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1
Wei, Hong, and Yutaka Fukui. "Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes." Zygote 8, no. 3 (August 2000): 267–74. http://dx.doi.org/10.1017/s0967199400001064.
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This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 °C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 μm and 28.3 μm vs 22.4 μm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 μm vs 24.7 μm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.
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Puspita, Satya Alysa Cahya, Suherni Susilowati, Trilas Sardjito, Abdul Samik, Indah Norma Triana, and Soeharsono Soeharsono. "Penambahan alfa-tokoferol dalam pengencer susu skim - kuning telur terhadap kualitas spermatozoa domba Sapudi yang disimpan pada suhu 5°C." Ovozoa Journal of Animal Reproduction 9, no. 3 (December 7, 2020): 69. http://dx.doi.org/10.20473/ovz.v9i3.2020.69-76.
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Spermatozoa in fresh semen of Sapudi ram has a limited life span. The storage of semen in cold temperatures (5 °C) is intended to prolong the spermatozoa's life. However, storage in cold temperatures can lead to increased production of reactive oxygen species (ROS). This condition reduces the quality of spermatozoa. The purpose of this study was to determine the effect of alphatocopherol supplementation in skim milk-egg yolk extender on viability, motility, and plasma membrane integrity of Sapudi ram spermatozoa. Fresh semen derived from Sapudi ram was divided into four treatment groups. Control treatment (P0): semen was added in the extender of skim milkegg yolk without alpha-tocopherol. Three other treatments: P1, P2, and P3 semen were added in skim milk-egg yolk extender with the supplementation of 0.25, 0.5, and 1 gram alpha-tocopherol/ 100 mL extender, respectively. The results showed that the viability, motility, and integrity of the spermatozoa plasma membrane decreased gradually according to the storage length. Supplementation of skim milk-egg yolk extender with 0.5 gram of alpha-tocopherol/100 mL (P2) was able to maintain spermatozoa quality longer (p <0.05) than the control group. It can be concluded that alpha-tocopherol with a concentration of 0.5 g/100 mL of skim milk-egg yolk extender effectively maintains the quality of Sapudi ram spermatozoa in storage at 5 ° C.
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Lloyd, R. E., R. M. A. Elliott, A. Fazeli, P. F. Watson, and W. V. Holt. "Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro." Reproduction, Fertility and Development 21, no. 3 (2009): 408. http://dx.doi.org/10.1071/rd08204.
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Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39°C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.
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Poulos, A., P. Sharp, D. Johnson, I. White, and A. Fellenberg. "The occurrence of polyenoic fatty acids with greater than 22 carbon atoms in mammalian spermatozoa." Biochemical Journal 240, no. 3 (December 15, 1986): 891–95. http://dx.doi.org/10.1042/bj2400891.
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Fatty acids with carbon chain lengths greater than 22 (VLCFA) have been detected in boar, ram, bull and human spermatozoa. Saturated and mono-unsaturated fatty acids were present in all spermatozoa but, except for human spermatozoa, polyenoic fatty acids were quantitatively the most important components. Marked differences in polyenoic fatty acid composition were observed. Whereas human spermatozoa contain predominantly di-, tri- and tetraenoic fatty acids with up to 32 carbon atoms, boar, ram and bull spermatozoa also contain pentaenoic and/or hexaenoic acids with up to 34 carbon atoms. Human and boar spermatozoa differ markedly from those of the ram and bull in that only n-6 series acids are present.
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RoyChoudhury, P. N., and G. Bhambhani. "Pellet Freezing of Ram Spermatozoa." Zentralblatt für Veterinärmedizin Reihe A 24, no. 8 (May 13, 2010): 696–700. http://dx.doi.org/10.1111/j.1439-0442.1977.tb01748.x.
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Gillan, L., G. Evans, and W. M. C. Maxwell. "Capacitation status and fertility of fresh and frozen - thawed ram spermatozoa." Reproduction, Fertility and Development 9, no. 5 (1997): 481. http://dx.doi.org/10.1071/r96046.
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The effect of cryopreservation on the capacitation status and fertility of ram spermatozoa was observed. After the chlortetracycline staining technique was validated for ram spermatozoa, it was applied to fresh or long-term frozen-stored spermatozoa. Fresh spermatozoa displayed mainly the F pattern (non-capacitated; 61·3%), becoming B pattern (capacitated; 54%) and AR pattern (acrosome reacted; 41%) with incubation (6 h at 37°C). In contrast, frozen spermatozoa displayed the B pattern (65· 9%), becoming the AR pattern (64·2%) with incubation. This demonstrates that cryopreservation may cause membrane changes in ram spermatozoa functionally equivalent to capacitation. The differences in capacitation status did not affectin vitro fertilization rates between fresh and frozen spermatozoa, but pregnancy rates at Day 18 after intrauterine artificial insemination were higher for fresh than for frozen spermatozoa. This difference was not evident at Day 50, possibly as a result of the high embryonic loss between Days 18 and 50 when fresh unincubated and frozen incubated spermatozoa were inseminated. Further research is necessary to determine what part of the cryopreservation process is responsible for the membrane changes in ram spermatozoa.
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Mortimer, S. T., and W. M. C. Maxwell. "Kinematic definition of ram sperm hyperactivation." Reproduction, Fertility and Development 11, no. 1 (1999): 25. http://dx.doi.org/10.1071/rd99019.
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Although it is known that ram spermatozoa exhibit hyperactivated motility under capacitating conditions, quantitative analyses of the head and flagellar movement of washed ram spermatozoa have not been published. Motile spermatozoa were recovered from semen by swim-up into HSOF medium, and their movement in 30-µm-deep chambers was videorecorded. Spermatozoa of interest were identified during tape playback (hyperactivated spermatozoa were identified by visual assessment of flagellar movement) and sequential head and tail images were traced onto overhead projector film attached to the video monitor. The flagellar movement characteristics beat angle (FBA), beat envelope (FBE) and curvature ratio (FCR) were determined by first principles, and head centroid kinematics were determined using Cartesian methods. Hyperactivated spermatozoa had significantly higher FBA and FBE and significantly lower FCR values than non-hyperactivated spermatozoa (all P<0.0001). The centroid kinematic values were also found to be significantly different, and kinematic criteria for ram sperm hyperactivation were developed. These criteria were refined by consideration of 60-Hz CASA-derived trajectories, and ram sperm hyperactivation was defined by: VCL > 250.0 µm s −1 and VSL ≤ 100.0 m s −1 and LIN ≤ 30% and ALHmax ≥ 9.0 µm.
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Nikolovski, Martin, Monika Dovenska, Ksenija Ilievska, Nikola Adamov, Branko Atanasov, Miroslav Radeski, Daniela Kirovski, Vladimir Petkov, and Toni Dovenski. "Homologous Seminal Plasma and Glutathione Promote Pre-Capacitation Motility and Structural Stability of Cryopreserved Ram Spermatozoa." Macedonian Veterinary Review 42, no. 2 (October 1, 2019): 169–79. http://dx.doi.org/10.2478/macvetrev-2019-0022.
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Abstract Reduced glutathione (GSH) and homologous ram seminal plasma (HSP), used as additives in cryopreserving (CP) media prior to freezing, showed conflicting results in retaining structural integrity and progressive motility in post-thawed ram spermatozoa. The aims of this research were: (1) to assess the effect of GSH and/or HSP supplementation via soybean-lecithin CP extender on cryopreserved ram spermatozoa viability, morphology and motility pattern; and (2) to assess the effect of incubation in the context of the previous aim. Quantitatively and qualitatively, homogenized and pooled ram ejaculates (N=10) were extended with one of the following extenders: control (C) – tris-based, GSH and HSP-free, experimental-1 (E1) – C + GSH 5 mM, experimental-2 (E2) – C + HSP 20 % and experimental-3 (E3) - GSH 5 mM + HSP 20 %. Following thawing, samples were taken at 0- and 3-hours from each group (n=10) and were assessed for spermatozoa viability, morphology, and motility pattern. C-0h samples yielded a spermatozoa population with low viability, altered head morphology and highly deviated motility pattern. E3-3h samples yielded spermatozoa with unaffected viability, head morphology and high progressive motility. In conclusion, E3 extender added to cryopreserved-thawed ram spermatozoa is most efficient in obtaining high viability, unaltered head morphology, and progressive motility.
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Kozubska-Sobocinska, A., and B. Rejduch. "Identification of heterosomes in spermatoza of rams with 54,XX/54,XY chimerism." Veterinární Medicína 53, No. 5 (June 13, 2008): 250–54. http://dx.doi.org/10.17221/1855-vetmed.
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The aim of the study was to identify heterosomes in the semen of three Romanov rams – carriers of leukocyte chimerism (FISH technique) and to determine the proportions between spermatozoa with X and Y chromosomes. The choice of bovine probes for hybridization with ram heterosomes was dictated by genetic conservatism of bovine and ovine heterosomes. The ratio between spermatozoa with a yellow fluorescent signal containing the X chromosome in the haploid set and spermatozoa with a red-purple signal indicating the presence of the Y chromosome, taking into account spermatozoa with no signal, was 52%:43%:5% in ram No. PL100006077676; 47%:44%:9% in ram No. PL100006078031; and 48%:46%:6% in ram No. PL100006078895. The results obtained lead us to conclude that the 54,XX/54,XY chimerism has no effect on sex ratio in offspring.
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Lloyd, R. E., E. Badia, A. Fazeli, P. F. Watson, and W. V. Holt. "Temporal dynamics of ram sperm binding and survival during 48-h coculture with oviducal epithelial cells." Reproduction, Fertility and Development 20, no. 7 (2008): 835. http://dx.doi.org/10.1071/rd08027.
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Following insemination, ram spermatozoa bind to oviducal epithelial cells (OEC) in vivo and remain viable for several hours before fertilisation. In the present study, we investigated whether OEC monolayers reproduce this effect in vitro, performing an analysis of ram sperm binding and survival over an extended (48 h) period at 39°C. We wanted to determine whether the reproductive cycle phase and/or oviducal region would influence ram sperm binding and survival in coculture with OEC and whether reproductive and non-reproductive epithelial cells bound and maintained the viability of ram spermatozoa equivalently. Oviducts were separated into groups based on their ovarian state (follicular or luteal) and then divided into two parts (isthmus and ampulla) for OEC isolation. Sheep kidney epithelial cells (Madin-Darby ovine kidney; MDOK) were purchased commercially. Reproductive cycle phase, but not oviducal region, affected sperm binding to OEC. Although more spermatozoa bound to luteal OEC than to follicular OEC at 1 h, at 24 h follicular OEC had bound more spermatozoa than luteal OEC. Generally, spermatozoa that were bound to OEC and MDOK had enhanced viability at each of the time points investigated (1, 6, 24 and 48 h), but the viability of the OEC-bound spermatozoa was greater than that of the MDOK-bound spermatozoa at 48 h. In conclusion, ram sperm–epithelial cell interactions are temporal, dynamic and depend on the origin of the epithelial cells.
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Bernecic, Naomi C., Bart M. Gadella, Simon P. de Graaf, and Tamara Leahy. "Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm." Reproduction 160, no. 2 (August 2020): 269–80. http://dx.doi.org/10.1530/rep-19-0430.
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Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20–23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.
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Maxwell, WM, GR Welch, and LA Johnson. "Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma." Reproduction, Fertility and Development 8, no. 8 (1996): 1165. http://dx.doi.org/10.1071/rd9961165.
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Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.
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Mehr, Mohammad Roostaei-Ali, Reza Rajabi-Toustani, and Rasool Motamedi-Mojdehi. "Leptin Receptor in Ram Epididymal Spermatozoa." Engineering 04, no. 10 (2012): 121–23. http://dx.doi.org/10.4236/eng.2012.410b031.
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Widiantoro, Krisna, Sri Pantja Madyawati, Trilas Sardjito, Tatik Hernawati, Indah Norma Triana, and Sunaryo Hadi Warsito. "Kualitas post-thawing semen domba Merino dalam bahan pengencer berbasis susu skim-kuning telur yang ditambah isolat crude protein Tirosine Kinase." Ovozoa : Journal of Animal Reproduction 10, no. 2 (August 17, 2021): 39. http://dx.doi.org/10.20473/ovz.v10i2.2021.39-45.
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This study was conducted to determine the effect of crude protein tyrosine kinase (PTK) isolates supplementation into skim milk-egg yolk based diluent to maintain the quality of Merino ram spermatozoa. Four ejaculates of two Merino rams were divided into two groups for the control group (P0): Merino ram semen was diluted in skimmed milk-egg yolk based diluent, and the treatment group (P1): Merino ram semen was diluted in skim milk-egg yolk based diluent contained crude PTK 1,597 mg/ml diluent. All diluted semen was equilibrated for 2 hours at 5 °C and filled into 0.25 mL French straws. The filled straws were placed on steel racks (Cooltop, Minitube) held in liquid nitrogen vapour for 10 minutes at –140 °C, immersed immediately in liquid nitrogen at –196 °C, and stored for 48 hours for later assessment. Post-thawed semen samples were evaluated for spermatozoa motility, viability, and morphological abnormality. The results showed that the spermatozoa motility of fresh semen of Merino ram was 82.5 ± 2.89, which was qualified for freezing. The post-thawing spermatozoa motility, viability, and morphological abnormalities of Merino ram in the P1 group were 34.11 ± 3.26%, 38.00 ± 3.00%, and 12.89 ± 4.54%, respectively. It were higher (p <0.05) than the control group of 24.44 ± 2.9%, 26.67 ± 3.32%, and 21.11 ± 3.02%. It was concluded that the addition of crude PTK isolates of 1.597 mg/ml skim milk-egg yolk diluent improved the quality of post-thawed spermatozoa of Merino ram.
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Leahy, T., J. P. Rickard, N. C. Bernecic, X. Druart, and S. P. de Graaf. "Ram seminal plasma and its functional proteomic assessment." Reproduction 157, no. 6 (June 2019): R243—R256. http://dx.doi.org/10.1530/rep-18-0627.
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Ejaculation results in the confluence of epididymal spermatozoa with secretions of the accessory sex glands. This interaction is not a prerequisite for fertilisation success, but seminal factors do play a crucial role in prolonging the survival of spermatozoa bothin vitroandin vivoby affording protection from handling induced stress and some selective mechanisms of the female reproductive tract. Reproductive biologists have long sought to identify specific factors in seminal plasma that influence sperm function and fertility in these contexts. Many seminal plasma proteins have been identified as diagnostic predictors of sperm function and have been isolated and appliedin vitroto prevent sperm damage associated with the application of artificial reproductive technologies. Proteomic assessment of the spermatozoon, and its surroundings, has provided considerable advances towards these goals and allowed for greater understanding of their physiological function. In this review, the importance of seminal plasma will be examined through a proteomic lens to provide comprehensive analysis of the ram seminal proteome and detail the use of proteomic studies that correlate seminal plasma proteins with ram sperm function and preservation ability.
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Takdir, Moh, Ismaya Ismaya, and Sigit Bintara. "THE PROPORTION OF X AND Y SPERM, VIABILITY AND MOTILITY OF RAM SPERMATOZOA AFTER SEPARATED WITH WHITE EGG ALBUMIN." Buletin Peternakan 41, no. 1 (February 27, 2017): 1. http://dx.doi.org/10.21059/buletinpeternak.v41i1.9130.
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ABSTRACT The aim of this research was to determine the proportion, viability and motility of X and Y ram spermatozoa separated with egg white albumin. Sperm samples derived from Garut ram, which was collected by using an artificial vagina. Observations were made on spermatozoa fraction above and below each medium fraction treatment. There are treatment egg white albumin as separation medium, each medium consisting of fractions top and bottom fraction with different concentration: 1) P0 = sperma before separation (control); 2) P1 = 10% above fraction + 30% lower fraction; P2 = 25% + 45%; P3 = 25% + 75%. Data proportion of X and Y, viability and motility were analyzed statistically by Completely Randomized Design patern in the direction followed by Duncan’s Multiple Range Test for data with a real difference. Separation with egg white albumin affect significantly increased the proportion of spermatozoa X and Y (P≤0.05), but tends to decrease the viability and motility of spermatozoa.The proportion of spermatozoa X and Y was highest in treatment P3,76.76% of spermatozoa X (fraction above 25%) and 79.81% spermatozoa Y (75% lower fraction), with an average viability obtained respectively 68,9% (fraction above) and 59,7% (bottom fraction), motility 77,5% (fraction above) dan 84,0% (bottom fraction). It was concluded that the egg white albumin is very effective in changing the proportions of X and Y ram sperm with the quality of spermatozoa after separation feasible for applications insemination or processed into frozen semen. (Keywords: Garut ram, White egg albumin, Spermatozoa X and Y)
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Gimeno-Martos, S., M. González-Arto, A. Casao, M. Gallego, J. A. Cebrián-Pérez, T. Muiño-Blanco, and R. Pérez-Pé. "Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa." Reproduction 154, no. 4 (October 2017): 469–81. http://dx.doi.org/10.1530/rep-17-0177.
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This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes duringin vitrocapacitation and the actions of progesterone (P4) and 17β-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40–45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERβ). ERα was located in the postacrosomal region of all the spermatozoa and ERβ on the apical region of 63.7% of the cells. The presence of ERβ was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848,P < 0.001). This significantly decreased afterin vitrocapacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 beforein vitrocapacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.
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Luna, Carolina, Marc Yeste, María M. Rivera del Alamo, Juan Domingo, Adriana Casao, Joan E. Rodriguez-Gil, Rosaura Pérez-Pé, José A. Cebrián-Pérez, and Teresa Muiño-Blanco. "Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates." Reproduction, Fertility and Development 29, no. 2 (2017): 394. http://dx.doi.org/10.1071/rd15231.
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It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics. Four sperm subpopulations (SP) were defined after the application of a principal component analysis procedure. Progressive spermatozoa with high straightness (STR) were found in SP1, reflected in the high linearity (LIN) and STR values and low amplitude of lateral head movement (ALH; rapid, non-hyperactivated spermatozoa). SP2 spermatozoa seemed to be starting to acquire hyperactivated motility, while the SP3 group consisted of rapid, hyperactivated spermatozoa. SP4 showed less-vigorous spermatozoa, with non-linear motility. The addition of SPP before in vitro capacitation with EGF induced a decrease in SP1 and an increase in SP3. However, a reduction in the chlortetracycline-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates with the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. These findings allow us to deduce that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation in spermatozoa but maintain them in a decapacitated state.
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Hollinshead, F. K., J. K. O'Brien, W. M. C. Maxwell, and G. Evans. "Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen - thawed sorted X- or Y-chromosome-bearing spermatozoa." Reproduction, Fertility and Development 14, no. 8 (2002): 503. http://dx.doi.org/10.1071/rd02034.
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The fertilizing ability of sex-sorted frozen–thawed ram spermatozoa was assessed after insemination of mature Merino ewes at a synchronized oestrus. Ewes were inseminated into the uterus or utero–tubal junction (UTJ) with a total of 140 × 106 unsorted (control) or 2–4 × 106 sorted (X or Y) frozen–thawed ram spermatozoa 54�to 57 hours after removal of progestagen-impregnated pessaries and an injection of 400 IU of pregnant mare serum gonadotrophin (Folligon®, Intervet). The spermatozoa were separated into X- and Y-chromosome-bearing spermatozoa after analysis with a modified high-speed cell sorter (SX MoFlo®). The number of ewes pregnant after insemination with unsorted frozen–thawed spermatozoa was significantly higher (26/48; 54.3%) than for ewes inseminated with either X- (12/48; 25.0%) or Y-sorted spermatozoa (7/48; 14.6%) (P<0.05). Seventeen of the eighteen lambs produced by ewes inseminated with X-sorted spermatozoa were female (94.4%) and 8/8 lambs from ewes inseminated with Y-sorted spermatozoa were male (100%). The sex ratio of the lambs born to ewes inseminated with sex-sorted spermatozoa was significantly skewed from the 51.3% male and 48.7% female ratio in the control group (P<0.05). This study showed, for the first time, that lambs of predicted sex can be produced after insemination with low numbers of sex-sorted cryopreserved ram spermatozoa.
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Ollero, M., N. García-López, R. Pérez-Pé, J. A. Cebrián-Pérez, and T. Muiño-Blanco. "Surface changes of ram spermatozoa by adsorption of homologous and heterologous seminal plasma proteins revealed by partition in an aqueous two-phase system." Reproduction, Fertility and Development 9, no. 4 (1997): 381. http://dx.doi.org/10.1071/r96037.
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Ram spermatozoa freed from seminal plasma by a dextran ‘swim-up’ procedure were incubated with Tween 20 and fractionated into motile (PEG-rich) and stationary (dextran-rich) fractions by centrifugal countercurrent distribution (CCCD) in an aqueous dextran–Ficoll– polyethylene glycol (PEG) two-phase system. Increasing concentrations of Tween 20 resulted in greater amounts of extracted protein and lower cell viability. Addition of bull seminal plasma increased the proportion of live cells, whereas ram seminal plasma increased the proportion of stationary cells. Proteins isolated from each type of seminal plasma restored the CCCD profile of treated spermatozoa to the right, this effect being reduced when proteins were thermally denatured. Bovine serum albumin induced a slight displacement to the left. No restoration of profile was achieved when ram spermatozoa were exposed to proteins from bull seminal plasma in the presence of protein-free ram seminal plasma. Adsorption of seminal plasma proteins by spermatozoa previously stripped of surface proteins by exposure to detergent reversed the detergent effect on motility. The findings are consistent with the concept that ram seminal plasma contains a factor that interferes with protein adsorption on the cell surface and prevents the protective effect of seminal plasma proteins on maintenance of cell viability
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Leahy, T., J. P. Rickard, R. J. Aitken, and S. P. de Graaf. "Penicillamine prevents ram sperm agglutination in media that support capacitation." REPRODUCTION 151, no. 2 (February 2016): 167–77. http://dx.doi.org/10.1530/rep-15-0413.
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Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7±2.7% to 2.8±1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.
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Maxwell, WM, and S. Salamon. "Liquid storage of ram semen: a review." Reproduction, Fertility and Development 5, no. 6 (1993): 613. http://dx.doi.org/10.1071/rd9930613.
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Research from 1930 to 1992 is reviewed with regard to storage of semen at reduced (0-5 degrees C or 10-15 degrees C) and at ambient temperatures. Diluents investigated have included synthetic buffers combined with sugars and egg yolk or its fractions, milk from various sources, glycine, and other substances. Irrespective of the diluent, dilution rate, temperature or conditions of storage, the spermatozoa deteriorate with time of storage. Changes include reduction in motility and morphological integrity of spermatozoa, accompanied by a decline in their survival in the female reproductive tract, reduction in fertility and increased embryonic loss. In critical studies, fertility declined rapidly when semen stored for more than 24 h was used for cervical insemination, but after intrauterine insemination some spermatozoa maintained their fertilizing capacity up to 10 days. Laparoscopic intrauterine or transcervical inseminations could be the means of improvement of fertility. These methods may eliminate the problem of sperm transport through the cervix and ageing of spermatozoa in the reproductive tract, thereby improving fertilization of ova and reducing embryonic loss.
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Druart, Xavier, Juliette Cognié, Gérard Baril, Frédérique Clément, Jean-Louis Dacheux, and Jean-Luc Gatti. "In vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe genital tract." REPRODUCTION 138, no. 1 (July 2009): 45–53. http://dx.doi.org/10.1530/rep-09-0108.
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The fertility of ram semen after cervical insemination is substantially reduced by 24 h of storage in liquid form. The effects of liquid storage on the transit of ram spermatozoa in the ewe genital tract was investigated using a new procedure allowing direct observation of the spermatozoa in the genital tract. Ejaculated ram spermatozoa were double labeled with R18 and MitoTracker Green FM, and used to inseminate ewes in estrus either cervically through the vagina or laparoscopically into the base of the uterine horns. Four hours after insemination, the spermatozoa were directly observedin situusing fibered confocal fluorescence microscopy in the base, middle and tip of the uterine horns, the utero-tubal junction (UTJ) and the oviduct. The high resolution video images obtained with this technique allowed determination of the distribution of spermatozoa and individual motility in the lumen of the ewe's genital tract. The results showed a gradient of increasing concentration of spermatozoa from the base of the uterus to the UTJ 4 h after intra-uterine insemination into the base of the horns. The UTJ was shown to be a storage region for spermatozoa before their transfer to the oviduct. Thein vitrostorage of spermatozoa in liquid form decreased their migration through the cervix and reduced the proportion of motile spermatozoa and their straight line velocity at the UTJ and their transit into the oviduct.
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Li, Y., M. Sun, J. Zhu, G. Jiao, J. Lin, H. Dong, J. Tan, and J. Zhou. "Species difference in the expression of Fas and Fas ligand in mature mammalian spermatozoa." Czech Journal of Animal Science 62, No. 12 (November 2, 2017): 519–26. http://dx.doi.org/10.17221/18/2017-cjas.
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Although it has been proposed that the Fas and Fas ligand (FasL) may protect ejaculated spermatozoa against apoptosis induced by lipoperoxidative damage and against lymphocytes present in the female genital tract, studies reported conflicting results on the presence of Fas receptors in ejaculated human spermatozoa. Furthermore, the expression of Fas/FasL on mature spermatozoa has not been observed in several important mammals. Using seven species, we observed the possibility for species difference in Fas/FasL expression on mature spermatozoa by both immunofluorescence microscopy and western blot analysis. Whereas intensive signals of Fas immunolabelling were detected in sperm head and middle piece and weak signals observed in the tail in 86–100% of the mouse, rat, bull, ram, and buck spermatozoa, only weak signals were detected on the whole body of 27% boar spermatozoa and in the head of 21% human spermatozoa. The pattern of FasL localization was identical to that of Fas in spermatozoa from human, mouse, rat, ram, and buck, but boar and bull spermatozoa showed weak and intensive FasL signals, respectively, only in the head. Western blotting further confirmed the Fas and FasL expression in mouse, rat, bull, ram, and buck, but not in human and boar spermatozoa. Taken together, the results revealed a marked species difference in Fas/FasL expression and an extensive co-expression of Fas and FasL among mature mammalian spermatozoa, suggesting that whereas spermatozoa from most species may be protected by Fas/FasL, those from human and boar may not use the Fas system for protection.
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Lloyd, R. E., P. F. Watson, and W. V. Holt. "15 HEAT SHOCK COGNATE 70 PROTEIN IMPROVES THE LONG-TERM SURVIVAL OF RAM SPERMATOZOA DURING STORAGE AT 17°C IN A COMMERCIAL EXTENDER." Reproduction, Fertility and Development 20, no. 1 (2008): 88. http://dx.doi.org/10.1071/rdv20n1ab15.
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Heat shock proteins (HSPs) are highly represented among oviductal epithelial cell (OEC) plasma membrane proteins (Sostaric et al. 2006 J. Proteome Res. 5, 3029–3027); their abundance increases in oviductal fluid in response to the presence of spermatozoa (Georgiou et al. 2005 Mol. Cell. Proteomics 4, 1785–1796), and they are capable of binding directly to spermatozoa (Boilard et al. 2004 Biol. Reprod. 71, 1879–1889). As a result of these observations, a role for HSPs in prolonging the functional life of spermatozoa in the oviduct prior to fertilization has been proposed. Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical AI (Salamon and Maxwell 2000 Anim. Reprod. Sci. 62, 77–111). This is due, in part, to extenders being unable to maintain the functional life of ram spermatozoa to the same extent as the oviduct. Therefore, in this study, we aimed to determine the effect of supplementing extenders with the HSPs, HSC70 and HSP70, on the maintenance of ram sperm function during storage. Eight freshly collected ram semen ejaculates were split and diluted in the extenders, INRA96 and RSD-1, to 25 � 106 spermatozoa mL–1, either alone (control) or supplemented with HSC70, HSP70, or α-tubulin (protein with a molecular weight similar to that of the two HSPs). The final concentration of protein in each case was 4 µg mL–1, and the samples were stored at 17�C for 48 h. At 1 h, 6 h, 24 h, and 48 h, the viability and DNA integrity of the stored spermatozoa was determined using the live and dead stains, SYBR�-14 and ethidium homodimer, and the Sperm-Ovis-Halomax kit, respectively. In each case, the mean percentage (%) of viable and DNA-fragmented (DF) spermatozoa was determined and log-transformed prior to analysis using factorial ANOVA. Statistical significance was defined as P < 0.05. In INRA96, but not RSD-1, the beneficial effect of protein supplementation on the % of viable spermatozoa observed during storage was significant (P < 0.001). Most noteworthy was INRA96 supplemented with HSC70, which maintained the % of viable spermatozoa observed during storage significantly (P < 0.001) better than INRA96 alone. Protein supplementation had no significant effect on the % of DF spermatozoa observed during storage. However, the % of DF spermatozoa observed increased significantly (P < 0.001) during storage, irrespective of the presence or absence of protein. On the basis of these findings, it is not implausible to suggest that supplementing the commercially available INRA96 extender with HSC70 would improve the fertility rates observed following cervical AI using stored ram semen. Furthermore, the increase in the incidence of DNA-fragmented spermatozoa observed during storage offers a potential explanation as to why poor fertility rates are often observed when ram semen stored in conventional extenders is used for cervical AI.
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Hollinshead, F. K., L. Gillan, J. K. O'Brien, G. Evans, and W. M. C. Maxwell. "In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing." Reproduction, Fertility and Development 15, no. 6 (2003): 351. http://dx.doi.org/10.1071/rd03060.
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The effect of sex sorting and freeze–thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen–thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen–thawed spermatozoa (60.9 ± 2.9% v. 57.0 ± 3.3% and 4.0 ± 0.1 v. 3.5 ± 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37°C). Sorted and non-sorted (control) frozen–thawed spermatozoa had similar acrosome integrity (73.7 ± 1.8% v. 75.2 ± 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 ± 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 ± 2.6% B pattern) before freezing. Overall, more sorted frozen–thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 ± 0.7%; P < 0.01) and less were uncapacitated (35.5 ± 0.6%; P < 0.05) than non-sorted (control) frozen–thawed spermatozoa (7.7 ± 0.8% and 38.6 ± 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen–thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 ± 11.9 v. 73.9 ± 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 ± 7.8 v. 38.6 ± 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen–thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen–thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen–thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 × 106 (commercial control) frozen–thawed spermatozoa (59%) than for 5, 10, 20 and 40 × 106 total sorted frozen–thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 × 106 resulted in a higher pregnancy rate (31%) than 106 (17%; P < 0.05), but was similar to ewes that received 4 × 106 sorted frozen–thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 × 106 than 5 or 20 × 106 non-sorted (control) or sorted frozen–thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen–thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen–thawed spermatozoa.
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San Agustin, Jovenal T., and George B. Witman. "Reactivation of demembranated, cytosol-free ram spermatozoa." Cell Motility and the Cytoskeleton 24, no. 4 (1993): 264–73. http://dx.doi.org/10.1002/cm.970240407.
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Tsantarliotou, M. P., I. A. Taitzoglou, P. Goulas, and N. A. Kokolis. "Dexamethasone reduces acrosin activity of ram spermatozoa." Andrologia 34, no. 3 (June 2002): 188–93. http://dx.doi.org/10.1046/j.1439-0272.2002.00491.x.
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Upreti, G. C., S. R. Payne, D. M. Duganzich, J. E. Oliver, and J. F. Smith. "Enzyme leakage during cryopreservation of ram spermatozoa." Animal Reproduction Science 41, no. 1 (January 1996): 27–36. http://dx.doi.org/10.1016/0378-4320(95)01442-x.
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Ahangari, Y. J., M. Nowrozi, and A. Soleimani. "The effect of dilution rates and freezing methods on post-thawing motility of Baluchi ram spermatozoa." Proceedings of the British Society of Animal Science 2002 (2002): 130. http://dx.doi.org/10.1017/s1752756200007869.
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For the effective use of Artificial insemination technique in sheep industry, investigation on the methods of ram semen dilution and freezing is necessary. Ahangari (1992) showed that various rates of dilution from 1:1 to 1:4 did not affect p>0.05 post thawing survival of Cambridge ram spermatozoa. Fiser et al (1987) achieved a 73% c.f. 67% pregnancy rate using thawed semen, previously frozen to -100 and -79 respectively. The objective of this study was to investigate the effect of two rates of dilution of semen and two methods of freezing on post-thawing motility of ram spermatozoa.
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NTEMKA, A., I. A. TSAKMAKIDIS, E. KIOSSIS, A. MILOVANOVIĆ, and C. M. BOSCOS. "Current status and advances in ram semen cryopreservation." Journal of the Hellenic Veterinary Medical Society 69, no. 2 (July 18, 2018): 911. http://dx.doi.org/10.12681/jhvms.18014.
Full textAbstract:
Ram semen cryopreservation contributes to genetic improvement through artificial insemination, eliminates geographical barriers in artificial insemination application and supports the preservation of endangered breeds thus the conservation of biodiversity. Sperm freezing process induces ultrastructural, biochemical and functional changes of spermatozoa. Especially, spermatozoa’s membranes and chromatin can be damaged, sperm membranes’ permeability is increased, hyper oxidation and formation of reactive oxygen species takes place, affecting fertilizing ability and subsequent early embryonic development. Aiming to improve ram frozen-thawed semen’s fertilizing capacity, many scientific investigations took place. Among them the composition of semen extenders, was a main point of interest. Semen preservation extenders regulate and support an environment of adequate pH and buffering capacity to protect spermatozoa from osmotic and cryogenic stress. Therefore, permeating (glycerol, dimethyl sulfoxide) and non-permeat ing (egg yolk, skimmed milk) cryoprotectants, sugars (glucose, lactose, trehalose, raffinose), salts (sodium citrate, citric acid) and antioxidants (amino acids, vitamins, enzymes) have been added and tested. Moreover, semen dilution rate, storage temperature, cooling rate and thawing protocol, are also some key factors that have been studied. The research results of this scientific topic are encouraging, not only about the freezing and thawing procedures, but also about the improvement of the additives’ properties. However, further research is needed to enhance the fertilizing ability of ram frozen-thawed semen, making its use practical in sheep reproductive management by the application of cervical artificial insemination.
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Gimeno-Martos, Silvia, Adriana Casao, Marc Yeste, José A. Cebrián-Pérez, Teresa Muiño-Blanco, and Rosaura Pérez-Pé. "Melatonin reduces cAMP-stimulated capacitation of ram spermatozoa." Reproduction, Fertility and Development 31, no. 2 (2019): 420. http://dx.doi.org/10.1071/rd18087.
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The presence of melatonin receptors on the surface of ram spermatozoa has led to speculation about melatonin having a role in sperm functionality. The aim of this study was to elucidate the mechanism through which melatonin regulates ram sperm capacitation induced by a cocktail containing cAMP-elevating agents. Cocktail samples capacitated in the presence of 1µM melatonin showed lower percentages of capacitated spermatozoa (chlortetracycline staining; P<0.001) together with a decrease in protein tyrosine phosphorylation (P<0.01) and lower levels of reactive oxygen species (ROS) and cAMP (P<0.05) compared with cocktail samples without the hormone. Determination of kinematic parameters, together with principal component and cluster analyses, allowed us to define four sperm subpopulations (SP). After 3h of incubation with cAMP-elevating agents, the percentages of spermatozoa belonging to SP1 (high straightness) and SP4 (less-vigorous spermatozoa with non-linear motility) increased while SP2 and SP3 (rapid spermatozoa starting hyperactivation or already hyperactivated) decreased compared with the control sample. The presence of melatonin at 100 pM and 10nM restored these subpopulations to values closer to those found in the control sample. These results indicate that melatonin at micromolar concentrations modulates ram sperm capacitation induced by cAMP-elevating agents, reducing ROS and cAMP levels, whereas at lower concentrations melatonin modifies motile sperm subpopulations. These findings warrant further studies on the potential use of melatonin for controlling capacitation in artificial insemination procedures.
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Ahangari, Y. J., R. F. E. Axford, and I. Ap Dewi. "A spectrophotometric method for the assessment of vigour of ram semen." Proceedings of the British Society of Animal Science 1995 (March 1995): 136. http://dx.doi.org/10.1017/s0308229600029020.
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An ideal method of assessment of semen would be simple, rapid and objective with a high correlation with the ability of semen sample to cause conception in the ewes (Ahangari, 1992). Currently the proportion of motile spermatozoa is determined by visual microscopic appraisal. Although, this method of assessment is rapid and easy, it is subjective. Jasko et al (1989) described a spetrophotometric procedure for measuring equine spermatozoan motility objectively. In the present study a modification of the method of Jasko et al (1989) was tested and applied to ram semen.
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Paul, R. K., K. Balaganur, S. V. Bahire, D. Kumar, and R. Singh. "Supplementation of cauda epididymal plasma improves sperm characteristics following liquid preservation of ram semen at 3–5°C." Reproduction, Fertility and Development 30, no. 11 (2018): 1389. http://dx.doi.org/10.1071/rd18063.
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Mammalian spermatozoa remain immotile and metabolically inactive in the cauda epididymidis, thus maintaining fertility for several weeks. The aim of this study was to functionally characterise and evaluate the effect of cauda epididymal plasma (CEP) on liquid preservation of ram spermatozoa. Four experiments were conducted to investigate the effects of: (1) CEP and its fractions on sperm motility; (2) CEP (10%, 15%, 20% v/v) on liquid preservation of ram spermatozoa; (3) seminal plasma (SP; 20%, 30%, 50% v/v) on liquid-preserved spermatozoa; and (4) both CEP and post-storage SP treatment on sperm characteristics. Biochemical characterisation of ram CEP revealed high protein (30.9 mg mL−1), catalase (68.9 IU mL−1), alkaline phosphatase (17.5 IU mL−1) activities and total antioxidant capacity (1112 µM Trolox equivalent). Progressive motility of prewashed cauda spermatozoa was reduced (P < 0.05) by CEP or its protein-rich fraction compared with protein-free plasma or phosphate-buffered saline. After 48 h storage, total motility, rapid motility (average path velocity >75 µm s−1; 53.9%, 73.5% and 71.8% with 0, 15% and 20% CEP respectively) and straight line velocity (86.3, 102.1 and 102.4 µm s−1 with 0, 15% and 20% CEP respectively) were significantly (P < 0.05) higher in the CEP-treated groups than the control. Viability and acrosomal integrity were similar between groups; however, functional membrane integrity was higher (P < 0.05) in the 15% CEP-treated group. Treatment of liquid-preserved spermatozoa with either 20%, 30% or 50% SP improved (P < 0.05) rapid motility and kinematics at each time point of storage compared with control. In conclusion, liquid preservation of ram spermatozoa in the presence of 15% or 20% CEP and post-storage treatment with SP significantly improve sperm characteristics.
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Rickard, J. P., T. Pini, C. Soleilhavoup, J. Cognie, R. Bathgate, G. W. Lynch, G. Evans, W. M. C. Maxwell, X. Druart, and S. P. de Graaf. "Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa." REPRODUCTION 148, no. 5 (November 2014): 469–78. http://dx.doi.org/10.1530/rep-14-0285.
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Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.
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Leahy, T., J. I. Marti, G. Evans, and W. M. C. Maxwell. "Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage." Reproduction, Fertility and Development 21, no. 4 (2009): 571. http://dx.doi.org/10.1071/rd08238.
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Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.
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Mortimer, Sharon T., and W. M. Chis Maxwell. "Effect of medium on the kinematics of frozen–thawed ram spermatozoa." Reproduction 127, no. 2 (February 2004): 285–91. http://dx.doi.org/10.1530/rep.1.00075.
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Cervically inseminated cryopreserved ram spermatozoa have reduced fertility due to poor mucus-penetrating ability. This effect is ameliorated by the addition of 20% (v/v) seminal plasma (SP) to the phosphate-buffered saline (PBS) thawing medium. The aims of this study were to determine whether the impaired mucus penetration was due to alterations in the sperm motility and, if so, whether these alterations were due to the SP or its viscosity, or to the medium components. To this end, artificial SP medium (ASP), a medium which supports motility but not capacitation, was compared with PBS and SP. Thawed, pooled semen from seven mature rams was layered under 1 ml each of PBS, SP and ASP and motile spermatozoa allowed to swim up (37 °C, 30 min). Upper regions of the overlays were harvested, and the capacitation status of the spermatozoa in each suspension determined by chlortetracycline (CTC) analysis. Sperm movement was videotaped in 300 μm chambers for both computer-aided sperm analysis assessment and manual flagellar curvature analysis. There was no effect of the culture medium on the concentration of spermatozoa recovered by swim up, nor on the proportion of motile spermatozoa. However, the spermatozoa resuspended in PBS did show changes associated with capacitation in both the CTC-binding patterns and in their movement patterns. These changes were significantly greater than those observed in spermatozoa resuspended in SP or ASP. These results indicated that the differences in sperm movement and function observed in SP medium were not due to changes in viscosity, but rather to components of the medium.
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Lloyd, R. E., E. Badia, P. F. Watson, and W. V. Holt. "325 PROLONGING THE LIFE OF RAM SPERMATOZOA IN VITRO USING OVIDUCTAL EPITHELIAL CELLS." Reproduction, Fertility and Development 19, no. 1 (2007): 278. http://dx.doi.org/10.1071/rdv19n1ab325.
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Although binding to oviductal epithelial cells (OEC) enhances both bull (Ellington et al. 1991 Theriogenology 35, 970–977) and boar (Fazeli et al. 1997 J. Reprod. Fertil. 20, 49 abst) sperm survival in vitro, the ram sperm-OEC interaction is largely uninvestigated. Consequently, this study aimed to determine (1) whether ram spermatozoa bind to OEC, and (2) whether this binding prolongs ram sperm survival in vitro. Four types of OEC (follicular isthmus, follicular ampulla, luteal isthmus, and luteal ampulla), isolated from the oviducts of ewes slaughtered locally, and sheep kidney epithelial (MDOK)cells, purchased commercially, were used in this study. Each cell type was grown in separate wells of chamber slides in a humidified incubator (39�C and 5 % CO2 in air) overnight. The next day, the growth medium was removed and the cells were washed once with PBS. Ficoll-washed ram spermatozoa, resuspended in Tyrode's medium, were added to the cells; as controls, spermatozoa were added to medium only (no OEC) in some wells. The viability of spermatozoa, unbound and bound to the cells, and in medium alone, was determined at 0, 1, 6, 24, and 48 h by incubating with the live and dead stains, Sybr-14 (100 nm) and ethidium homodimer (1 �m), respectively, for 15 min. In each case, 200 spermatozoa were counted and classified as live or dead using a fluorescence microscope. The experiment was replicated 4 times using an ejaculate from a different ram each time. The mean percentage (%) of viable spermatozoa was determined in each case by normalizing to the viability determined for each ram ejaculate at 0 h. Data were log transformed and analyzed using factorial ANOVA. Statistical significancewas defined as P < 0.05. Following 1, 6, 24, and 48 h of co-culture, the % of viable spermatozoa bound to the OEC for each of the culture periods (mean � SEM: 129.87 � 7.34, 101.21 � 7.34, 126.72 � 7.34, and 135.97 � 7.34, respectively) was significantly greater (P < 0.001) than the % unbound to the OEC (20.94 � 7.34, 9.55 � 7.34, 14.47 � 7.34, and 31.38 � 7.34, respectively), and in medium alone (35.44 � 14.67, 31.38 � 14.67, 24.45 � 14.67, and 28.49 � 14.67, respectively). This effect was irrespective of the oviductal region and the reproductive cycle of the ewe used to derive the OEC. Interestingly, the MDOK cells, like the OEC, selectively bound viable spermatozoa following 1 and 6 h of co-culture (136.75 � 39.41 and 153.31 � 39.41, respectively). However, the % of viable spermatozoa bound to the MDOK was significantly less than to the OEC following 24 h (P = 0.05) and 48 h (P < 0.01) of co-culture (93.52 � 39.41 and 72.85 � 39.41, respectively). These data show that epithelial cells originating from reproductive and non-reproductive tissues both are capable of binding and prolonging sperm viability over the first 6 h of co-culture, but only OEC are capable of prolonging sperm viability for longer periods of time. This work was funded by DEFRA (UK), Innovis (UK), and IMV (France).
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Gillan, L., G. Evans, and W. M. C. Maxwell. "The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro." Reproduction, Fertility and Development 12, no. 6 (2000): 237. http://dx.doi.org/10.1071/rd00032.
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In order to investigate the interaction of fresh and frozen–thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen—thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen—thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen–thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen—thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen—thawed spermatozoa. The findings reported in this study show that fresh and frozen—thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.
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Stojanov, T., S. J. Robinson, S. L. Rhodes, J. K. O'Brien, G. Evans, and W. M. C. Maxwell. "In vitro fertilisation with chilled-stored ram spermatozoa." Theriogenology 41, no. 1 (January 1994): 302. http://dx.doi.org/10.1016/s0093-691x(05)80212-2.
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Hollinshead, F. K., J. K. O’Brien, L. Gillan, M. Meyers, W. M. C. Maxwell, and G. Evans. "Liquid storage of flow cytometrically sorted ram spermatozoa." Theriogenology 62, no. 3-4 (August 2004): 587–605. http://dx.doi.org/10.1016/j.theriogenology.2003.11.020.
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Hinkovska, V. Tz, A. B. Momchilova, D. H. Petkova, and K. S. Koumanov. "Phospholipase A2 activity in ram spermatozoa plasma membranes." International Journal of Biochemistry 19, no. 6 (January 1987): 569–72. http://dx.doi.org/10.1016/0020-711x(87)90143-1.
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Arando, Ander, Juan V. Delgado, Francisco A. Arrebola, José M. León, Carlos J. Alcalá, and Carlos C. Pérez-Marín. "Vitrification induces critical subcellular damages in ram spermatozoa." Cryobiology 87 (April 2019): 52–59. http://dx.doi.org/10.1016/j.cryobiol.2019.02.005.
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Martí, E., R. Pérez-Pé, C. Colás, T. Muiño-Blanco, and J. A. Cebrián-Pérez. "Study of apoptosis-related markers in ram spermatozoa." Animal Reproduction Science 106, no. 1-2 (June 2008): 113–32. http://dx.doi.org/10.1016/j.anireprosci.2007.04.009.
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Al Dulaimi, Makki Khalaf Hussein, Paul Rodian Tapaloaga, Dana Tapaloaga, Ion Calin, Marius Popica, and Diana Moru. "Age related morphometric features of spermatozoa in ram." Journal of Biotechnology 208 (August 2015): S37. http://dx.doi.org/10.1016/j.jbiotec.2015.06.103.
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Oláh, János, Tamás Pécsi, András Kovács, Anna Pécsi, and András Jávor. "Tenability of ram semen." Acta Agraria Debreceniensis, no. 31 (November 24, 2008): 63–66. http://dx.doi.org/10.34101/actaagrar/31/3008.
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It could be stated that the diluted semen of Awassi rams taken in the breeding season preserved its fertilizing abilityat different temperatures for different periods of time. The motility of spermatozoa kept at 23 vs. 8oC was checked daily. The largest spread of data was observed 24 hours after taking the semen, then the motility rate of cells showed a linear decrease. Motility results of fresh and frozen-thawed samples were compared also after heat resistance test and significant differences were found between these groups. Significant individual differences were observedin the sperm motility after heat resistance test.
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Gillan, L., K. Skovgold, P. F. Watson, W. M. C. Maxwell, and G. Evans. "Fate and functional integrity of fresh and frozen - thawed ram spermatozoa following intrauterine insemination." Reproduction, Fertility and Development 11, no. 6 (1999): 309. http://dx.doi.org/10.1071/rd99074.
Full textAbstract:
Ewes in a synchronized oestrus were inseminated (intrauterine) with fresh and frozen–thawed spermatozoa and the spermatozoa were either recovered from each section of the reproductive tract after the animal was killed (Experiments 1a and 1b) or after they were voided from the cervix (Experiment 2). In Experiment 1a, only 1.2 0.27% of the original inseminate was recovered. Placing a ligature at the base of the uterine horn in Experiment 1b led to the recovery of 3.0 0.33% of the original inseminate, located mainly in each uterine horn (33.1 5.48%), and each isthmic and ampullary region of the oviduct (2.9 5.48% and 4.0 5.48%, respectively). A higher proportion of spermatozoa recovered from the isthmus were uncapacitated when observed by chlortetracycline staining than those recovered from the uterus (26.4 1.92% and 15.6 1.92%, respectively, P<0.05). Experiment 2 showed that large proportions of spermatozoa were voided from the tract through the vagina, with similar numbers of fresh and frozen–thawed spermatozoa lost from the tract. However, frozen–thawed spermatozoa were lost at a faster rate than fresh (P<0.05) and with a more advanced membrane state (66.8 1.30% and 53.2 1.30% were acrosome reacted respectively; P<0.001). Large numbers of recovered spermatozoa had lost their tails, with frozen–thawed spermatozoa more susceptible to tail loss than fresh spermatozoa (55.0 0.96% and 45.5 0.96% respectively; P<0.05).
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Pérez-Marín, Carlos Carmelo, Ander Arando, Francisco Maroto-Molina, Alberto Marín, and Juan Vicente Delgado. "Las subpoblaciones de espermatozoides y su calidad en fracciones producidas por la centrifugación de una sola capa en muestras frescas y normospérmicas de esperma de cordero." Revista Mexicana de Ciencias Pecuarias 12, no. 2 (September 15, 2021): 386–401. http://dx.doi.org/10.22319/rmcp.v12i2.5683.
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Single layer centrifugation (SLC) technique has been developed to select the best sperm population in the ejaculate in order to increase the fertilization rates by artificial insemination or in vitro fertilization. Normospermic ram semen samples containing 800 and 3,000 × 106 sperms/ml (C800 and C3000, respectively) were processed by SLC. Three sperm fractions were separated in each sample following silica-coloidal sperm centrifugation and sperm yield, quality and subpopulations were analyzed in each one. In C800 group, the sperm recovery rate did not vary in any studied fraction, but when samples were highly concentrated (C3000) the top fraction (F1) contained significantly higher spermatozoa than bottom fraction (F3). Also, it was observed that F1 in C3000 had got a significantly higher percentage of spermatozoa (53.2 %) than in C800, while the quantity of spermatozoa recovered in fraction 2 was lower (25.2 % vs 45.4 %). Based on the sperm motility parameters, three sperm subpopulations were identified: SP1, low velocity spermatozoa showing no progressive movement (19.1 %); SP2, rapid and progressive spermatozoa (43.7 %); and SP3, rapid spermatozoa but non-linear movement (37.2 %). While SLC has been implemented for sperm separation in suboptimal and/or low concentrated sperm samples, this trial demonstrates that SLC is not efficient to separate different sperm populations in normospermic ram sperm samples containing high concentrations of spermatozoa.
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El-Shahat, K., T. Ismail, M. Badr, and K. Zaki. "20 EFFECT OF PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE TREATMENT ON MOTILITY, HYPERACTIVITY AND ACROSOME REACTION OF RAM SPERMATOZOA." Reproduction, Fertility and Development 29, no. 1 (2017): 117. http://dx.doi.org/10.1071/rdv29n1ab20.
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The aim of this study was to determine the effects of in vitro treatment of freshly ejaculated ram spermatozoa with different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) at different incubation times on motility, hyperactivity (HA) and acrosome reaction (AR). Freshly ejaculated spermatozoa collected from three rams were pooled and then subjected to swim up technique in modified sperm Tyrode’s albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of PHE (0, 10, 20, 30, 40, 50, 75, and 100 mM). Following incubation (0, 1, 2, 3 and 4 h), sperm motility and hyperactivity were examined under the phase contrast microscope and acrosome reaction was detected by staining of the spermatozoa with silver nitrate. Results showed that treatment of spermatozoa with high concentrations of PHE (30, 40, 50, 75, and 100 mM) significantly increased the motility when compared with the control immediately after dilution (82.0, 82.0, 81.0, 82.0, 82.0 v.76.0%, respectively).This increase existed for the first and second hours of incubation. However, when the incubation time was increased for more than 2 h total motility significantly decreased (P < 0.05, ANOVA) as compared with the control. The same trend was observed in hyperactive motility. Treatment of spermatozoa with 50 and 75 mM of PHE for 1 h significantly increased the percentage of sperm with incomplete AR (20.0 and 27.0% respectively). At 4 h incubation, (49.0%) of spermatozoa treated with 75 mM PHE had undergone complete AR. Furthermore, the maximum value of total acrosome reaction (73.0%) was achieved at 4 h post incubation after addition of 75 mM PHE to ram sperm. In conclusion, under our experimental conditions, 75 mM PHE for 4 h was considered the best concentration of PHE for treatment of ejaculated ram spermatozoa for in vitro induction of acrosome reaction.
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Rickard, J. P., R. E. Schmidt, J. W. Maddison, R. Bathgate, G. W. Lynch, X. Druart, and S. P. de Graaf. "Variation in seminal plasma alters the ability of ram spermatozoa to survive cryopreservation." Reproduction, Fertility and Development 28, no. 4 (2016): 516. http://dx.doi.org/10.1071/rd14123.
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Variation in the effect of seminal plasma on sperm function and fertility has been hypothesised to be due to differences between males and their seminal plasma composition. The freezing resilience of individual rams (n = 17) was investigated to characterise inter-male variation. This was determined by measuring the degree of change in motility induced by cryopreservation (Experiment 1). Experiment 2 examined the effect of pooled seminal plasma from rams identified as having high or low resilience to freezing on the cryosurvival of washed spermatozoa from either high (n = 3) or low (n = 3) sperm freezing resilience rams. Immediately after thawing and throughout the incubation period (0–4 h), spermatozoa from high-resilience rams frozen with high-resilience seminal plasma demonstrated superior motility to spermatozoa from high-resilience rams frozen with low-resilience seminal plasma (P < 0.001). Similarly, spermatozoa from low-resilience rams frozen with high-resilience seminal plasma exhibited higher motility than spermatozoa from low-resilience rams frozen with low-resilience seminal plasma immediately after thawing (0 h; P < 0.001). The present study shows that variation in freezing resilience of ram spermatozoa is related to the source and composition of the seminal plasma.