181 FERTILIZATION CHARACTERISTICS OF SPERMATOZOA FROM BULLS GRAZING TALL FESCUE PASTURES

2012 ◽  
Vol 24 (1) ◽  
pp. 202
Author(s):  
J. P. Harris ◽  
J. L. Edwards ◽  
L. A. Rispoli ◽  
R. R. Payton ◽  
N. R. Rohrbach ◽  
...  
Keyword(s):  
Tall Fescue ◽  
Ergot Alkaloid ◽  
Mixed Models ◽  
Semen Analysis ◽  
Stress Test ◽  
In Vitro Assays ◽  
Serum Prolactin ◽  
Computer Assisted ◽  
In Vitro Production

Fertilization with spermatozoa from bulls ingesting elevated concentrations of ergovaline [i.e. consumption of toxic endophyte-infected (E+) tall fescue] results in reduced cleavage rates. Thus, the objectives of the current study were to evaluate motility and penetration rates of spermatozoa from bulls grazing tall fescue pastures and intracellular calcium (Ca2+) parameters of presumptive zygotes (PZ). During a 3-month study, 6 Angus bulls (average age = 15.1 ± 0.04 months) were appointed to graze Kentucky 31 tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum, an ergot alkaloid-producing endophyte (n = 3), or Jesup tall fescue infected with non-ergot alkaloid-producing endophyte (NTE) MaxQ™ (n = 3). Bulls were grouped by bodyweight (BW) and scrotal circumference (SC) to graze pastures from April 18 to June 26. Blood samples, BW, SC, semen and rectal temperatures (RT) were collected every 7 days. Semen was evaluated for gross motility, morphology and computer assisted semen analysis (CASA) parameters. Semen from a subset of bulls (n = 2 per treatment; acceptable motility after 3-h stress test) was used to assess spermatozoa function using in vitro assays; that is, in vitro production of embryos, penetration rates obtained 6.5 to 7.5 h post-insemination (hpi) and intracellular Ca2+ measured 8.0 to 10.0 hpi. Data were analyzed using a mixed models procedure (semen data), generalized linear mixed models (in vitro data) and nonlinear regression (intracellular Ca2+ data) of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Concentrations of serum prolactin were higher in bulls grazing NTE compared with those of the bulls grazing E+ tall fescue pastures (123.43 ± 9.23 ng mL–1 vs 97.13 ± 9.23 ng mL–1, respectively; P = 0.05). Gross motility of spermatozoa (90.95 ± 2.67% vs 85.62 ± 2.67%, NTE and E+ respectively; P = 0.17) and percent normal morphology (77.14 ± 1.93% vs 77.61 ± 1.93%, NTE and E+ respectively; P = 0.87) before cryopreservation did not differ. However, motility immediately post-thaw (58.27 ± 2.81% vs 43.84 ± 5.30%, NTE and E+, respectively; P = 0.02) and following a 3-h stress test (51.13 ± 3.88% vs 23.33 ± 3.23%, NTE and E+, respectively; P < 0.0001) were decreased for spermatozoa from bulls grazing E+ tall fescue. The percentage of PZ that cleaved was higher for oocytes fertilized with spermatozoa from bulls grazing NTE (76.30 ± 3.93%) compared with that of the bulls grazing E+ tall fescue pastures (58.92 ± 3.9%; P = 0.007; n = 1539 PZ, n = 10 replicates). Penetration rates were higher in oocytes fertilized with spermatozoa from NTE bulls (87.42 ± 1.63%) compared with that of E+ bulls (64.54 ± 3.28%; P < 0.0001; n = 2547 oocytes, n = 33 replicates). Meiotic progression of maternal chromatin was hastened in oocytes penetrated by spermatozoa from bulls grazing E+ compared to that from bulls grazing NTE tall fescue. Intracellular Ca2+ parameters (baseline, P = 0.01; amplitude, P = 0.0002; and area under the curve, P = 0.006) were reduced in PZ fertilized with spermatozoa from E+ bulls (n = 192 oocytes, n = 4 replicates). These findings indicate impaired spermatozoa function in bulls grazing E+ tall fescue pastures that extends beyond gross semen characteristics and may provide direction for future studies.

137 Assessment of fertilizing ability of Merino ram semen cold stored up to 48h by heterologous IVF of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 194 ◽  
Author(s):  
D. A. Galarza ◽  
M. Ladrón de Guevara ◽  
P. Beltrán-Breña ◽  
M. J. Sánchez-Calabuig ◽  
A. López-Sebastián ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Skim Milk ◽  
Semen Parameters ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Straight Line ◽  
Fertilizing Ability ◽  
Pronuclear Formation ◽  
Bovine Oocytes

The use of cold-stored ram semen has been applied in sheep AI programs, because it preserves its fertilizing ability similar to fresh. Besides, the heterologous IVF has been successfully employed to assess semen fertilizing ability in several species. Hence, we aimed to evaluate the fertilizing ability of ram semen cold stored up to 48h at 5°C by assessing heterologous IVF using bovine oocytes. Fifteen pools of 3 normospermic Merino ram (2-7 years) ejaculates were collected using artificial vagina, diluted to 200×106 spermatozoa mL−1 with ultra-heat-treatment-based extender (skim milk-6% egg yolk) and cold stored up to 48h. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF using fresh semen (FS, n=707), semen cold stored to 24h (CS24, n=832) or semen cold stored to 48h (CS48, n=611). In parallel, homologous IVF (control, n=1356) and parthenogenesis (parth control non-fertilized oocytes, n=334) were performed. Ram non-selected and selected (BoviPure, Nidacon International, Mölndal, Sweden) semen parameters were evaluated by computer-assisted semen analysis. Sperm-oocyte interaction was assessed at 2.5h post-insemination (hpi) by evaluating the number of bound spermatozoa, whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using phase contrast and confocal microscopy. Cleavage rate was evaluated in all groups at 48 hpi. Data obtained from 5 replicates were analysed using one-way ANOVA. Data was expressed as mean±standard error of the mean. In terms of sperm storage time, non-selected semen showed a significant decrease (P&lt;0.05) for CS24 and CS48 compared with FS on progressive motility [SPM (%): 52.30±4.1 and 36.9±5.5v. 71.3±1.6] and straight-line velocity (mm s−1: 132.2±6.1 and 109.7±6.3v. 176.7±4.3), respectively. However, selected semen showed a decrease (P&lt;0.05) only for CS48 when compared with CS24 or FS on SPM (35.6±3.9v. 56.1±6.91 and 59.3±2.6) and straight-line velocity (83.5±4.4v. 105.3±6.5 and 110±2.0), respectively. No differences were observed between heterologous IVF groups in all parameters evaluated. Homologous IVF showed a higher percentage of penetration only when compared with heterologous FS group (44.4±6.8v.12.5±4.5%; P&lt;0.01). The polyspermy was higher in heterologous CS24 group when compared with homologous IVF (11.4±3.4v. 3.8±2.2; P&lt;0.05). The homologous IVF group, as expected, showed the higher percentage of pronuclear formation at 18 hpi compared with heterologous IVF with FS (67.3±5.8v. 35.2±5.6%), CS24 (72.1±4.5 v. 37.2±5.7%) and CS48 (63.0±6.0 v. 27.0±5.6%), respectively (P&lt;0.001). Likewise, cleavage rate was higher in homologous group compared with heterologous IVF and parthenogenetic groups for FS (78.3±2.6.8v. 46.3±3.2 and 7.0±2.3%), CS24 (78.4±2.6 v. 48.3±3.2 and 4.9±2.0%), and CS48 (78.4±3.3 v. 43.3±3.5 and 4.3±1.2%), respectively (P&lt;0.001). In conclusion, Merino ram semen cold stored up to 48h maintains its fertilization ability to the same extend as fresh and can be used for sheep crossbreeding programs.

scholarly journals 12PREDICTION OF BULL FERTILITY BY COMPUTER ASSISTED SEMEN ANALYSIS

2004 ◽  
Vol 16 (2) ◽  
pp. 128 ◽  
Author(s):  
S. Cseh ◽  
T. Polichronopoulos ◽  
L. Solti
Keyword(s):  
Sperm Motility ◽  
Semen Analysis ◽  
Computer Assisted ◽  
Reproductive Process ◽  
Return Rates ◽  
Frozen Semen ◽  
Chi Square Analysis ◽  
Fertilizing Capability ◽  
Weighted Values

Sperm motility is clearly essential for fertilization both in vivo and in vitro. Motility is necessary for successful sperm transport, a step that is bypassed with in vitro fertilization. Recently, increasing attention has been paid to the objective evaluation and characterization of sperm motility more than simply determining the total proportion of motile spermatozoa. The purpose of computerassisted semen analysis (CASA) is to provide values for sperm concentration and sperm motility more rapidly and accurately than those obtained with traditional semen analyses methods. The objective of our experiment was to investigate the effect of specific aspects of sperm movement, such as the velocity of progression and the actual pattern of movement, to the fertilizing capability of sperm. Frozen semen samples of 10 HF breeding bulls were used in the study. For the motility analyses, Medealab CASA system (Medealab, Germany, Ver. 4.1) was used, and the velocity parameter of VCL (curvalinear velocity, μms−1), VSL (straight line velocity, μms−1), and VAP (average path velocity, μms−1) were evaluated and compared with the Day 30 and 75 non−return rates (NR30 and NR75). For every sample, a total of 10 fields were examined for 8s using a disposable 20 micron capillary chamber (CellVision, USA) giving a total of 1165 to 2831 cells evaluated. Chi square analysis, analyses of variance and linear correlation coefficient was applied to the statistical evaluation and comparison of the results. Data are based on weighted values. From the same batch of the analyzed frozen semen, a total of 8099 females were inseminated in more than 100 farms with a total of 6590 animals being positive for pregnancy at Day 30 and 4525 animals at Day 75. Within the bulls, differences were found in the values of NR30 and NR75 (P&lt;0.05). Our data indicate very strong differences between the males’ NR30 and NR75 values (NR30: 65.6%±13.04 to 79.6%±11.17; P&lt;0.001 and NR75: 37.8%±10.38 to 58.3%±15.53; P&lt;0.001) reflecting the individual differences in the fertilizing capability of the males. All velocity parameters show very high correlation with strong significance both non−return rates but the best values belong to VAP (NR30 and NR75; P&lt;0.02). Our data indicate that the bulls with lower VCL (25.51±33.04 to 79.54±58.03), VSL (11.35±19.45 to 36.36±35.71), and VAP (12.67±19.06 to 41.75±34.45) values showed lower fertilization rates both at NR30 and NR75. Computer and video technologies have advanced rapidly in recent years; thus the capability and accuracy of the latest versions of CASA systems are considerably better and they give more information about the different motion characteristics of spermatozoa. Because of the vital role of sperm motility in the reproductive process, such systems will enable us to move into a new era of diagnostic andrology and predict the fertilizing capability of semen. Supported by NKFP-Grants 4/040/2001 and 4/031/2001.

Effect of Panax notoginseng Extracts on Inferior Sperm Motility in vitro

1999 ◽  
Vol 27 (01) ◽  
pp. 123-128 ◽  
Author(s):  
Jung-Chou Chen ◽  
Ming-Xiong Xu ◽  
Leih-Der Chen ◽  
Yan-Nian Chen ◽  
Tsan Hung Chiu
Keyword(s):  
Sperm Motility ◽  
Sperm Count ◽  
Semen Analysis ◽  
Panax Notoginseng ◽  
World Health ◽  
Computer Assisted ◽  
Butanol Extract ◽  
The World ◽  
Health Organization

The purpose of this study was to investigate the effects of Panax notoginseng extracts on inferior sperm motility in vitro. Semen samples were collected from 23 patients with sperm motility between 20% and 40%. The sperm count was over 20 × 106/ml in accordance with the World Health Organization standard. 1.0 mg/ml and 2.0 mg/ml of Panax notoginseng extracts including aqueous extract, n-butanol extract, and polysaccharide fraction on sperm motility and progression were evaluated by computer assisted semen analysis. The results demonstrated that sperm motility as well as progression on inferior sperm motility were enhanced at 1 hour and 2 hours after incubation with all three types of extracts.

scholarly journals Asthenozoospermia in Mice with Targeted Deletion of the Sperm Mitochondrion-Associated Cysteine-Rich Protein (Smcp) Gene

2002 ◽  
Vol 22 (9) ◽  
pp. 3046-3052 ◽  
Author(s):  
Karim Nayernia ◽  
Ibrahim M. Adham ◽  
Elke Burkhardt-Göttges ◽  
Jürgen Neesen ◽  
Mandy Rieche ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Computer Assisted ◽  
Microscopical Examination ◽  
Structural Protein ◽  
Wild Type ◽  
Vitro Fertilization ◽  
In Vivo Experiments ◽  
Cysteine Rich Protein

ABSTRACT The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J × 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp−/− female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp−/− 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp−/− mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.

scholarly journals A Review on Animals Semen Characteristics: Fertility, Reproduction and Development

2019 ◽  
pp. 1-9
Author(s):  
Farzad Moradpour
Keyword(s):  
Structural Integrity ◽  
Current Knowledge ◽  
Semen Analysis ◽  
Kinetic Property ◽  
Genetic Material ◽  
Computer Assisted ◽  
Semen Characteristics ◽  
The Individual

In this research, the goal of review was summarizing the current knowledge of the methods available to assess in vitro quality of frozen-thawed bovine spermatozoa also, a review on animal’s semen characteristics: fertility, reproduction and development after AI with that semen. Artificial insemination (AI) is the first generation reproductive biotechnology that has made a deep contribution to the genetics improvement in several animals. A fertile ejaculate must meet certain semen characteristics quality standards, such as: normal morphology, active energy metabolism, progressive motility, structural integrity and functionality of the membrane, penetration capacity and optimum transfer of genetic material. The percentage of total motile spermatozoa in normal canine ejaculates is between 70 to 90%. By the way, there are a lot of parameters that able to change on the composition and structure of various sperm plasma member domains, such as change temperature and sensitive to any theirs environments in vivo and vitro (tropical climates), season also nutrition. Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements that gives extensive information about the kinetic property of the ejaculate based on measurements of the individual sperm cells.

163 HYPERACTIVATION OF STALLION SPERM IN FOLLICULAR FLUID FOR IN VITRO FERTILIZATION OF EQUINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 193 ◽  
Author(s):  
A. Lange-Consiglio ◽  
F. Cremonesi
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Motility ◽  
Follicular Fluid ◽  
Zona Pellucida ◽  
Semen Analysis ◽  
Computer Assisted ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
Low Incidence

In vitro fertilization has remained elusive in the horse, as evidenced by low sperm penetration rates when IVF has been attempted with in vivo- or in vitro-matured oocytes. It is likely that the low sperm penetration rates observed in IVF studies are due to the inability to appropriately capacitate or hyperactivate, or both, stallion sperm in the laboratory. The acquisition of hyperactivated sperm motility has been observed within the oviducts of mammals at the time of fertilization and is required for zona pellucida penetration in conjunction with the acrosome reaction (AR). Although the zona pellucida is considered the prime physiological inducer of AR, previous studies have shown a low incidence of AR in zona pellucida-bound stallion spermatozoa after 1 h of in vitro binding. This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for AR. Protein-bound progesterone, present in equine follicular fluid (FF), has been demonstrated to induce AR in stallion spermatozoa. In this context, the aims of this study were (1) to hyperactivate stallion sperm in FF and (2) to verify whether this hyperactivation supports equine IVF. Pooled FF, aspirated from the preovulatory follicles of oestrous mares, was used and its progesterone concentration was determined by immune enzymatic assay. Spermatozoa from fertile stallions selected by a swim-up procedure were pre-incubated for 6 h in capacitating medium (modifed Whittens's medium (WM) supplemented with 25 mM NaHCO3 and 7 mg mL–1 of BSA) and then incubated for 6 h at 37°C in either FF or capacitating WM. Sperm motility was assayed by computer-assisted semen analysis, rates of AR were assessed by fluorescein isothiocyanate-PNA staining and rate of apoptosis was assessed by an annexin V test. For IVF, spermatozoa were incubated at 10 × 106 sperm mL–1 in capacitating WM for 6 h and then diluted to 1 × 106 sperm mL–1 in capacitating WM with or without 10% of FF. Five mature mare oocytes were transferred into droplets (100 μL) of the sperm suspensions covered with mineral oil and then incubated for 18 h at 38.5°C in 5% CO2 in humidified air. After that, oocytes were transferred to an embryo culture medium (DMEM/F-12) for an additional 3 days. Data were analysed by ANOVA. Treatment of sperm with FF resulted in a significant (P ≤ 0.05) decrease of 3 motility variables indicative of hyperactivation: straight line velocity, straightness and linearity. The highest rate of AR (29.44%) and a lower rate of apoptosis (16.93%) were obtained after 4 h of incubation in follicular fluid. By coupling capacitating conditions with the induction of hyperactivation using follicular fluid, we have obtained reproducible percentages of 8-cell-stage embryos (18.56%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with FF did not fertilize (0%). It is concluded that mare FF does not impair sperm viability, stimulates equine sperm hyperactivation in vitro, induces the AR and supports equine IVF.

15 QUANTIFICATION OF BULL SPERM TRAITS AS ASSESSED BY COMPUTER-ASSISTED SEMEN ANALYSIS AND THE RELATIONSHIP TO PREGNANCY RATE FOLLOWING CONTROLLED BREEDING

2017 ◽  
Vol 29 (1) ◽  
pp. 115
Author(s):  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
F. V. Ramukhithi ◽  
Z. C. Raphalalani ◽  
T. R. Netshirovha ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Pregnancy Rate ◽  
Semen Analysis ◽  
Computer Assisted ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Bull Sperm ◽  
Sperm Traits

The bull’s contribution through artificial insemination to reproductive efficiency is of great biological importance. The objectives were (1) to compare the oestrous synchronization response of Bonsmara and Nguni cows; and (2) to find the relationship between cow’s conception rate (in vivo and in vitro fertilization) and bull sperm motility rate assessed by computer-assisted semen analysis (CASA) following AI. For the in vivo sperm fertility test, 100 Bonsmara and 482 Nguni cows were randomly selected and subjected to oestrous synchronization protocol and AI with frozen–thawed assessed semen by CASA before AI. Briefly at Day 0, cows were inserted with an intravaginal CIDR® (1.9 g), which was removed on Day 7. Prostaglandin was then administered (2 mL) on Day 8 and a heatmount detector was placed on the hindquarter of each cow. For the in vitro sperm fertility test, collected oocytes from slaughterhouse were in vitro matured (n = 360) and in vitro fertilized (sperm/mL) in 100-µL droplets (final volume) of BO-IVF medium per treatment bulls (Bonsmara or Nguni bull). The frozen/thawed semen straws of Bonsmara and Nguni bulls were randomly selected and used under the same IVF conditions. The thawed bull’s sperm characteristics were examined by CASA before in vitro fertilization. Data were analysed using ANOVA. Treatment means were compared using the Fisher’s protected least significant difference t-test. There was no significant difference in oestrous response for the Bonsmara (83.0%) and Nguni (90.8%) cows, respectively. The Bonsmara cows recorded a significantly higher pregnancy rate (59.0%) compared with the Nguni (37.1%) cows (P < 0.05). Sperm traits such as total motility (TM), progressive motility and rapid were found to be positively correlated with conception rate (r = 0.06, 0.03, and 0.08, respectively; P < 0.01), although correlations were low. There was no difference in the average frozen–thawed sperm TM rate of Nguni (92.2%) and Bonsmara (81.0%). There was a lower fertilization rate following IVF with Bonsmara and Nguni bull sperm. In conclusion, Nguni cows had similar oestrous response as Bonsmara cows. The sperm traits from Bonsmara and Nguni bulls were found to be related to in vivo conception and in vitro fertilization rate when sperm cells were assessed by CASA technology. However, the pregnancy rate was lower in Nguni cows.

180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

2016 ◽  
Vol 28 (2) ◽  
pp. 221
Author(s):  
D. Le Bourhis ◽  
S. Camugli ◽  
P. Salvetti ◽  
L. Schibler ◽  
E. Schmitt
Keyword(s):  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Fluid Medium ◽  
And Control

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

371 DOES Percoll™ GRADIENT CENTRIFUGATION CAUSE HYPERACTIVATION ON CRYOPRESERVED BOVINE SPERMATOZOA?

2010 ◽  
Vol 22 (1) ◽  
pp. 342
Author(s):  
L. Z. Oliveira ◽  
R. P. Arruda ◽  
E. C. C. Celeghini ◽  
A. F. C. Andrade ◽  
A. C. Lucio ◽  
...  
Keyword(s):  
Density Gradient ◽  
Calcium Influx ◽  
Ph Monitoring ◽  
Semen Analysis ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Computer Assisted ◽  
Head Displacement ◽  
Bovine Spermatozoa

The density difference of 0.06% between X- and Y-bearing bovine spermatozoa has the potential to provide for separation X-bearing spermatozoa on specific gradient solutions of Percoll™ (Hossepian de Lima VFM et al. PCT/BR2004/000009). The question remains whether Percoll™ density gradient centrifugation induces the hyperactivation of spermatozoa, because it is believed that some decapacitating proteins are removed from the sperm surface during the process. Thus, the objective of this study was to evaluate if Percoll™ centrifugation causes hyperactivation of cryopreserved bovine spermatozoa. Semen doses were collected from six bulls of different breeds, including three taurine and three Zebu animals, and four ejaculates per bull were evaluated. The semen samples were thawed and evaluated before (control) and after (sexed group) centrifugation (500 g) in discontinuous Percoll™ density gradient (Hossepian Lima VFM et al. PCT/BR2004/000009). Sperm motility was assessed by Computer-Assisted Semen Analysis (CASA, Hamilton Thorne Biosciences, Beverly, MA, USA) and the results (mean ± SEM) were submitted to ANOVA. Higher (P < 0.0001) total and progressive sperm motilities were observed in sexed (85.0 ± 1.8 and 75.9 ± 1.7%, respectively) than in the control group (69.9 ± 1.7 and 59.2 ± 1.6%, respectively). The average path velocity, straight-line velocity and curvilinear velocity (VCL) were lower (P < 0.01) in sexed (91.3 ± 1.1, 79.8 ± 0.8, and 137.6 ± 3.5 (im/s, respectively) than in control group (107.6 ± 4.8, 91.1 ± 4.1, and 173.2 ± 8.9 (im/s, respectively). The amplitude of lateral head displacement (ALH) was higher (P < 0.0001) in control (6.7 ± 0.3 μm) than in sexed semen (5.1 ± 0.2 μm) and beat cross frequency was higher (P < 0.05) in sexed (35.9 ± 0.9 Hz) than in control semen (33.1 ± 1.0 Hz). The straightness and linearity (LIN) were higher (P < 0.01) in sexed (87.4 ± 0.7 and 61.4 ± 1.4%, respectively) than in control semen (84.4 ± 0.7 and 55.5 ± 1.2%, respectively). Regarding the results obtained in the present study, it was not possible to infer that the method used induced sperm hyperactivation. According to Marquez and Suarez (2007 Biol. Reprod., 76, 660-665), an increase in pH and intraflagellar calcium plays a key role in the axoneme changes, promoting an increase in ALH (from 9 to 13 μm) and VCL (from 200 to 315 μm/s), as well as a decrease in LIN (from 57 to 25%), which characterizes the presence of hyperactivated cells in the sample. In the present study, centrifuged spermatozoa presented significantly lower ALH and VCL and higher LIN compared to control semen samples. Therefore, the presence of glucose in the Percoll™ stock solution and constant pH monitoring could have prevented alkalinization of the medium, thereby preventing the occurrence of intraflagelar calcium influx and the consequent triggering of in vitro sperm hyperactivation.

13 MOTILITY CHARACTERISTICS OF SPERMATOZOA FROM BULLS GRAZING TALL FESCUE PASTURES

2014 ◽  
Vol 26 (1) ◽  
pp. 121
Author(s):  
J. P. Harris ◽  
J. L. Edwards ◽  
L. A. Rispoli ◽  
N. R. Rorhbach ◽  
T. M. Prado ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Tall Fescue ◽  
Ergot Alkaloid ◽  
Festuca Arundinacea ◽  
Fixed Effects ◽  
Block Design ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Scrotal Circumference ◽  
Cold Room

Fertilisation is less than expected with spermatozoa from bulls consuming toxic endophyte-infected (E+) tall fescue. The objective of this study was to evaluate motility characteristics of spermatozoa from bulls grazing tall fescue pastures using computer-assisted sperm analysis (CASA). Semen was collected from six Angus bulls (average age = 15.1 ± 0.04 months) during a three-month grazing study. Bulls grazed Kentucky 31 tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum, an ergot alkaloid-producing endophyte (n = 3), or Jesup tall fescue with Max-Q™ (NTE), a non-ergot alkaloid producing endophyte (n = 3), and grouped by body weight and scrotal circumference to graze pastures from April 18 to June 26. Semen was collected once per week between 0600–0800 h beginning in mid-May and ending the last week of June. Gross motility and morphology was evaluated before extending with Bioxcell® animal protein-free formula (IMV, Aigle, France) and antibiotics (CSS 100, 2% of total volume). Extended semen was then evaluated using CASA to determine final dilution and packaged into straws (20 million sperm/straw), where equilibration occurred over 3 h in a cold room at 4°C. Straws were frozen for 7 min in static vapor of liquid nitrogen and plunged into goblets filled with liquid nitrogen. Semen was thawed and assessed using CASA at 0 and 3 h post-thaw. Data were analysed as a randomised block design with the fixed effects of treatment, blocking on semen collection date, utilising the mixed models procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Data were tested for normality (Shapiro-Wilk W ≥ 0.90), and treatment differences were determined using F-protected least significant differences. Path velocity (P = 0.001) and progressive velocity (P = 0.003) were lower in spermatozoa from bulls grazing E+ during the last 2 weeks of collection in June independent of time of assessment post-thaw. Sperm head area decreased in size in spermatozoa from E+ grazing bulls at 3 h post-thaw (P = 0.04) compared with NTE grazing bulls. Percent of rapid (progressive % with path velocity >50 μm s–1) and medium (progressive % with path velocity <50 μm s–1 but > 30μm s–1) velocity spermatozoa was decreased for E+ grazing bulls compared to NTE grazing bulls (P < 0.0001 and P = 0.004, respectively) and was accompanied by an increase in static (immobile) spermatozoa from E+ bulls (P < 0.0001). These findings indicate that spermatozoa movement and velocity are impaired in bulls grazing E+ tall fescue pastures compared to bulls grazing NTE tall fescue pastures after the freeze and thaw process, which may explain decreased fertilisation and cleavage rates of oocytes co-incubated with these spermatozoa.

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