scholarly journals Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 111-117 ◽  
Author(s):  
Florencia Ardon ◽  
Susan S Suarez
Keyword(s):  
Seminal Plasma ◽  
Seminal Vesicles ◽  
Outer Leaflet ◽  
Protein Coat ◽  
Frozen Semen ◽  
Custom Made ◽  
Sperm Plasma Membrane ◽  
Molecular Effects ◽  
Bsp Proteins ◽  
Live Sperm

Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

69 REACTIVE OXYGEN SPECIES EVALUATION OF DONKEY FROZEN SEMEN ADDED TO HOMOLOGOUS SEMINAL PLASMA ON POST-THAW

2015 ◽  
Vol 27 (1) ◽  
pp. 127
Author(s):  
P. V. L. Oliveira ◽  
J. V. Oliveira ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
C. P. Freitas-Dell'aqua ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Kinetic Parameters ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Reactive Oxygen ◽  
Computer Assisted ◽  
Oxygen Species ◽  
Inflammation Response ◽  
Frozen Semen ◽  
Uterine Inflammation

For many years the pregnancy rate of donkey frozen semen presented lower results in donkey jennies; however, a recent study showed an increase in pregnancy rates using frozen semen added to seminal plasma on post-thaw. A hypothesis for this result is the higher uterine inflammation response after breeding when using seminal plasma. The same studies demonstrated higher uterine inflammation in the presence of higher reactive oxygen species concentration. The aim of the present study was to evaluate the content of reactive oxygen species in donkey frozen semen added to homologous seminal plasma on post-thaw. Five ejaculates from each 3 donkeys were used. Semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The semen was frozen with Botu-CryoTM extender (Botupharma, Brazil) in an isothermal box in straws containing 100 × 106 of total sperm. The samples were thawed at 46°C for 20 s. After this, the straws of each donkey were divided in 2 group: control group (CG), in which the semen was incubated at 37°C for 5 min, and plasma seminal group (PG), in which the semen was incubated at 37°C for 5 min with 70% of homologous seminal plasma. Sperm kinetic parameters were evaluated by computer-assisted semen analysis, and the plasma membrane integrity (propidium iodide and fluorescein isothiocyanate -PSA) and reactive oxygen species (5–6-carboxi-2,7-diclorodihidrofluoresceindiacetate) were evaluated by flow cytometer. Comparison of sperm parameters was performed by t-test. Total motility (%, CG = 75.4 ± 8.2a v. PG = 57.5 ± 16.4b), progressive motility (%, CG = 42.0 ± 8.7a v. PG = 33.3 ± 13.2b), progressive angular velocity (μm/s, CG = 95.8 ± 10.8a v. PG = 88.9 ± 10.9b), and percentage of rapid sperm (%, CG = 58.4 ± 12.5a v. PG = 41.0 ± 17.3b) were higher in CG compare with PG. No difference (P < 0.05) was observed in membrane integrity (%, CG = 20.7 ± 7.4 v. PG = 20.6 ± 7.8); however, reactive oxygen species (%, CG = 12.3 ± 10.6a v. PG = 81.8 ± 32.5b) were higher in PG. The results of this study showed that the addition of homologous seminal plasma on post-thaw decreases the sperm kinetic parameters and viability of donkey frozen semen but increases reactive oxygen species, and this may cause higher uterine inflammation response in donkey jennies and increase their fertility.

67 EFFECTS OF ANTIOXIDANTS LACTOFERRIN AND CATALASE ON STALLION FROZEN SEMEN

2015 ◽  
Vol 27 (1) ◽  
pp. 126
Author(s):  
H. S. Martins ◽  
M. F. Brito ◽  
I. B. M. Sampaio ◽  
R. Stahlberg ◽  
M. R. Souza ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Seminal Plasma ◽  
Skim Milk ◽  
Straight Line ◽  
Frozen Semen ◽  
Significant Difference ◽  
Sperm Membrane ◽  
Positive Effect ◽  
Artificial Vagina ◽  
Motility Parameters

During cryopreservation, the sperm were submitted to an increased generation of reactive oxygen species. Furthermore, because of the large portion of seminal plasma removal, there is a decrease of sperm antioxidant protection. Addition of antioxidants proteins found in seminal plasma, such as lactoferrin (Lf) and catalase (Cat), to the freezing semen extenders could protect the sperm during cryopreservation. Lactoferrin is a transferrin, which prevents the hydroxyl radicals generation, and Cat plays an antioxidant role. The aim of this study was to determine the effects of Lf and Cat supplementation to the INRA 82 freezing extender (Battelier et al. 1997) on sperm motility parameters and membrane functionality of stallion frozen semen. Semen from 6 stallions was collected with an artificial vagina, diluted with Kenney extender (1 : 1), and centrifuged (500 × g, 10 min). The supernatant was discarded, and sperm number per milliliter was calculated. Semen was resuspended with 3 extenders to 100 × 106 sperm mL–1. The treatments were distributed in (F1) control, INRA 82 freezing extender (Battelier et al. 1997), (F2) F1+ 500 μg mL–1 lactoferrin, and F3) F1 + 200 IU mL–1 catalase. Semen samples were packaged in 0.5-mL straws and cooled to 5°C (0.27°C min–1). For semen freezing, the straws were laid over the LN vapor for 20 min and plunged into the LN. The straws were thawed at 37°C for 30 s. Motility parameters of frozen semen were determined using a computer sperm cell analysis, and sperm membrane functionality was assessed by the hyposmotic swelling test (Lagares et al. 1998). The data were analysed using Friedman test using stallion as a block. A probability of P < 0.05 was considered significant. There was no significant difference between the percentage of total sperm motility (median, minimum-maximum value; F1: 29.9, 11.0–82.7; F2: 49.8, 7.7–55.2; F3: 39.8, 5.7–92) and progressive sperm motility (F1: 7.1, 3.2–23.3; F2: 13.4, 2.6-22.4; F3: 15.6, 1.1–29.6), and functional sperm membrane (F1: 26.7, 14.7–56.2; F2: 50.5, 15.7–61.7; F3: 46.6, 13.8–50.9) with regard to the treatment. However, the velocity parameters: velocity average path (F1: 29.3, 22.1–33.80; F2: 34.6, 24.8–44; F3: 35.7, 18.2–42.6), velocity curvilinear (F1: 36.9, 30.5–45.1; F2: 42.5, 34.7–51; F3: 44.6, 25.5–50.9), and velocity straight line (F1: 23.4, 17–3.60; F2: 28.9, 18.8–38.2; F3: 26.6, 13.6–37.2) in the treatment with Lf (F2) were higher compared with the control (F1; P < 0.05). These results corroborate with studies reporting the lack of positive effect on equine sperm motility when antioxidants were added to skim milk-based extenders. Although the addition of Lf or Cat to skim milk-based extenders did not improve the motility sperm characteristics and sperm membrane functionality, more studies about the positive effect of Lf on the velocity parameters are necessary. Lactoferrin could then play an important role on the oxidative metabolism, which provides energy to the sperm movement.Acknowledgments to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, for the financial support.

scholarly journals Pharmacokinetic Profiles of Nevirapine and Indinavir in Various Fractions of Seminal Plasma

2001 ◽  
Vol 45 (10) ◽  
pp. 2902-2907 ◽  
Author(s):  
Rieneke M. E. van Praag ◽  
Sjoerd Repping ◽  
Jan W. A. de Vries ◽  
Joep M. A. Lange ◽  
Richard M. W. Hoetelmans ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Seminal Vesicles ◽  
Sexual Abstinence ◽  
Limited Data ◽  
Dosing Interval ◽  
Drug Ingestion ◽  
Drug Concentrations ◽  
Antiretroviral Drug ◽  
Immunodeficiency Virus

ABSTRACT Limited data are available on antiretroviral drug concentrations in seminal plasma during a dosing interval. Further, since human ejaculate is composed of fluids originating from the testes, the seminal vesicles, and the prostate, all having different physiological characteristics, drug concentrations in total seminal plasma do not necessarily reflect concentrations in the separate compartments. Five human immunodeficiency virus type 1-infected patients on nevirapine (NVP; 200 mg twice a day [b.i.d.]) and/or indinavir (IDV; 800 mg b.i.d. with ritonavir, 100 mg b.i.d.) regimens used a split ejaculate technique to separate seminal plasma in two fractions, representing fluids from the testes and prostate (first fraction) and fluids from the seminal vesicles (second fraction). Split-ejaculate samples were provided at 0, 2, 5, and 8 h after drug ingestion, on separate days after 3 days of sexual abstinence. NVP and IDV showed time-dependent concentrations in seminal plasma, with peak concentrations in both fractions at 2 and 2 to 5 h, respectively, after drug ingestion. The NVP concentrations were not significantly different between the first and second fractions of the ejaculate at all time points measured and were in the therapeutic range, except for the predose concentration in two patients. The median (range) predose IDV concentrations in the first and second fractions of the ejaculate were 448 (353 to 1,015) ng/ml and 527 (240 to 849) ng/ml, respectively (P = 0.7). In conclusion, NVP and IDV concentrations in seminal plasma are dependent on the time after drug ingestion. Furthermore, our data suggest that NVP and IDV achieve therapeutic concentrations in both the testes and prostate and the seminal vesicles throughout the dosing interval.

THE COMPOSITION OF THE STALLION'S SEMEN

1956 ◽  
Vol 13 (3) ◽  
pp. 279-NP ◽  
Author(s):  
T. MANN ◽  
E. LEONE ◽  
C. POLGE
Keyword(s):  
Citric Acid ◽  
Seminal Plasma ◽  
Seminal Vesicles ◽  
Chemical Methods ◽  
Quantitative Indication ◽  
Final Composition ◽  
Vesicular Secretion

SUMMARY The composition of the semen of the stallion was studied by microscopic and chemical methods and the extent of fluctuations in the same animal has been determined. Two characteristic constituents of the seminal plasma of the horse, ergothioneine and citric acid, have been shown to originate in the ampullae and seminal vesicles respectively. With the aid of chemical methods for the analysis of semen it has been possible to obtain a general quantitative indication of the contribution of the ampullar and vesicular secretions towards the final composition of normal stallion ejaculates. By the method of fractionate collection of semen, an attempt was made to determine the sequence with which the different portions of the semen of the stallion are ejaculated. It has been demonstrated that the sperm-containing fraction is rich in ergothioneine but not in citric acid, and that it is followed by a post-sperm fraction, which has a high content of citric acid and consists mainly of the vesicular secretion. The seminal characteristics of the jackass have been shown to resemble those of the stallion.

scholarly journals Major proteins of bovine seminal plasma inhibit phospholipase A2

1994 ◽  
Vol 303 (1) ◽  
pp. 121-128 ◽  
Author(s):  
P Manjunath ◽  
S Soubeyrand ◽  
L Chandonnet ◽  
K D Roberts
Keyword(s):  
Enzyme Activity ◽  
Phospholipase A2 ◽  
Seminal Plasma ◽  
Inhibitory Activity ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Pla2 Activity ◽  
Sperm Capacitation ◽  
Bsp Proteins

We have recently shown that the major proteins of bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called BSP proteins) bind to spermatozoa and that the binding sites on the plasma membrane of spermatozoa are choline phospholipids. In view of the fact that these phospholipids are substrates for phospholipase A2 (PLA2), a key enzyme in sperm capacitation and the acrosome reaction, the effect of BSP proteins on this enzyme activity was investigated. Since these BSP proteins are ubiquitous, the effect on pig pancreatic PLA2 was also studied. In contrast with control proteins, when preincubated with phosphatidylcholine as substrate, all BSP proteins inhibited both pancreatic and sperm PLA2 activity in a dose-dependent manner and in the presence of 1-6 microM BSP protein the enzyme activity was completely abolished. When phosphatidylethanolamine was used as substrate, only pancreatic PLA2 was inhibited. On the other hand, when the BSP proteins were preincubated with the enzyme followed by addition of substrate, a biphasic effect was observed; there was stimulation of enzyme activity below 1.3 microM BSP followed by an inhibition above this concentration. The inhibitory activity was trypsin-sensitive but heat-resistant. The effect of co-incubation of heparin, which is implicated in sperm capacitation and which also interacts with BSP proteins, was studied. Heparin (10 microM) had no effect on the PLA2 inhibitory activity exhibited by all BSP proteins. The PLA2 inhibitory effect exhibited by BSP proteins was abolished with excess substrate. The BSP proteins were adsorbed on PLA2-agarose and could be affinity cross-linked to the enzyme, indicating a direct interaction of enzyme with the inhibitor. These results suggest that these BSP proteins modulate PLA2 activity and therefore, phospholipid metabolism.

Characterisation of the progesterone receptor on canine spermatozoa

2005 ◽  
Vol 17 (7) ◽  
pp. 733 ◽  
Author(s):  
Jui-Te Wu ◽  
Pei-Shiue Tsai ◽  
Shuang-Lin Lee ◽  
Feng-Pang Cheng
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Progesterone Receptor ◽  
Seminal Plasma ◽  
High Capacity ◽  
Fluorescein Isothiocyanate ◽  
Dependent Manner ◽  
Sperm Plasma Membrane ◽  
Pre Treatment ◽  
Dose Dependent

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone–bovine serum albumin–fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 ± 4%), but this labelling was markedly reduced (33 ± 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 ± 10% (onset) to 59 ± 7% by 5 h, where it plateaued. Progesterone (P4) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 µg/mL P4 corresponding to 10 ± 5%, 16 ± 9%, 23 ± 7% and 30 ± 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P4-induced AR (12 ± 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P4-induced AR (10 µg/mL) in capacitated ejaculated spermatozoa from 19 ± 6% to 11 ± 4% (h151, 1 : 10) and 12 ± 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

330 CORRELATION BETWEEN TWO APOPTOTIC MARKERS IN FRESH BOAR SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 280 ◽  
Author(s):  
M. Trzcinska ◽  
M. Bryla ◽  
Z. Smorag
Keyword(s):  
Room Temperature ◽  
Annexin V ◽  
Molecular Probes ◽  
Green Fluorescence ◽  
Research Committee ◽  
Outer Leaflet ◽  
Chi Squared ◽  
Apoptosis Assay ◽  
Live Sperm ◽  
Boar Spermatozoa

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. It is a well-characterized mechanism that allows eukaryotes to eliminate unneeded, senescent, or aberrant cells, but the significance of apoptosis in ejaculated animal sperm is still unresolved. In this experiment, we designed 2 methods to detect early changes in the membranes of boar spermatozoa based on the slight increase in membrane permeability (Vybrant Apoptosis Assay Kit #4; Molecular Probes, Eugene, OR, USA) and on translocation of the phospholipid phosphatidyserine (PS) from the inner to the outer leaflet of the plasma membrane (Annexin-V-FLUOS Staining Kit; Roche, Mannheim, Germany). Detection of early changes in the sperm plasma is very important when designing storage protocols. Three ejaculates from 3 boars were used in the experiment. After collection and separation of the gel, the semen was analyzed using the following assays: (1) Annexin-V-FITC/PI assay: Sperm (2 � 106) were washed and diluted in 100 �L of HEPES buffer; 6 �L of Annexin-V-FITC and 4 �L of PI were added to the sample. The tubes were incubated for 15 min at room temperature in the dark. (2) YO-PRO-1/PI assay: YO-PRO-1 stock solution (1 �L) and 1 �L of PI stock solution were added to the samples. The tubes were gently mixed and incubated for 20 min at room temperature in the dark. After the incubation period, the sperm cell suspensions were analyzed under a fluorescence microscope at 40� magnification. At least 200 spermatozoa per sample were evaluated. Using the YO-PRO-1/PI assay, we observed 3 groups of sperm: apoptotic sperm showed green fluorescence (2–8%), necrotic sperm showed red and green fluorescence (9–37%), and live sperm showed no fluorescence (58–89%). Using the Annexin V-PI assay, 4 sperm subpopulations were easily detectable: apoptotic sperm showed green fluorescence (0.5–7%), early necrotic sperm showed red and green fluorescence (10–35%), necrotic sperm showed only red fluorescence (2–6%), and live sperm showed no fluorescence (57–87%). The results were compared by a chi-squared test. Significant differences (P &lt; 0.01) in the percentage of all sperm subpopulations (apoptotic, necrotic, and live sperm) among boars and among ejaculates from the same boar were observed. We also observed a strong correlation between these 2 methods. Using the Annexin-V-FITC/PI assay, we detected more sperm subpopulations, and this method seems to be more sensitive than the YO-PRO-1/PI assay. However, these 2 methods detect changes in membrane spermatozoa but in different aspects of apoptosis, and this may also cause differences in the frequencies of apoptotic cells found by the different assays. This study was supported by the Polish Research Committee, grant no. 2 P06D 023 30.

46 Effect of Bioxcell® and Triladyl® Extenders on Washed Semen of South African Indigenous Bucks

2018 ◽  
Vol 30 (1) ◽  
pp. 162
Author(s):  
L. P. Nethenzheni ◽  
M. L. Mphaphathi ◽  
N. C. Negota ◽  
T. L. Nedambale
Keyword(s):  
Treatment Group ◽  
South African ◽  
Liquid Nitrogen ◽  
Seminal Plasma ◽  
Semen Parameters ◽  
Mean Values ◽  
Treatment Groups ◽  
Frozen Semen ◽  
The Mean

Semen extenders and seminal plasma are vital for cryopreservation of buck semen. The objectives of the study were to evaluate the effect of 2 extenders: Triladyl® (Minitube, Tiefenbach, Bavaria) and Bioxcell® (IMV, L’Aigle, France) and the removal of seminal plasma on buck semen. Six indigenous bucks were used in this study and 6 ejaculates were collected from individual bucks. The semen was pooled and then randomly allocated into 6 groups: (1) raw-washed, (2) raw-non-washed, (3) Triladyl®-washed, 4) Triladyl®-non-washed, (5) Bioxcell®-washed, and (6) Bioxcell®-non-washed. Spermatozoa viability was assessed using Eosin-Nigrosin and morphology using Spermac® (Vitrolife, Göteborg, Sweden) stains. The washed semen samples were all diluted into (1:4 v/v) with PBS and centrifuged at 1500 × g for 10 min. Semen samples were then extended with Triladyl® or Bioxcell® per treatment groups and equilibrated for 2 h at 5°C. The semen samples were loaded into straws per treatment groups and placed 5 cm above a liquid nitrogen vapour for 10 min and then stored at –196°C until use. After 1 month of storage, frozen semen straws per treatment group were thawed at 37°C for 30 s, and spermatozoa parameters were analysed post-thaw. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010 (SAS Institute Inc., Cary, NC, USA). There was a higher (P < 0.05) live and normal spermatozoa percentage in non-washed semen extended with Bioxcell® (45.7 ± 21.2) than the semen extended with Triladyl® (24.5 ± 22.2%). Live and normal spermatozoa percentages were drastically reduced in the Bioxcell® (5.2 ± 4.9) and Triladyl® (6.9 ± 8.6%) washed semen groups. There was a higher (P < 0.05) percentage of spermatozoa with head abnormalities in non-washed semen extended with Triladyl® (20.4 ± 10.2), compared with the semen extended with Bioxcell® (18.3 ± 12.4%) following freeze-thawing. There was a higher (P < 0.05) percentage of spermatozoa with head abnormalities in washed semen samples extended with Triladyl® (34.0 ± 16.0) compared with the semen extended with Bioxcell® (10.1 ± 7.0%). There were higher (P < 0.05) percentages of spermatozoa with coiled tail abnormalities in washed semen extended with Bioxcell® (65.4 ± 25.0) compared with Triladyl® (35.9 ± 21.6%). In conclusion, the liveability of spermatozoa was negatively affected by washing of semen extended with Bioxcell® and Triladyl® extender. Bioxcell® significantly increased tail abnormalities and Triladyl® gave less protection against head abnormalities following cryopreservation of South African unimproved indigenous bucks’ semen.

21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

2019 ◽  
Vol 31 (1) ◽  
pp. 136
Author(s):  
M. M. Tshabalala ◽  
K. A. Nephawe ◽  
M. L. Mphaphathi ◽  
C. M. Pilane ◽  
T. L. Nedambale
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Semen Parameters ◽  
Dna Integrity ◽  
Low Density ◽  
Bull Semen ◽  
Frozen Semen ◽  
Live Sperm

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P&lt;0.05. Sperm motility and membrane integrity were significantly higher (P&lt;0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P&lt;0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION

2016 ◽  
Vol 28 (2) ◽  
pp. 152
Author(s):  
M. A. Lagares ◽  
H. S. Martins ◽  
M. R. Souza ◽  
C. F. A. M. Penna ◽  
F. O. P. Leme ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Propidium Iodide ◽  
Acrosome Reaction ◽  
Fluorescent Microscopy ◽  
Calcium Ionophore ◽  
Peanut Agglutinin ◽  
Binding Assays ◽  
Frozen Semen ◽  
Fertilizing Ability

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P < 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P > 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P < 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P < 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen. Acknowledgments are extended to CAPES, Brazil, for the financial support.

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