362 STORAGE OF BOVINE SPERM FOR 20 H BETWEEN SEMEN COLLECTION AND SEXING

2010 ◽  
Vol 22 (1) ◽  
pp. 337
Author(s):  
J. L. Anema ◽  
J. K. Graham ◽  
R. W. Lenz ◽  
G. E. Seidel
Keyword(s):  
Flow Cytometry ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Ph Measurements ◽  
Sperm Analysis ◽  
Bovine Sperm ◽  
Semen Collection ◽  
Dairy Bulls ◽  
And Control ◽  
Better Than

The objective of this study was to optimize bovine sperm storage for up to 20 h between semen collection and sex sorting followedby cryopreservation. Two successive ejaculates were obtained from mature dairy bulls (Holstein, n = 5; Jersey, n = 3) via artificial vagina. Treatments were then applied to the neat semen to which antibiotics were added as recommended by Certified Semen Services (Columbia, MO). Nothing further was added to the control samples until staining with Hoechst 33342 for sorting. For Treatment 1, semen was diluted 9:1 with a MOPS solution resulting in 24 mM MOPS and similarly, Treatment 2 resulted in 24 mM MOPS +2% egg yolk. A subsample of each treatment and control was sorted by flow cytometry shortly after collection, and sperm then were frozen following standard processing procedures. The other subsample was stored at 15-18°C and sorted 20 h after collection followed by cryopreservation. pH measurements were made before staining samples for sorting. Samples were evaluated post-thaw for subjective progressive and total sperm motility, by computer-assisted sperm analysis (CASA, Berkeley, CA, USA), and by flow cytometry for sperm viability using propidium iodide and SYBR-14. Treatment 1 performed better than the control (Table 1), while results for Treatment 2 were similar to the control. Second ejaculates were superior to first ejaculates. pH measurements showed that addition of MOPS kept the pH about 0.2 units higher than the control, but pH declined similarly over time in all groups. While responses for the 20 h sort were numerically lower than the 0 h sort (P > 0.1), the majority of responses were acceptable for most, but not all bulls. In conclusion, storing sperm in 24 mM MOPS was beneficial. Surprisingly, 2% egg yolk negated the beneficial effect of MOPS, possibly due to increasing osmolarity by ∼15mOsM/kg due to pH adjustment. Addition of MOPS provided better results than the control for both the 0 h and 20 h sorts. Table 1.Main effect means of semen characteristics

scholarly journals The Quality of Frozen-thawed Canine Semen With Respect to Semen Extender Composition and Sequence of Ejaculate Collection in Dogs

2016 ◽  
Vol 64 (1) ◽  
pp. 169-175
Author(s):  
Jiří Šichtař ◽  
Ondřej Šimoník ◽  
Petra Folková ◽  
Adéla Dokoupilová ◽  
Radko Rajmon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Time Interval ◽  
Sperm Analysis ◽  
Semen Collection ◽  
Movement Characteristics ◽  
Semen Extender

The aim of this study was to evaluate the effect of clarified egg yolk addition to semen extender, and the semen collection sequence on the quality of frozen-thawed semen in dogs. Semen was collected from 6 dogs in a time interval of 24 hours. As parameter of the quality of frozen-thawed (F-T) semen, the motility by computer assisted sperm analysis (CASA) and plasma membrane integrity by hypo-osmotic swelling test (HOS) were evaluated. All kinematic parameters of sperm motility were higher in F-T samples containing the whole in comparison to the clarified egg yolk. The sequence of semen collection affected sperm movement characteristics of native as well as F-T semen, but it was not possible to determine whether the fresh semen from the 1st or 2nd collection is of higher quality. All motility parameters of sperms frozen with extender containing the whole egg yolk were significantly higher in the case of the 2nd collection. The situation was not so clear in the case of clarified egg yolk addition, but the velocity values were higher in F-T samples from the 2nd collection. In contrast to proven differences in motility, the effect of the addition of clarified egg yolk and the sequence of semen collection were not projected at all on the quality of plasma membrane of canine sperms evaluated by HOS test.

scholarly journals Evaluación de la motilidad espermática a través del sistema C.A.S.A de semen caprino criopreservado bajo diferentes medios diluyentes

Respuestas ◽  
2013 ◽  
Vol 18 (2) ◽  
pp. 16-27
Author(s):  
Leonardo Hernández-Corredor ◽  
Alexander Nivia-Osuna ◽  
Daniel Hernández-Villamizar ◽  
Jorge Alexander Rubio-Parada ◽  
Armando Quintero-Moreno
Keyword(s):  
Lateral Displacement ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Complete Medium ◽  
Sperm Analysis ◽  
Straight Line ◽  
Nitrogen Vapor ◽  
Computer Assisted Sperm Analysis ◽  
The University ◽  
Casa System

 El estudio evaluó la motilidad espermática y su efecto postdescongelación en semen caprino, en dos medios comerciales (Andromed® y TwoStep®) y diferentes protocolos de congelación (medio completo, con adicción del 10% de yema de huevo, semen centrifugado y sobrenadante seminal), se utilizaron machos de la raza alpina de la Universidad Francisco de Paula Santander Ocaña, el semen fue colectado con electroeyaculador, una vez los medios terminados y parte de los contenidos seminales enteros o centrifugados mezclados, se estabilizó por 2 horas, se envasó en pajillas de 0,5 cc y se congela en vapores de nitrógeno por 10 minutos, las pajillas se llevaron al laboratorio de Andrología de la Universidad del Zulia y por medio del sistema C.A.S.A.(Computer Assisted Sperm Análisis) se evaluaron los parámetros de motilidad como velocidad curvilínea (VCL), velocidad rectilínea (VSL), velocidad lineal (VAP), índice de linealidad (LIN), índice de rectitud (STR), índice de oscilación (ALH), Amplitud media del desplazamiento lateral de la cabeza del espermatozoide (BCF), los datos fueron analizados por medio del procedimiento GLM de SAS versión 9.0; los mejores índices de motilidad (VCL, ALH, BCF) fueron expresados enel tratamiento de contenido seminal centrifugado en medio Andromed®. (p≤0,001))La mejor progresividad espermática (VSL,LIN,STR)se presentó el tratamiento de Semen completo de caprino, criopreservado en medio comercial TwoStep®. ABSTRACT  The study evaluated the effect sperm motility and sperm post-thawing in goats, two commercial means (Andromed ® and Two Step ®) and different freezing protocols (complete medium with 10% addition of the egg yolk, semen centrifuged supernatant and seminal ), we used males of the Alpine race of the University Francisco de Paula Santander Ocaña, semen was collected with electroejaculator once finished media and part of the whole and centrifuged seminal contents mixed, stabilized by two hours, packed in 0.5 cc straws and frozen in nitrogen vapor for 10 min, the straws were taken to the laboratory of Andrology at the University of Zulia and through CASA system (Computer Assisted Sperm Analysis) were evaluated motility parameters such as curvilinear velocity (VCL), straight line velocity (VSL), linear velocity (VAP), linearity index (LIN), straightness index (STR) Oscillation Index (ALH ) average amplitude of the lateral displacement of the sperm head (BCF), the data were analyzed by the GLM procedure of SAS version 9.0, the highest rates of motility (VCL, ALH, BCF) were expressed in the treatment of seminal content centrifugation Andromed ® medium. (p ≤ 0.001)) The best progressive sperm (VSL, LIN, STR) will present the full Semen treatment goats, cryopreserved at Two Step ® commercial medium.Keywords: semen, buck, Andromed, Two step.

scholarly journals Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽  
2020 ◽  
Vol 15 (20) ◽  
pp. 1965-1980
Author(s):  
Teresa Vilanova-Perez ◽  
Celine Jones ◽  
Stefan Balint ◽  
Rebecca Dragovic ◽  
Michael L Dustin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Sperm Function ◽  
Sperm Analysis ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

scholarly journals Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen

2020 ◽  
pp. 2209-2218
Author(s):  
Fernando Evaristo da Silva ◽  
Jaqueline Candido Carvalho ◽  
Camila de Paula Freitas Dell'Aqua ◽  
Frederico Ozanam Papa ◽  
Marc Roger Jean Marie Henry ◽  
...  
Keyword(s):  
Cell Damage ◽  
Membrane Integrity ◽  
Sodium Caseinate ◽  
Egg Yolk ◽  
Freezing Process ◽  
Computer Assisted ◽  
Semen Cryopreservation ◽  
Sperm Analysis ◽  
Frozen Semen ◽  
Buffalo Semen

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.

scholarly journals Effect of two freezing extenders on characteristic of fresh and frozen-thawed semen in endangered Old Kladruber stallions – A pilot study

2017 ◽  
Vol 62 (No. 6) ◽  
pp. 227-233 ◽  
Author(s):  
J. Šichtař ◽  
A. Nehasilová ◽  
O. Šimoník ◽  
F. Bubeníčková
Keyword(s):  
Egg Yolk ◽  
Quality Parameters ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Fluorescence Staining ◽  
Sperm Analysis ◽  
Sperm Characteristic ◽  
Computer Assisted Sperm Analysis ◽  
Fresh Semen

The aim of the study was to evaluate the effect of two different extenders on sperm characteristic before equilibration and post-thaw in the endangered Old Kladruber stallions. Also, the response of individual stallions to the extenders used was tested. Semen was collected from six stallions every other day within one week. After centrifugation of the collected sperm-rich fraction, the supernatant was removed and sperm pellets were divided to two aliquots; these were diluted either with Gent (Minitube, Germany) or privately manufactured lactose-EDTA-egg yolk extender (Lact). Three cryopreserved insemination doses (IDs) from each extender (Gent and Lact) were prepared for each stallion from one collection (108 samples from six stallions in total). As a parameter of quality, the motility (computer assisted sperm analysis), viability (fluorescence staining), and morphology (eosin/nigrosine staining) were evaluated after dilution with freezing extenders (fresh) and after thawing (frozen-thawed). The different effects of chosen extenders on the quality of fresh semen were only manifested in higher kinematic parameters of sperm when the Lact extender was used. However, in frozen-thawed samples, the Gent extender yielded significantly better results in all of the evaluated parameters. The representation of sperm subpopulation was significantly influenced by extender in fresh as well as frozen-thawed samples; moreover, we found a significant effect of freezing on the distribution of these subpopulations. The response of individual stallions to chosen extenders was evident in the different quality of fresh as well as frozen-thawed IDs; Gent extender yielded better frozen-thawed IDs. Based on our results, among others describing quality parameters of ejaculate in endangered Old Kladruber stallions, we can recommend using Gent extender for the production of frozen-thawed IDs.  

Swim-up of tammar wallaby (Macropus eugenii) spermatozoa in Biggers, Whitter and Whittingham (BWW) medium: maximisation of sperm motility, minimisation of impairment of sperm metabolism and induction of sperm hyperactivation

2017 ◽  
Vol 29 (2) ◽  
pp. 345 ◽  
Author(s):  
Minjie Lin ◽  
Xiyi Zhang ◽  
Ray N. Murdoch ◽  
R. John Aitken
Keyword(s):  
Sperm Motility ◽  
First Record ◽  
Tammar Wallaby ◽  
The Other ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Rapid Induction ◽  
Marsupial Species ◽  
Accurate Quantification ◽  
Better Than

A variety of media were compared for their ability to sustain the motility of tammar wallaby spermatozoa over an 8-h period following swim–up from coagulated semen. The study demonstrated that a modified Tyrode’s solution, Biggers, Whitter and Whittingham medium (BWW) was significantly better than any of the other assessed media in supporting wallaby sperm motility. After 8 h of incubation in BWW, motility was maintained at 79.3 ± 9.3%, with 77.0 ± 10.4% rapid and 65.7 ± 8.7% progressively motile spermatozoa. By contrast, motility was <10% at the same 8-h time point in all of the other media assessed. After 2 h of incubation in BWW, tammar spermatozoa consumed more oxygen than their counterparts in PBS (52.0 ± 2.7 vs 75.0 ± 6.6 μL per 108 spermatozoa per 2 h; P < 0.001). Motility was not enhanced in any of these media by the addition of 5 mM N-acetyl-D-glucosamine, the major energy substrate in wallaby semen. However, addition of dibutyryl cAMP and pentoxifylline in BWW resulted in the extremely rapid induction of hyperactivated motility in the entire sperm population. This burst of hyperactivated motility was entirely dependent on calcium in BWW and significantly inhibited by calmidazolium, a calmodulin inhibitor. A set of computer-assisted sperm analysis parameters were identified that permitted the accurate quantification of hyperactivation rates in this species. This is the first comparative analysis of media for harvesting and incubating marsupial spermatozoa and the first record of hyperactivated motility in any marsupial species.

201 EFFECTS OF REMOVING SEMINAL PLASMA DURING 8-h STORAGE BEFORE SEX-SORTING BOVINE SPERM

2012 ◽  
Vol 24 (1) ◽  
pp. 213 ◽  
Author(s):  
C. A. Burroughs ◽  
K. M. Evans ◽  
R. W. Lenz ◽  
G. E. Seidel
Keyword(s):  
Seminal Plasma ◽  
Sperm Concentration ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Long Term Storage ◽  
Bovine Sperm ◽  
Standard Procedures ◽  
Term Storage ◽  
Artificial Vagina

We evaluated sex-sorting parameters and post-thaw motility for sperm stored with or without seminal plasma for 8 h before sorting. One first ejaculate was collected from each of 6 bulls routinely collected via artificial vagina; ejaculates contained at least 70% motile and 75% morphologically normal sperm and sperm concentrations ranged from 0.75 to 2.21 × 109 sperm mL–1. Ejaculates were divided into 2 samples and centrifuged at 1000 × g for 15 min. Seminal plasma from 1 sample was replaced with TALP (pH 7.4) to a sperm concentration of 1.4 × 109 sperm mL–1. The seminal plasma/sperm admixture of the other sample (control) was suspended to initial ejaculate sperm concentration. Both samples were stored for 8 h at 16°C before being subjected to standard sex-sorting procedures. Sperm were analyzed and bulk sorted on a MoFlo SX (XY Inc., Navasota, TX, USA) flow cytometer/cell sorter for percentage of live-oriented cells, percentage of membrane-impaired sperm (cell membranes permeable to red food colouring, which were discarded during sorting) and resolution between X- and Y-bearing sperm populations (peak to valley ratio). Sorted sperm were frozen according to standard procedures and post-thaw motility was determined immediately after thawing using computer-assisted sperm analysis. Treatments were compared using a paired t-test. Control sperm stored with seminal plasma resulted in a higher percentage of live-oriented cells (55%) versus those stored without seminal plasma (51%; P = 0.02). The percentage of membrane-impaired sperm was lower for control sperm (19%) than that of samples without seminal plasma (28%; P < 0.001). Resolution was greater for sperm stored without seminal plasma (34%) than for control sperm (10%; P = 0.04). Post-thaw, both total and progressively motile sperm were higher for samples without seminal plasma (63 and 53%, respectively) compared with those of the control samples (52 and 45%, respectively; P < 0.04). In conclusion, sperm stored for 8 h without seminal plasma had greater resolution between X- and Y-bearing populations and higher post-thaw motility than control sperm. However, these samples had a higher percentage of membrane-impaired sperm that were removed during sorting. Long-term storage of sperm in their seminal plasma before sex-sorting appears to be detrimental to post-sorting, post-thaw sperm motility.

74 EFFECT OF POWDERED EGG YOLK IN THE EXTENDER AND DONOR AGE ON FRESH AND THAWED SPERM MORPHOMETRY IN SMALL RUMINANTS

2015 ◽  
Vol 27 (1) ◽  
pp. 130
Author(s):  
M. J. Palomo ◽  
W. Garcia ◽  
A. Tabarez
Keyword(s):  
Small Ruminant ◽  
Small Ruminants ◽  
Egg Yolk ◽  
Sperm Head ◽  
Computer Assisted ◽  
Donor Age ◽  
Mean Values ◽  
Sperm Analysis ◽  
Sperm Morphometry ◽  
Ram Sperm

Our aim was to reduce heterogeneity and microbiological contamination risk on small ruminant semen cryopreservation by replacement of fresh egg yolk by pasteurized powdered egg yolk, assessing simultaneously the effect of the donor age (1 year v. 2 years old) on sperm head morphometry of fresh and thawed sperm. Briefly, fresh ejaculates from 8 rams and from 8 bucks were collected in autumn during 2 consecutive years. Immediately after collection, ejaculates from each species were mixed in equal quantities, and pooled semen was centrifuged twice (600 g for 10 min). Then the pellet was split into 2 aliquots and resuspended in an extender containing 15% (v/v) of fresh or powdered egg yolk supplemented with 5% glycerol in a Tris-based medium. Afterward, sperm samples were refrigerated at 5°C for 4 h before being frozen in nitrogen liquid vapours. Buck and ram sperm-head morphometry was analysed by computer-assisted sperm analysis (ISAS®) on fresh and thawed sperm previously stained with Diff Quick®, and the data were analysed using ANOVA (mean ± s.e., n = 6). From ram sperm studies, no differences were found between fresh and post-thaw sperm, neither between egg yolk-based extenders or donor ages, showing the following mean values of head length (8.4 ± 0.0 μm), width (4.9 ± 0.0 μm), area (34.2 ± 0.1 μm2), perimeter (23.4 ± 0.1 μm), ellipticity (1.7 ± 0.0), elongation (0.2 ± 0.0), rugosity (0.8 ± 0.0), and regularity (0.9 ± 0.0). Likewise buck semen did not show significant differences on sperm-head dimensions after cryopreservation, only on head-shape parameters as ellipticity, elongation, and regularity between fresh and thawed sperm from 2-year-old donors, independently of egg yolk used as cryoprotectant. However, the age of the buck had a significant effect on all assessed morphometric parameters in fresh and thawed sperms, except regularity (Table 1). Our results suggest that, from a morphometric point of view, the powdered egg yolk can be used satisfactory on small ruminant sperm cryopreservation, but in goats, the head changes due to the donor age should be considered. Table 1.Effect of fresh v. powdered egg yolk (EY) in the extender and donor age on fresh and thawed buck-sperm morphometry Research supported by INIA (RZ2009–00008–00–00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundación Carolina.

58 EFFECT OF MELATONIN IMPLANT ON BLANCA de RASQUERA BUCKS DURING SPRING ON SPERM MORPHOMETRY BEFORE AND AFTER CRYOPRESERVATION

2015 ◽  
Vol 27 (1) ◽  
pp. 122
Author(s):  
A. Tabarez ◽  
W. García ◽  
M. J. Palomo
Keyword(s):  
Egg Yolk ◽  
Sperm Head ◽  
Head Width ◽  
Computer Assisted ◽  
Sperm Cryopreservation ◽  
Sperm Analysis ◽  
Know How ◽  
Before And After ◽  
Sperm Morphometry ◽  
Computer Assisted Sperm Analysis

In order to improve sperm cryopreservation throughout the year and accelerate the process of preservation of this Catalonian goat breed in extinction danger, we proposed to assess the effect of melatonin implant application in Blanca de Rasquera males during spring on sperm head morphometry of fresh and thawed sperm. Therefore 8 bucks of 30 months old approximately were divided into 2 groups. In one of the groups, 2 melatonin implants (Melovine®, CEVA) were inserted into bucks 60 days before starting the collection of semen, and the other group was kept untreated. Briefly, fresh ejaculates from each group of 4 bucks were collected in spring, immediately mixed in equal quantities, and centrifuged twice (600 g for 10 min). Then the pellet was resuspended in a Tris-based medium containing 15% (v/v) of powdered egg yolk supplemented with 5% glycerol. Afterward, sperm samples were refrigerated at 5°C for 4 h before being frozen in LN vapour. Buck sperm head morphometry was analysed by computer-assisted sperm analysis (ISAS®) on fresh and thawed sperm previously stained with Diff Quick®. Data were analysed by GLM multivariate procedure (IBM SPSS, 2011; mean ± s.e., n = 6), showing significant differences among treatments in all the morphometric parameters except head perimeter and rugosity (Table 1). Our results suggest that melatonin application in bucks increases the ellipticity and elongation of fresh and thawed sperm, meanwhile the cryopreservation process reduces both parameters. Likewise melatonin implants increase significantly the head length only on thawed sperms as cryopreservation process increases the head width, area in sperms from implanted males and regularity only in sperms from nonimplanted bucks. These head changes on fresh and thawed sperm morphometry should be deeply investigated in order to know how they could affect sperm cryosurvival and fertility. Table 1.Effect of melatonin implant on Blanca de Rasquera bucks during spring on morphometry of fresh and thawed sperm This research was supported by INIA (RZ2009–00008–00–00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundación Carolina.

53 FREEZING BULL SEMEN IN A SYNTHETIC MEDIUM

2017 ◽  
Vol 29 (1) ◽  
pp. 134
Author(s):  
L. Gavin-Plagne ◽  
P. Bodranghien ◽  
A. Vachet ◽  
L. Commin ◽  
S. Buff ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Synthetic Medium ◽  
Calf Serum ◽  
Egg Yolk ◽  
Wilcoxon Test ◽  
Computer Assisted ◽  
Sperm Cells ◽  
Bull Semen ◽  
Significant Difference

Animal-derived products are currently used to cryopreserve sperm cells. However, these products represent potential risks of contamination by pathogens. Optidyl® (IMV Technologies, L’Aigle, France), containing egg yolk, is a reference product in Europe used routinely in bovine insemination centers. Commercial media such as soy lecithin or liposome-based media have been used to replace extenders containing products derived from animals. However, their protective effect could be called into question because of their non-synthetic or unstable properties. Despite these innovative extenders on the market, it might be necessary for sanitary reasons to cryopreserve bull semen in a stable and synthetic extender. CRYO3 (Stem Alpha, Saint-Genis l’Argentière, France), a serum-free and protein-free medium used for cryopreserving somatic and stem human cells, is a potential medium to cryopreserve reproductive cells. Recently, CRYO3 improved cryopreservation of in vitro-produced bovine embryos compared with fetal calf serum and BSA-based media. Thus, it could be interesting to test this medium on sperm cells. The objective of this study was to compare 2 in vitro freezing media on bull semen: a commercial egg yolk-based medium and a synthetic medium containing 20% CRYO3. Sperm from 5 bulls were collected in Auriva station (Brindas, France). A sample of each ejaculate was used to assess fresh semen quality. The remaining sperm of each bull was split and diluted in 2 media: Optidyl® and a CRYO3-based medium. Semen was equilibrated at least 4 h at 4°C before being packaged in 0.25-mL French straws, and then frozen into a programmable freezer and plunged into liquid nitrogen. Osmolarity and pH were respectively 1462 mOsm/kg and 6.9 for Optidyl® and 1286 mOsm/kg and 6.8 for CRYO3 medium. Viability (with SYBR-14 and propidium iodide) and high mitochondrial membrane potential (hMMP) (with JC-1) were assessed using flow cytometry. A hypo-osmotic swelling test was performed to assess functional membrane integrity (FMI). The motility parameters were evaluated by computer-assisted sperm analysis. Statistical analysis was performed using Wilcoxon test with the R software. Fresh sperm showed 52% viability, 64% hMMP, 74% FMI, and 62 and 76% progressive and total motility, respectively. For all parameters measured, no significant difference was observed between extenders and between fresh and frozen–thawed sperm (P > 0.05). However, Optidyl® showed clearly better survival than CRYO3 (45% v. 16% for viability, 58% v. 20% for hMMP, 67% v. 25% for FMI, 51% v. 14% for progressive motility and 72% v. 32% for total motility, respectively). These results show that it is possible to freeze bovine semen in synthetic extender though the low survival rate after freezing-thawing. Indeed, it is known that motility and flow cytometry parameters are not necessarily good indicators of fertility. Artificial inseminations will be done to verify the fertility of the sperm cryopreserved in CRYO3. Repetitions with more bulls from different breeds will be performed to complete this preliminary work. This work was supported by grant CRB-ANIM ANR-11-INBS-0003.

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