Pituitary Extract Research Articles

Comparative efficacy of carp pituitary extract and Ovaprim during captive breeding of Cat Fish, Pabda (Ompok pabda).

Captive breeding was conducted to compare the efficacy of CPE and Ovaprim on O. pabda and it was observed that both the inducing agents were quite effective. However, R3 (CPE) dose (5 mg/kg bd.wt. to male and 16 mg/kg bd.wt. to female) registered less fertilization and hatchlings compared to the T3 dose of Ovaprim (0.5 ml/kg bd.wt. to male and 1.5 ml/kg bd.wt. to female). As compared to the conventional hypopysation, which requires high quantity of carp pituitary extract (CPE), Ovaprim is quite effective at low quantity for inducing breeding under captivity. Vijaya kumar reported induce spawning of O. malabaricus with single dose Ovaprim 0.5 ml/ kg bw while Banik et al. reported induce spawning of O. bimaculatus with single dose Ovaprim 0.5 ml/kg bd.wt. of male and 1.5 ml/kg bd.wt. to female. Haniffa et al. advocated three different dosage of Ovaprim (0.3 ml, 0.5 ml and 0.7 ml) for induce breeding of O. malabaricus where the variations in fertilization rate were 70.87 and 91% and survival no. and rate was 58.62 and 70% respectively. Three different treatments, during the experiment ensures a high rate of fertilization (66.88 & 65.40) and hatching (78.40%), when highest survival rate was 59.8, 62.1 and 61.4% respectively with three treatments of Ovaprim. The results of the experimental data indicate that, for successful spawning and seed production of Pabda, Ovaprim can be a better option than Carp pituitary extract. It is envisaged from the data that Ovaprim not only induce complete spawning but fertilization, hatching and survival is more ensured than CPE. Further study should be conducted for better management and understanding about the reproductive physiology, breeding behavior and larval rearing of Pabda to save this unique fresh water species.

The Effect of Solithromycin, a Cationic Amphiphilic Drug, on the Proliferation and Differentiation of Human Meibomian Gland Epithelial Cells

ABSTRACTPurpose: We previously discovered that azithromycin (AZM) acts directly on immortalized human meibomian gland epithelial cells (IHMGECs) to stimulate their lipid and lysosome accumulation and overall differentiation. We hypothesize that this phospholipidosis-like effect is due to AZM’s cationic amphiphilic drug (CAD) nature. If our hypothesis is correct, then other CADs (e.g., solithromycin [SOL]) should be able to duplicate AZM’s action on IHMGECs. Our purpose was to test this hypothesis.Materials and Methods: IHMGECs were cultured in the presence of vehicle or SOL (2, 10, or 20 µg/ml) for up to 7 days under proliferating or differentiating conditions. Positive (epidermal growth factor and bovine pituitary extract for proliferation; AZM for differentiation) and negative (vehicle) controls were included with the experiments. IHMGECs were evaluated for cell number, neutral lipid content, and lysosome accumulation.Results: Our results demonstrate that SOL induces a rapid and dose-dependent increase in the accumulation of neutral lipids and lysosomes in HMGECs. The lysosomal effects were most prominent with the 10 and 20 µg/ml doses, and occurred earlier (i.e., 1 day) with SOL than with the AZM (10 µg/ml) control. The effects of SOL and AZM on IHMGEC differentiation were essentially the same after 3 days of culture. SOL did not influence the proliferation of HMGECs during a 7-day time period.Conclusions: Our results support our hypothesis that SOL, a CAD, is able to reproduce AZM’s impact on lysosome and lipid accumulation, as well as the differentiation, of HMGECs. The effect of SOL on lysosome appearance was faster than that of AZM.

Mating calls of three prochilodontid fish species from Brazil

Passive acoustic monitoring (PAM) of fish sounds has been used as a means of detecting the presence and abundance of fishes. Prior to PAM, bioacoustical analyses of sounds are needed to characterize species-specific characteristics of calls. With hydrophones placed in outdoor ponds, we recorded the mating calls of three species of prochilodontid fishes (Prochilodus argenteus, P. costatus, P. lineatus). Fish were induced to spawn and call by injection of carp (Cyprinus carpio) pituitary gland extract. We recorded a total of 394 pulse train calls and additional single pulse calls that were not associated with trains. The trains of all three species were similar in nature - series of low frequency pulses that lasted from 1 to 11 s, often with an initial rapid rise followed by a slow tapering of pulse amplitude. Dominant frequency of single pulses and trains was greatest in P. lineatus, with P. costatus and P. argenteus exhibiting lower single pulse dominant frequencies, and P. costatus having lower train dominant frequency. With data from the three species combined, the dominant frequency of pulses significantly increased with fish standard length. There was also a significant linear relationship between the dominant frequency of pulses and trains. Discriminant function analysis showed that differences in train dominant frequency, pulse duration, and pulse period between the three species were significant enough to discriminate between them. This study was the first to fully characterize the sounds of these three Prochilodus species, and should assist fisheries biologists monitoring spawning behavior in these species.

Successful, induced propagation of African catfish (Clarias gariepinus) by ovarian lavage with sperm and hormone mixture

The in vitro fertilisation method is widespread in fish aquaculture. In general, calculated doses of different hormones are injected intramuscularly or intraperitoneally, by using a syringe and needle, to induce ovulation and spermiation. We tested a novel approach to delivering sperm and hormone together to eggs, in African catfish, and concluded that sperm plus carp pituitary extract injection into the ovary lobes (through the oviduct) leads to successful ovulation and recovery of developing embryos in all of the experimental conditions applied. These results indicate that injected spermatozoa with suspended carp pituitary are stored in the ovary lobes for up to 10h without loss of biological activity and that the ovulated eggs could be fertilised by the injected sperm, after being released from the body cavity. In addition, the seminal fluid was an appropriate delivery vehicle for the ovulation inducing hormone (i.e. carp pituitary) via ovarian lavage.

Characterization of carp gonadotropins: Structure, annual profile, and carp and zebrafish pituitary topographic organization

Two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are important players in the hypothalamic-pituitary-gonadal axis of vertebrates. In the present work, we describe the construction of recombinant (r) common carp (Cyprinus carpio; c) FSH (rcFSH) and LH (rcLH) using the Pichia pastoris system, the generation of specific antibodies against their respective β subunits, and their use in the development and validation of specific ELISAs. We produced carp rLH and rFSH as single-chain polypeptides, wherein the GTH subunit α was joined with either cLHβ or cFSHβ mature protein-coding sequences to form a fusion gene that encodes a yoked polypeptide, in which the GTH β-subunit forms the N-terminal part and the α-subunit forms the C-terminal part. Competitive ELISAs were developed, using primary antibodies against rcLHβ or rcFSHβ, respectively, and rcLHβα or rcFSHβα for the standard curves. The standard curves for cLH paralleled those of pituitary extracts of the homologous fish and also those of other cyprinids species like the black carp (Mylopharyngodon piceus), goldfish (Carassius auratus), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella). We used the specific antibodies raised against cFSH and cLH to study the specific localization of the different GTH cells in the pituitary of carp and its taxonomic relative species – the zebrafish. Both FSH and LH cells are localized in the center of the proximal pars distalis enveloping both sides of the neurohypophysis. LH cells form a continuous population throughout the PPD, while FSH cells are more loosely distributed throughout the same area and form small aggregations. Marked annual changes were encountered in gonadosomatic index (GSI), follicle diameter, mRNA levels and protein levels of FSH and LH. From September to November, all fish had low GSI, and the ovary contained previtellogenic follicles. From December, the GSI level increased and remained high until March, the follicular diameter reached its maximum in January, where the ovary contained large fully grown follicles. Thereafter, spawning occurred through March and April and ended in May, and GSI level and follicle diameter increased again; and the ovary contained mid-vitellogenic follicles. LH pituitary content and mRNA levels were low at pre- and early vitellogenesis, increasing gradually during this process to reach a peak of LH mRNA levels in mid vitellogenic ovary and a peak of LH content in fully grown ovarian follicles. However, no significant change occurred in FSH pituitary content and mRNA levels in vitellogenic fish and in fish during final maturation stages. A dramatic difference was found in the total content of each gonadotropin in the pituitary, with higher LH than FSH. Moreover, follicle diameter was positively and significantly correlated with LH pituitary content and its transcript levels – but not with the pituitary content or mRNA levels of FSH. Taken together, these results indicate that in carp, LH alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.

Levels of 17β estradiol, vitellogenin, and prostaglandins during the reproductive cycle of Oreochromis niloticus

Circulating levels of estradiol, vitellogenin and prostaglandins could be references of oocytes quality in the aquaculture industry. This study evaluated the performance of 17β-estradiol (E2) and vitellogenin (Vtg) levels in plasma and prostaglandins in oocytes (PGE 2) during the reproductive period of Oreochromis niloticus in a commercial farm in Ecuador. Adults fish females were treated with estradiol-synthetic (ES) and tilapia pituitary extracts (TP) during 25 days. For quantification of E2 and PGE 2, an enzyme-linked immunosorbent assay (ELISA) was used, while for Vtg relative quantification a Western blot analysis was performed. Circulating E2 concentrations were different during the first week of follow-up ( P < 0.05) resulting in different levels of Vtg per treatment ( P < 0.05) affecting on gonadal growth. The ovaries had an asynchronous development, where the mature oocytes proportions were higher with ES and TP than control. Relative fecundity was different between treatments ( P < 0.05), where TP generated 15.7 ± 5.8 oocytes per g of spawning female. Furthermore, concentrations of PGE 2 were different between treatments ( P < 0.05). The information reported in this study will be useful to improve the reproduction methods and have some biological signals before ovulation in tilapia commercial culture.

Morfología de testículos de la tuvira (Gymnotus spp.) Sometida a la reproducción por diferentes protocolos hormonales

The aim of study was to analyze the testis morphology of males tuviras (Gymnotus spp.) submitted to reproduction by different hormonal inducers. Forty male tuviras (40g ± 5.0) were used approximately in a completely randomized design with four treatments and 10 replicates; each animal was considered a repetition. For hormonal protocol establishment were tested three inducers: crude extract of carp pituitary (EBHC), equine chorionic gonadotropin (ECG), synthetic analogue of gonadotropin-releasing hormone (GnRH-a) and the control group was administered saline solution at 2%. The three hormones and control were applied in two doses (preparatory and final induction). Ten males per treatment were euthanized for macroscopic and microscopic analysis. It was determined volumetric proportion occupied by the components of testicular parenchyma (seminiferous tubules and intertubular tissue) and quantified populations of spermatogonia type A, type B, primary spermatocytes, secondary spermatocytes, spermatids, Sertoli cells (S) and lumen measurement . The results indicated that the hormone GnRH-a showed a higher capacity to promote the final maturation of the testes (P &lt; 0.05), followed by eCG hormone. The EBHC hormone failed to promote the final maturation in males tuviras.

Comparative Studies on Clarias gariepinus Pituitary Extracts and Synthetic Hormone on Induced Spawning and Growth Performance of the African Catfish Clarias gariepinus Fry

Comparative Studies on Clarias gariepinus Pituitary Extracts and Synthetic Hormone on Induced Spawning and Growth Performance of the African Catfish Clarias gariepinus Fry

Production of live larvae following in vitro maturation of zebrafish oocytes

This study aimed to assess the effects of carp pituitary extract (CPE), follicle stimulating hormone (FSH) and luteinizing hormone (LH) on zebrafish oocyte maturation and the ability of these mature oocytes to be fertilized and developed until hatching. Stage III follicles were matured in eight treatments: five concentrations of CPE (16, 32, 48, 64 and 80 μg/mL), one of FSH (0.5 μg/mL), one of LH (0.5 μg/mL), or one combination of FSH (0.5 μg/mL) and LH (0.5 μg/mL). Maturation rates in CPE treatments were 12.8% (16 μg/mL), 24.8% (32 μg/mL), 27.0% (48 μg/mL), 22.7% (64 μg/mL) and 9.6% (80 μg/mL); in FSH was 15.7% (0.5 μg/mL), in LH was 31.8% (0.5 μg/mL) and in FSH (0.5 μg/mL) combined with LH (0.5 μg/mL) it was 50.4%. In vitro fertilization was performed in all treatments; however, only the treatment combining FSH and LH resulted in fertilized oocytes. After maturation using FSH combined with LH, the cleavage rate was 33.3% and hatching rate of live larvae was 20.0%. These results showed that FSH combined with LH was effective in IVM of zebrafish oocyte.

Gonadorelin increases semen production and does not affect its quality in Leporinus obtusidens

Use of carp pituitary extract (CPE) as a hormone inducer for Leporinus obtusidens is cost-prohibitive; moreover, CPE contains unknown concentrations of gonadotropins. We evaluated the efficacy of gonadorelin as a substitute by analyzing its effect on sperm characteristics of L. obtusidens and cost-effectiveness (20, 40, 60, 80, and 100μgkg−1). Untreated and CPE-treated (4mgkg−1) fish were used as negative and positive controls, respectively. Following a single intramuscular dose of hormones, semen was collected at 200°-h. At doses from 60μgkg−1, all fish produced higher milt volume (P<0.05) than the negative control (0.2–0.8mLkg−1), equal (P>0.05) to the positive control (3.52±0.97mLkg−1). Gonadorelin did not affect sperm concentration (P>0.05). The percentage of morphologically normal spermatozoa was >64% in all treatments. The number of spermatozoa with intact membrane (70%–85%) was equal (P>0.05) to that in the controls. Relative to the negative control, gonadorelin did not affect the motility rate (P>0.05). Curvilinear velocity was equal (P>0.05) to that in both controls. The sperm swimming path was linear (0.9) 10s after activation and was not (P>0.05) dose-dependent. Considering the response rate of gonadorelin-induced fish and the similarity of semen quality compared to controls, the lowest effective dose to produce a higher volume of semen in L. obtusidens is 60μgkg−1, and the cost to induce 1kg of L. obtusidens is 14.0% lower than that with CPE.

Comparative researches concerning artificial reproduction with pituitary extract and GnRH in Carp

Comparative researches concerning artificial reproduction with pituitary extract and GnRH in Carp

&lt;b&gt;Captive breeding for conservation of Dussumier’s Catfish (Actinopterygii: Siluriformes: Clariidae: &lt;I&gt;Clarias dussumieri&lt;/I&gt;) a Near Threatened endemic catfish of peninsular India&lt;/b&gt;

The peninsular Indian endemic Dussumier’s Catfish Clarias dussumieri once abundant in the wetlands and other water bodies of Kerala is now in rapid decline. The present paper focuses on an approach towards the conservation of this rare catfish through artificial propagation. Fishes were bred in captivity by the administration of fish pituitary extract at the rate of 20–40 mg.kg-1. Spawning occurred after 12–14 hr of injection and fertilized eggs hatched after 16:30hr. Larval rearing was carried out in cement cisterns and the larvae attained a mean size of 51.6±1.6mm in 60 days. Since brooders of this species have become extremely rare in nature, the present study on captive breeding by hormonal manipulation methods without sacrificing the male fishes, assumes significance for conserving this endemic species.

Crioconservación de semen de dorada Brycon moorei con dimetilsulfóxido

El objetivo fue evaluar la calidad del semen descongelado de dorada Brycon moorei crioconservado con dimetilsulfóxido (DMSO) a tres porcentajes de inclusión. El semen se obtuvo de nueve machos mantenidos en cautiverio en la Estación Piscícola Repelón (Atlántico, Col), inducidos con extracto pituitario de carpa (4,5 mg/kg). El semen fue diluido en proporción 1:3 con un diluyente compuesto de DMSO a tres porcentajes 5%, 10% y 15%; glucosa al 6% y yema de huevo al 12%; empacado en macrotubos de 2,5 ml, congelados en vapores de nitrógeno y después de tres meses descongelados a 35°C durante 90 s. Semen fresco fue considerando como tratamiento control. En semen descongelado se evaluó movilidad total, tipos de movilidades, progresividad, velocidades y concentración espermática con el programa Sperm Class Analyzer SCA®; adicionalmente en semen fresco se determinó volumen, color y tiempo de activación. El semen fresco presentó movilidad mayor a 80% y tiempo de activación entre 28,5 y 41 s; mientras que, la concentración espermática osciló entre 10188,1 y 14590,2 millones/ml. La movilidad total del semen descongelado fue mayor cuando DMSO se incluyó a 5% (40,1±5,0%) o 10% (43,3±8,7%) (p&gt;0,05); pero a 15% registró la menor movilidad (30,6±7,9%) y el mayor porcentaje de espermatozoides inmóviles (69.4±7.9%) (p&lt;0,05); lo cual sugiere que inclusiones de DMSO por encima de 10% ocasionan mayores daños al espermatozoide de dorada. Los resultados permiten concluir que DMSO debe ser incluido entre 5 y 10%, junto con glucosa al 6% y yema de huevo al 12% para crioconservar semen de dorada.

Efficiency of three reproductive hormones in artificial propagation of Heterobranchus longifilis (valenciennes, 1840)

Efficiency of three reproductive hormones: ovaprim, ovulin and carp pituitary extract (C.P.E) in artificial propagation of Heterobranchus longifilis was investigated. Total of twelve broodstock fish, comprising nine females and three males at eighteen months of age were used. Females were divided into three groups. Each group was induced to spawn with different hormones. Pooled sperm from the three males were used to fertilize the eggs obtained from individual female. The effect of the hormones on latency period, fecundity, fertilization, hatchability and larval survival was recorded. Latency period (P&lt;0.05) lasted for nine (ovaprim), ten (ovulin) and eleven hours (C.P.E.) at 26 oC. Fecundity (P&lt;0.05) mean values were 57.67±2.186 (ovaprim), 43.00±10.970 (C.P.E.) and 35.00±8.544 eggs (ovulin). Fertilization rate (P&lt;0.05) mean values were 94.00±1.15% (ovaprim), 82.00±2.31% (ovulin) and 82.00±1.73% (C.P.E.). Hatchability success (P&lt;0.05) mean values were 84.33±3.48% (ovulin), 94.67±1.45% (ovaprim) and 89.33±0.88% (C.P.E.) as survival rate was not significant. The study has provided data, comparing the effectiveness of the hormones, revealing that the three hormones can be used for artificial propagation of H. longifilis with varied degree of success.Keywords: Artificial breeding, ovaprim, ovulin, carp pituitary extract, broodstock, Heterobranchus longifilis

Spawning performance of Clarias gariepinus (Burchell, 1822) induced with ethanol preserved and fresh catfish pituitary extract.

This study was conducted to determine the efficacy of preserved and fresh pituitary extract of Clarias gariepinus (Burchell, 1822) to induced spawning in the same species. Growth performance of fry was also monitored for 15 days to determine the possible effect of natural hormone treatment on the fry. Pituitary glands (PG) were obtained from 12 male broodstocks, six extracted PG were preserved in 96% ethanol 24 h before injection, while the other were extracted and used in their fresh state. The PG solutions (1 g ml-1 of saline water) were injected at 1 ml kg-1 of the female broodstock. Ovaprim® (a synthetic hormone) was used as the control and administered at a rate of 0.5 ml kg -1 of fish. After a uniform latency period of 9 h 30 min, fish were striped, fertilized, incubated and the performance evaluated. Results obtained revealed better hatching performance using Ovaprim® synthetic hormone (64.52%), however, preserved PG gave better hatchability (59.74%) than fresh PG extract (51.39%). After 15 days of feeding ad libitum with shell-free Artemia cysts, the growth of fry spawn with preserved PG was comparable with that of the control, while least performance was observed using the fresh PG. It was concluded that preservation of PG in ethanol 24 h before injection had a positive effect on breeding performance and could be exploited in the commercial production of C. gariepinus fingerlings.

Molecular markers of oocyte differentiation in European eel during hormonally induced oogenesis

Reproduction in captivity is a key study issue in Anguilla anguilla as a possible solution for its dwindling population. Understanding the mechanisms controlling the production of ribosomal building blocks during artificially induced oocyte maturation could be particularly interesting. Transcription levels of ribosomal biogenesis associated genes could be used as markers to monitor oogenesis. Eels from the Albufera Lagoon were injected with carp pituitary extract for 15weeks and ovaries in previtellogenic (PV) stage (non-injected), in early-, mid-, late-vitellogenesis (EV, MV, LV), as well as in migratory nucleus stage (MN) were analysed. 5S rRNA and related genes were highly transcribed in ovaries with PV oocytes. As oocytes developed, transcriptional levels of genes related to 5S rRNA production (gtf3a), accumulation (gtf3a, 42sp43) and nucleocytoplasmic transport (rpl5, rpl11) and the 5S/18S rRNA index decreased (PV>EV>MV>LV>MN). On the contrary, 18S rRNA was at its highest at MN stage while ubtf1 in charge of activating RNA-polymerase I and synthesising 18S rRNA behaved as 5S related genes. Individuals that did not respond (NR) to the treatment showed 5S/18S index values similar to PV females, while studied genes showed EV/LV-like transcription levels. Therefore, NR females fail to express the largest rRNAs, which could thus be taken as markers of successful vitellogenesis progression. In conclusion, we have proved that the transcriptional dynamics of ribosomal genes provides useful tools to characterize induced ovarian development in European eels. In the future, such markers should be studied as putative indicators of response to hormonal treatments and of the quality of obtained eel oocytes.

276 - Creating gmp epithelial and fibroblast cells from a single biopsy for the potential use in tissue engineering

Background: Tissue engineering of human airways is an emerging field where an organ or tissue is created to replace a diseased/ non-functional one. Most of these are manufactured, at least partially, with patient's own starting materials (autologous). In the particular case of airways, there is a clinical need for epithelialisation, both to reduce infection and pain and aid in the restoration of organ function. The production of epithelial cells for clinical use needs to be carried out under the control of good manufacturing practices (GMP). The major hurdles to overcome are the use of a murine fibroblast (3T3 J2F) feeder layer or bovine pituitary extract (BPE) in the culture medium. Here we describe a method to obtain both squamous epithelial cells and fibroblasts from a single autologous biopsy in a GMP compliant manner.

Ovopel and carp pituitary extract for the reproductive induction of Colossoma macropomum males

The objective of this study was to evaluate Ovopel and carp pituitary extract (CPE) in the reproductive induction of Colossoma macropomum males. Nine treatments were tested in triplicate, totaling 27 experimental units. C. macropomum breeders were subjected to the following treatments: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg; 2.5 mg CPE/kg (traditional protocol); and a control treatment (no hormone). Breeders under hormone treatment produced a larger (P < 0.05) semen volume (2.4 ± 0.7 to 4.2 ± 0.3 mL) compared with the control (0.9 ± 0.4 mL). Sperm concentration did not differ significantly among treatments (7.2 × 109 ± 1.7 to 10.8 × 109 ± 2.6 spermatozoa/mL). Total sperm count was higher (P < 0.05) after treatment with 0.3, 0.4, and 0.6 Ovopel pellet/kg (41.6 ± 9.3 to 42.3 ± 10.5 × 109 spermatozoa) than the other Ovopel treatments (20.0 ± 2.4 to 26.9 ± 8.2 × 109 spermatozoa) and control (6.6 ± 1.1 × 109 spermatozoa), but did not differ significantly from CPE (33.7 ± 3.2 × 109 spermatozoa). Sperm motility was higher (P < 0.05) in the CPE treated, and 0.2, 0.3, and 0.7 Ovopel pellet/kg (88.3 ± 2.9 to 90.0 ± 5.0) breeders when compared with the other treatments (70.0 ± 10.0 to 78.3 ± 5.8), except for the 0.4 pellet/kg (81.7 ± 2.9) treatment, which did not differ significantly from any of the treatments. The motility period of the spermatozoa did not differ significantly among treatments (93.5 ± 15.7 to 120.0 ± 7.6 s). For the sperm morphological analysis, occurrence of normal spermatozoa was similar across the treatments, with three sperm abnormalities (short tail, bent tail, and detached head) differing (P < 0.05) among the treatments. Ovopel efficiently induced reproduction of C. macropomum breeders, with treatment using 0.3 and 0.4 Ovopel pellet/kg and CPE providing the best semen characteristics.

Semen characteristics of Colossoma macropomum from three successive sample collections in the same reproductive cycle

The objective of this work was to evaluate the semen characteristics of C. macropomum fish from three successive semen sample collections taken during the same reproductive cycle. Six breeders (6.4 ± 1.5 kg) induced with crude carp pituitary extract (2.5 mg kg−1) were used. Samples of semen were collected in graduated syringes after a period of 260 degree-hours. The semen was collected from the same males in September 2014 (sample 1), December 2014 (64 days after the first collection – sample 2) and February 2015 (86 days after the second collection – sample 3). The semen volume was higher (P < 0.05) in the sample 1 (6.7 mL) and sample 2 (3.4 mL) than in the sample 3 (1.0 mL). Sperm concentration and normal sperm did not significantly differ between samples. Likewise, sperm motility, curvilinear velocity, displacement velocity, oscillation and beat cross frequency did not vary, whereas straight-line velocity, straightness and progression were higher (P < 0.05) in sample 2 (December). In general, despite lower amounts, the semen appeared to be of good quality for all three samples collected during the same reproductive period.

Influence of Pilocarpine and Timolol on Human Meibomian Gland Epithelial Cells.

Investigators have discovered that topical antiglaucoma drugs may induce meibomian gland dysfunction. This response may contribute to the dry eye disease commonly found in patients with glaucoma taking such medications. We hypothesize that drug action involves a direct effect on human meibomian gland epithelial cells (HMGECs). To test this hypothesis, we examined the influence of the antiglaucoma drugs, pilocarpine and timolol, on the morphology, survival, proliferation, and differentiation of HMGECs. Immortalized (I) HMGECs (n = 2-3 wells/treatment/experiment) were cultured with multiple concentrations of pilocarpine or timolol for up to 7 days. Experiments included positive controls for proliferation (epidermal growth factor and bovine pituitary extract) and differentiation (azithromycin). Cells were enumerated using a hemocytometer and evaluated for morphology, neutral lipid staining, and lysosome accumulation. Our results demonstrate that pilocarpine and timolol cause a dose-dependent decrease in the survival of IHMGECs. The clinically used concentrations are toxic and lead to cell atrophy, poor adherence, or death. By contrast, drug levels that are known to accumulate within the conjunctiva, adjacent to the meibomian glands, do not influence IHMGEC survival. These latter concentrations also have no effect on IHMGEC proliferation or differentiation, and they do not interfere with the ability of azithromycin to stimulate cellular neutral lipid and lysosome accumulation. This dose of pilocarpine, though, did suppress the epidermal growth factor+bovine pituitary extract-induced proliferation of IHMGECs. Our results support our hypothesis and demonstrate that these antiglaucoma drugs, pilocarpine and timolol, have direct effects on HMGECs that may influence their morphology, survival, and proliferative capacity.