in spermatozoa during semen storage and significantly improved the fertilising capacity of spermatozoa after 72h semen storage compared with the other MLT concentrations. To expand wild resources of O. macrolepis, 1µM MLT can be used as a semen additive to maintain better sperm function and enhance sperm fertilising capacity in artificial insemination (AI).

Effects of follicular ablation and GnRH on synchronization of ovulation and conception rates in embryo recipient heifers.

Two experiments were performed to determine effects of follicular ablation (FA) and GnRH treatment on conception rate and synchronization in timing of ovulation among Holstein heifers. In Experiment 1, heifers were randomly allocated to four groups: Control (n = 84): prostaglandin F2α (PGF) IM on Day 0; FA-5/GnRH (n = 43): FA 5 days before PGF and GnRH on Day 2; FA-4/GnRH (n = 48):FA 4 days before PGF and GnRH on Day 2; andFA-3/GnRH (n = 21): FA 3 days before PGF and GnRH on Day 2. Ultrasonography was performed to determine follicular size, ovulation occurrence, and size of CL. In Experiment 2, heifers were assigned to three groups: Control (n = 264), FA-5/GnRH, and FA-4/GnRH. Pregnancy diagnosis was performed at Days 30 and 60. In Experiment 1, size of largest follicle at time of PGF was less variable (P ≤ 0.05) in all FA groups compared to the Control group. With the FA-5/GnRH and FA-4/GnRH treatments, there were greater (P ≤ 0.05) proportions of timing of ovulation synchronization (86 % and 85 %, respectively) compared to the Control (61 %) and FA-3/GnRH (62 %) groups. In Experiment 2, conception rates did not differ among groups, however, there were more pregnancies per cow when timing-of-ovulation treatments were imposed. In conclusion, follicular ablation combined with GnRH treatment resulted in an increased proportion of heifers having synchronized ovulation and, therefore, number of recipient heifers available for embryo transfer. Additionally, there was no effect on conception rate when there was greater synchronization in timing of ovulation among heifers.

Effects of human chorionic gonadotropin and intravaginal progesterone device treatment after artificial inseminations on the reproductive performance of normal and repeat breeder lactating dairy cows.

We examined the effects of human chorionic gonadotropin (hCG) treatment on Day 5 (Day 0 = day of artificial insemination: AI) and intravaginal progesterone device (IVPD) treatment from Day 5 to 19 on the conception and detection rates of return to estrus (re-estrus) in lactating dairy cows. A total of 306 cows from a commercial dairy farm were divided into the following three groups on Day 5: non-treatment group (n = 128), untreated; hCG group (n = 71), 3,000 IU hCG was administered (intramuscularly); IVPD group (n = 107), IVPD was inserted into the vagina from Day 5 to 19. Re-estrus detection was performed up to Day 25. Pregnancy was diagnosed by rectal palpation between Day 50 and 60. There was an interaction between treatment and AI number (P < 0.01) on the conception rate of first-AI. For cows with more than three AIs, the IVPD treatment (66.7%) was more effective than the non-treatment (23.1%) (P < 0.05). The re-estrus detection rate was significantly (P < 0.05) higher in the IVPD group (60.7%) than that in the non-treatment group (41.4%) and tended (P < 0.1) to be higher than that in the hCG group (37.8%). Our results suggested that the conception rate can be improved by IVPD treatment, especially in cows with more than three AIs. In addition, IVPD treatment can induce higher estrus expression up to 25 days after AI in non-pregnant cows.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Pre-conception exposure to THC fails to impact nicotine reward in adult offspring.

Exposure to environmental stimuli in one generation can produce altered behavioral and neurobiological phenotypes in descendants. Recent work has shown that parental exposure to cannabinoids alters the rewarding properties of other abused drugs in the subsequent generation. However, whether preconception Δ9-tetrahydrocannabinol (THC) administration modifies the affective properties of nicotine in offspring is unknown. To address this question, male and female rats (F0) received THC (0 or 1.5 mg/kg) throughout the adolescent window and were bred on PND 65. In Experiment 1, adult F1-THC and F1-Veh progeny (males and females) underwent nicotine locomotor sensitization procedures during which nicotine (0 or 0.4 mg/kg) was administered every other day for five exposures, and locomotor activity was recorded on each exposure followed by a final nicotine challenge. There was no cross-generational effect of THC on nicotine locomotor sensitization, although acute exposure to nicotine produced greater activity in females relative to males independent of THC history. In Experiment 2, adult F1-THC and F1-Veh progeny (males and females) were implanted with jugular catheters and trained to self-administer nicotine (0.03 mg/kg/infusion). Following acquisition, all subjects were allowed to self-administer nicotine on a number of reinforcement schedules, e.g., FR2, FR5 and PR, followed by dose response and extinction procedures. Across all indices, F1-THC and F1-Veh subjects displayed similar IVSA of nicotine with no sex differences. The fact that there was no evidence of cross-generational effects of THC on nicotine suggests that such effects are drug-specific.

Maternal conjugated linoleic acid alters hepatic lipid metabolism via the AMPK signaling pathway in chick embryos.

The effects of maternal conjugated linoleic acid (CLA) on embryonic development and hepatic lipid metabolism were investigated in chick embryos. A total of 180 Arbor Acres female broiler breeders (36 wk old) were randomly divided into the following 3 dietary treatment groups: a basic diet (control), a basic diet containing 0.5% CLA (CLA1), and a basic diet containing 1.0% CLA (CLA2). The females were fed for 8 wk, and the eggs from each group were collected and hatched during the last 2 wk. The results showed that the addition of dietary CLA increased the broken egg rate and reduced the fertilization rate and the egg hatchability (P < 0.05). CLA enrichment decreased the polyunsaturated and monounsaturated fatty acids and increased the saturated fatty acids in the yolk sac (P < 0.05). The yolk sac weight, body weight, and body length had a linear decrease with CLA supplementation (P < 0.05). In the developing chick embryo (at E14) and newly hatched chick (D0), the serum triglyceride concentration decreased with maternal CLA supplementation and was accompanied by a reduction in subcutaneous adipose tissue deposition. In addition, maternal CLA supplementation mediated the hepatic lipid metabolism by decreasing the mRNA expression of sterol regulatory element-binding proteins-1c (SREBP-1c), fatty acid synthase and acetyl-CoA carboxylase, and increasing the mRNA expression of adenosine 5'-monophosphate-activated protein kinase α (AMPKα), peroxisome proliferator-activated receptors α (PPARα), liver fatty acid-binding protein, adipose triglyceride lipase and carnitine palmitoyltransferase in embryonic chick livers (P < 0.05). A drop in SREBP-1c protein expression and an increase in the protein expression of p-AMPKα and PPARα were also observed in the liver of chick embryo (P < 0.05). In conclusion, maternal CLA supplementation regulated the fatty acid composition in the yolk sac, and mediated embryonic chick development and hepatic lipometabolism, and these effects may be related to the AMPK pathway. These findings suggest the potential ability of maternal CLA supplementation to reduce fat deposition in chick embryos.

Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization.

Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and in vivo fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycycle are presented. Through a structure-based approach applied on our in-house library, compound 1r was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a KD value of 21 µM and the latter an IC50 value of 13.2 µM. Further investigations highlighted that compound 1r induced 20% sperm death at 25 µM and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of 1r and N-acetyl cysteine (NAC). Interestingly, while 1r was not able to hamper the ZP/sperm binding, it markedly decreased the in vitro oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by 1r/NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA's biological role in fertilization as well as provide a starting point for developing 1r structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.

Effect of dimethylformamide on sperm quality and fertilizing ability of Indian red jungle fowl (Gallus gallus murghi).

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.

Effect of treatment of phantom cows with a progesterone-based synchrony programme.

Aim: To determine the effect of a progesterone-based synchrony programme on the daily hazard of conception and the probability of being pregnant at the end of the seasonal mating period in cows not observed in oestrus within 35-49 days of insemination and that were diagnosed non-pregnant (phantom cows) on seasonally calving New Zealand dairy farms. Secondary aims were to determine the prevalence of phantom cows and estimate the proportion of phantom cows with a functional corpus luteum (CL) at enrolment. Methods: Phantom cows from 14 New Zealand commercial dairy farms were enrolled in a randomised, controlled trial. Cows that were artificially inseminated ≤14 days after mating start date and were not subsequently detected in oestrus, were presented for pregnancy diagnosis approximately 49 days after mating start date. Non-pregnant cows were diagnosed as phantom cows and randomly allocated to treatment and control groups. A milk sample was collected for progesterone assay to determine the presence of a functional CL. Treatment consisted of an injection of buserelin and insertion of an intravaginal device containing progesterone on Day 0, injections of dinoprost and equine chorionic gonadotrophin, and removal of the intravaginal device on Day 7, injection of buserelin on Day 9, and fixed time artificial insemination on Day 10. Treatment group cows were then mixed with bulls for the remainder of the seasonal mating period. Cows allocated to the control group were mated naturally by bulls. Statistical models were constructed to determine the effect of treatment on the daily hazard of conception and the probability of being pregnant at the end of the seasonal mating period. Results: A total of 378/4,214 (9.0%) cows presented for pregnancy diagnosis were diagnosed as phantom cows. A functional CL was diagnosed in 257/362 (71.0%) phantom cows. Median predicted enrolment to conception intervals were 33 (95% CI = 30-45) and 30 (95% CI = 28-33) days, for cows in the control and treatment groups, respectively. The odds of being pregnant at the end of mating were 1.70 (95% CI = 1.34-2.17) times greater for treated phantom cows than untreated phantom cows. Estimated marginal mean proportion pregnant at mating end date were 59.5 (95% CI = 47.9-70.1)% and 71.5 (95% CI = 62.6-79.0)% for control and treatment group cows, respectively. Conclusions: Treatment with a progesterone-based synchrony programme significantly increased the probability of phantom cows being pregnant at the end of the seasonal mating period.

Potential of Auraptene in Improvement of Oocyte Maturation, Fertilization Rate, and Inflammation in Polycystic Ovary Syndrome Mouse Model.

Polycystic ovary with poor-quality oocytes has remained problematic in polycystic ovary syndrome (PCOS) patients. It is well documented that the inflammation and production of reactive oxygen species (ROS) in PCOS ovaries are significantly higher than normal voluntaries. In this study, we hypothesized that auraptene (AUR), as a coumarin derivative with anti-inflammatory properties, may be effective in improvement of oocyte maturation and fertilization rate in PCOS patients. For this purpose, PCOS model was induced in NMRI mice and confirmed by ovarian histopathology observations and hormonal assays. PCOS-induced mice were administrated with AUR (PCOS-AUR) and metformin (PCOS-MET), and their effects on inflammation, apoptosis rate, oocyte maturation, and in vitro fertilization capacity were determined and compared with those normal and PCOS animals treated with sesame oil (PCOS-sesame oil) and no treatment (PCOS). Treatment with AUR and MET decreased the inflammation and apoptosis rates in PCOS mice compared with PCOS animals with no treatment. PCOS-AUR and PCOS-MET oocytes also showed higher intracellular glutathione and lower ROS concentrations compared with PCOS mice, indicating improved oocyte maturation rate. PCOS-AUR and PCOS-MET groups showed higher percentages of expansion rate and MII stage oocytes, and lower rate of abnormal oocytes compared with PCOS with no treatment. The rate of fertilization in the oocytes isolated from PCOS-AUR and PCOS-MET groups was higher than PCOS-sesame oil and PCOS groups. Our findings suggest that AUR can be considered as a potential candidate for improvement of oocyte maturation and fertilization capacity in PCOS patients, comparable to MET.