Insights into soy lecithin and egg yolk-based extenders for chilling canine spermatozoa

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Andressa Dalmazzo ◽  
Daniel de Souza Ramos Angrimani ◽  
João Diego A. Losano ◽  
Carolina C. Rocha ◽  
Carlos A. B. Sobrinho ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Compensatory Mechanism ◽  
Soy Lecithin ◽  
Sperm Analysis ◽  
Tbars Assay

SummaryThe aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3′3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.

240 FUNCTIONAL TRAITS OF CAT SPERM DURING DISTINCT MATURATION STATUS

2011 ◽  
Vol 23 (1) ◽  
pp. 218
Author(s):  
E. G. A. Perez ◽  
M. Nichi ◽  
C. A. Baptista Sobrinho ◽  
P. A. A. Góes ◽  
A. Dalmazzo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Structural Damage ◽  
Membrane Integrity ◽  
Domestic Cat ◽  
Lower Percentage ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Suitable Model ◽  
Sperm Analysis

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.

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 Addition Palmitic Acid and Vitamin E to the Tris-Egg Yolk Diluter in Canine Semen Cryopreservation

2021 ◽  
Vol 49 ◽  
Author(s):  
Marcos Antônio Celestino de Sousa Filho ◽  
Luanna Soares de Melo Evangelista ◽  
Filipe Nunes Barros ◽  
Jefferson Hallisson Lustosa da Silva ◽  
Anna Monallysa Silva de Oliveira ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Vitamin E ◽  
Citric Acid ◽  
Palmitic Acid ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Acrosomal Membrane

Background: Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal’s species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing.Materials, Methods & Results: Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog’s semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn´t exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2.Discussion: The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100 µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.Keywords: dog, semen, antioxidants, cryopreservation.Descritores: cão, sêmen, antioxidantes, criopreservação.

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 Effects of the addition of docosahexaenoic acid and ?-tocopherol on quality of equine spermatozoa stored at 5°C

2020 ◽  
Vol 41 (1) ◽  
pp. 167 ◽  
Author(s):  
Breno Fernandes Barreto Sampaio ◽  
Bruno Gomes Nogueira ◽  
Maria Inês Lenz Souza ◽  
Eliane Vianna da Costa-e-Silva ◽  
Carmem Estefânia Serra Neto Zúccari
Keyword(s):  
Lipid Peroxidation ◽  
Plasma Membrane ◽  
Docosahexaenoic Acid ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Membrane Composition ◽  
Artificial Vagina

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and ?-tocopherol (?-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; ?-Toh; DHA/?-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of ?-Toh; : 10 ng mL-1 of DHA + 2 mM of ?-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p < 0.05) when compared to control (62.1 ± 16.2), DHA/?-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p < 0.05) than the groups treated with DHA (2,036.0 ± 687.0), ?-Toh (1,890.8 ± 749.5) and DHA/?-Toh (1,821.1 ± 627.2). In conclusion, ?-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.

scholarly journals How to Increase Post-Thaw Semen Quality in Poor Freezing Stallions: Preliminary Results of the Promising Role of Seminal Plasma Added after Thawing

Animals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 414 ◽  
Author(s):  
Jiří Šichtař ◽  
Filipa Bubeníčková ◽  
Jitka Sirohi ◽  
Ondřej Šimoník
Keyword(s):  
Plasma Membrane ◽  
Seminal Plasma ◽  
Semen Quality ◽  
Membrane Integrity ◽  
Control Group ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Prolonged Incubation ◽  
Sperm Analysis ◽  
Spermatozoa Motility

The aim of this study was to evaluate the effect of the addition of two types of seminal plasma (SP) after thawing on the functional characteristics of frozen–thawed (F–T) spermatozoa of poor freezing stallions during prolonged incubation periods. Seminal plasma from stallions with 35–40% (standard seminal plasma, (S-SP)) and 60–70% (above standard seminal plasma, (A-SP)) progressively motile spermatozoa after thawing was used. The motility, kinematic parameters (Computer Assisted Sperm Analysis), distribution of spermatozoa into subpopulations, integrity (carboxyfluorescein diacetate/propidium iodide staining), and functionality (hypo-osmotic swelling (HOS) test) of the spermatozoa plasma membrane were evaluated after thawing (T0) and after 30 min (T30) of incubation at 37 °C. There was no effect of SP addition on spermatozoa motility, but there was a significant positive effect on the kinematic parameters at T0 and T30. The addition of SP significantly increased the percentage of spermatozoa in the fast subpopulation at T0 as well as at T30. Plasma membrane integrity was not affected by the treatment, but functionality significantly decreased by 5% compared to the control group when samples were incubated for 30 min with A-SP. In conclusion, generally, the post-thaw addition of seminal plasma positively affected the post-thaw quality of semen from poor freezing stallions.

scholarly journals The in vitro effect of kanamycin on the behaviour of bovine spermatozoa

2019 ◽  
Vol 1 (4) ◽  
pp. 36-40
Author(s):  
Michal Ďuračka
Keyword(s):  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
In Vitro Cultivation ◽  
Sperm Analysis ◽  
Time Dependent Effect ◽  
Bovine Spermatozoa ◽  
And Function ◽  
Vitro Effect

The use of antibiotics is a common part of animal biotechnologies. Especially, the use of antibiotics in semen extenders is necessary. However, the effect of antibiotics on the spermatozoa structure and function is still not completely examined. Therefore, the aim of our study was to investigate the effect of kanamycin on bovine spermatozoa at concentrations of 80 and 160 µg/mL during the 24 h in vitro cultivation. Semen samples were collected from clinically healthy Holstein-Friesian bulls. At times of 0, 2 and 24 h the motility assessment, mitochondrial activity, acrosomal and membrane integrity evaluation were performed. The sperm motility was measured using the Computer-assisted sperm analysis (CASA). Mitochondrial activity was evaluated through the Mitochondrial Toxicity Test (MTT). The acrosomal status was determined using the fast green/rose bengal staining on slides. Similarly, the membrane integrity was analysed using the eosin-nigrosin staining. Our results revealed the dose- and time-dependent effect of kanamycin under the in vitro conditions. In conclusion, the selected concentrations of kanamycin may have adverse effects on the motility, mitochondrial activity, acrosomal and membrane integrity during semen processing. Considering the relatively low concentrations used, we do not recommend to use kanamycin as a supplement in bovine semen extenders.

scholarly journals Characteristics of the post-thawed Balinese bull semen extended in three different extenders and equilibration times

2019 ◽  
Vol 44 (2) ◽  
pp. 135
Author(s):  
A. S. Amal ◽  
R. I. Arifiantini ◽  
M. A. Setiadi ◽  
S. Said
Keyword(s):  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Equilibration Time ◽  
Osmotic Swelling ◽  
Sperm Analysis ◽  
Bull Semen ◽  
Sperm Survival ◽  
Computer Assisted Sperm Analysis

The objectives of the present study were to compare and determine the best post-thawed characteristics of balinese bull sperm cryopreserved in three different extenders; animal based (Tris-clarified egg yolk (Tris-cEY)), and non-animal based extenders (Bioxcell® (lecithin based) and Optixcell® (liposome based)) in combination with three different equilibration times (30 minutes, 2 hours, 4hours). Thirty six ejaculates were collected from six Balinese bulls and frozen in three extenders (Tris-cEY, Bioxcell® and Optixcell®) after equilibration in three different times (30 minutes, 2hours and 4hours). Computer-assisted sperm analysis (CASA), hypo-osmotic swelling test (HOST) and eosin nigrosin staining were used in the post-thawed semen analysis. There was a significant interaction between equilibration time and extender type for sperm motility, viability and membrane integrity. Thirty minutes equilibration time had the lowest values (P<0.05) for all the evaluated parameters independent of extender type. Overall, semen extended in Tris-cEY, Bioxcell® and Optixcell® were similarly better when equilibrated at 4 hours (P>0.05). Moreover, post-thawed semen which were extended in Optixcell® for 2 hours equilibration showed a better motility compared with the other extenders (P<0.05). In conclusion, two hours equilibration of semen with Optixcell® is sufficient for semen freezing. Four hours equilibration has the best sperm survival, independent of the extender type.

scholarly journals Effect of crocin and naringenin supplementation in cryopreservation medium on post-thawed rooster sperm quality and expression of apoptosis associated genes

2019 ◽  
Author(s):  
Mahdieh Mehdipour ◽  
Hossein Daghigh Kia ◽  
Abouzar Najafi
Keyword(s):  
Lipid Peroxidation ◽  
Mrna Expression ◽  
Caspase 3 ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Ros Generation ◽  
Hatching Rate

AbstractThe aim of our research was to examine the effects of crocin (0.5 (C0.5), 1 (C1) and 1.5 (C1.5) mM) and naringenin (50 (N50), 100 (N100) and 150 (N150) µM) in cryopreservation extender for freezing rooster semen. Sperm motility, viability, abnormalities, membrane integrity, mitochondrial activity, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC, the mRNA expression of pro-apoptotic (CASPASE 3) and anti-apoptotic (Bcl-2) genes, fertility and hatchability rate were investigated following freeze-thawing. C1 and N100 resulted in the higher (P < 0.05) total motility and progressive motility in comparison to the control group. C1 and N100 improved viability, membrane integrity and reduced lipid peroxidation. We found much higher values for mitochondria activity with C1 and N100 respect to the control group. The C1 and N100 showed lower percentages of early apoptosis when compared with control group. Also, C1 and N100 had higher TAC when compared with control group. The mRNA expression of BCL-2 in the C1 and N100 group were significantly higher than that of other treatments. The expression of CASPASES 3 was significantly reduced in C1 and N100 group (P < 0.05) when compared to control group. Significantly higher percentage of fertility and hatching rate were observed in C1 and N100 compared to the control group. In conclusion, crocin at 1 mM and naringenin at 100 µM seem to improve the post-thawing rooster semen quality, fertility and could protect the sperm against excessive ROS generation by reducing the pro-apoptotic (CASPASE 3) and increasing anti-apoptotic (Bcl-2) genes.

12 FERTILITY OF FROZEN EQUINE SPERM IN SYSTEMS FOR CRYOPRESERVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 117
Author(s):  
R. R. D. Maziero ◽  
P. N. Guasti ◽  
I. D. P. Blanco ◽  
I. Martin ◽  
G. A. Monteiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Egg Yolk ◽  
Automated System ◽  
Epifluorescence Microscopy ◽  
Computer Assisted ◽  
Plasma Membrane Integrity ◽  
Sperm Analysis

Optimizing cryopreservation of equine sperm will facilitate genetic banking and propagation of important horse strains through assisted reproduction. This study aimed to evaluate the motility pattern using computer-assisted sperm analysis (CASA) and plasma membrane integrity by epifluorescence microscopy of equine semen frozen in 0.5 mL straws at different freezing rates; also, a fertility trial was performed according to the freezing protocol. Three ejaculates from four stallions of various breeds (Mangalarga Marchador, Westfallen, Hanovarian and Arabian) and ages (5 to 20 years) were collected and processed for cryopreservation. The stallions were housed at the CERBEQ, Reproduction Centre of the Department of Animal Reproduction and Veterinary Radiology, UNESP. The ejaculates were filtered and submitted to analysis by CASA (HTM IVOS 12, Hamilton Thorne Research, USA). In addition, the plasma membrane integrity was determined by fluorescent probes. After evaluation, the ejaculates were diluted at 1:1 (extender:semen) with skim milk extender Botu-Semen™ and centrifuged at 600 × g for 10 min. The supernatant was removed and the pellet resuspended to a final concentration of 100 × 106 sperm mL–1 with milk-egg yolk freezing extender (Botu-Crio™). Semen was packaged in 0.5-mL straws (IMV, LAigle, France) and was placed in nitrogen for 20 min and then from room temperature to 5°C and then frozen in two different cooling systems: an isothermic box (42 cm × 28 cm × 12.5 cm) was placed upon racks suspended 6 cm above liquid nitrogen or other 20 min then immersed into nitrogen and automated system Mini Digitcool™ (IMV Technologies, France), cooling at a –40°C min–1 rate. All straws were stored in liquid nitrogen until thawing and analysis. The straws were thawed in a water bath at 46°C for 20 s and the samples were evaluated for progressive motility, angular progressive velocity, progressive velocity, track speed, percentage of rapid sperm and percentage of sperm with plasma membrane integrity. For the fertility trial, 65 clinically healthy mares had their oestrous cycle monitored by ultrasound and inseminated postovulation with sperm into the uterus. Ovulation was induced with 1 mL of deslorelin acetate (GnRH) injected IM when a 35-mm follicle was detected. Thirty-six hours later, mares were monitored every 6 h until ovulation was detected. When it was detected, mares were inseminated with 800 × 106 total sperm. Pregnancy was confirmed via ultrasound examination 15 days after ovulation. Pregnancy rate was 52.2% using the isothermic box and 60% using the automated machine. Statistical analysis from the frozen–thawed semen evaluated parameters was performed using the statistics software Proc. MIXED of SAS 9.1 and for the fertility trial, logistic regression using the Proc GENMOD from SAS 9.1. The conventional method using the isothermic box was similar to the automated machine with a fast freezing rate. Additionally, AI with 800 × 106 sperm frozen in the isothermic box or automated system resulted in similarly acceptable conception rates.

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