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 PENGARUH METODE PENCUCIAN SPERMATOZOA SAPI ACEH TERHADAP MOTILITAS, PERSENTASE HIDUP, DAN INTEGRITAS MEMBRAN PLASMA UTUH SPERMATOZOA (The Infuence of Aceh Bull Spermatozoa Washing Method on Spermatozoa Motility and Plasma Membrane Integrity of Intact Spermatozoa)

2015 ◽  
Vol 9 (2) ◽  
Author(s):  
Listin Handayani ◽  
Dasrul Dasrul ◽  
Muslim Akmal ◽  
Cut Nila Thasmi ◽  
Hamdan Hamdan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Spermatozoa Motility ◽  
Sperm Washing ◽  
Fresh Semen

This study aimed to determine the effect of sperm washing by swim up and centrifugation in isotonic medium on sperm quality of aceh bull. In this study, fresh semen from healthy male aceh bull aged 3-4 months was collected using artificial vagina. Immediately after semen collection, fresh semen quality was examined macroscopically and microscopically. Subsequently, sperm washing was performed by centrifugation and swim up in sperm washing medium. Group 1 (P0) as control group, cement washed with isotonic solution (andromed medium: saline solution) with ratio of 1:8. 2. Group 2 (P1), cement was separated by centrifugation method, group 3 (P2), all cement was separated by swim up method then examined the sperm quality sperm washing results. Each treatment was repeated 5 times. Quality parameters measured were the percentage of spermatozoa motility, sperm viability, and plasma membrane integrity intact spermatozoa. Data were analyzed with analysis of variance one-way pattern, followed by Duncan's multiple test. The results showed the mean ± SD percentage of sperm motility of each treatment group (P0; P1; P2) respectively amounted to 72.00±3.74, 66.40±4.77, and 73.60±3.29%. The percentage of viability was 72.00 ±3.74%, 66.40±2.88%, 71.80±2.17%. The percentage of plasma membrane integrity is intact spermatozoa was 68.20±1.79%, 57.20±3.77%, 69.00±2.00%. Results of this study showed that the percentage of motility, live spermatozoa and plasma membrane integrity intact after separation by swim-up method were significantly different (P <0.05) compared with no separation.Key words: spermatozoa quality, aceh bulls, centrifugation, swim up

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 Semen Quality and Freezability Analyses in the Ejaculates of Two Poitou Donkeys in the Southern Hemisphere

2021 ◽  
Vol 8 ◽  
Author(s):  
Francisca Ebel ◽  
Omar Ulloa ◽  
Pablo Strobel ◽  
Alfredo Ramírez-Reveco
Keyword(s):  
Southern Hemisphere ◽  
Seminal Plasma ◽  
Semen Quality ◽  
Individual Variability ◽  
Computer Assisted ◽  
Plasma Analysis ◽  
Sperm Analysis ◽  
New Information ◽  
Chilean Army ◽  
Sperm Movement

The Baudet du Poitou is a vanishing donkey breed recognized for engendering robust working mules. In Chile, only two pure breed Poitou males exist, which belong to the Chilean army and are used for mule production. We performed an extensive sperm and seminal analysis of these two jackasses aged 3 and 6 years and investigated the use of a simple hypometabolic extender for sperm cryopreservation. Computer-assisted sperm analysis showed high motility, velocity, and linearity in sperm movement. The seminal plasma analysis revealed that sodium and chloride were the main electrolytes, and globulins were the main metabolites. Active and variable enzymatic activity was observed. New information is reported about gamma-glutamyltransferase, aspartate aminotransferase, zinc, and magnesium concentrations in seminal plasma of Poitou donkeys. Ejaculates among jackasses showed some variability due to individual variability and different stages in sexual maturation according to age. The freezability index analysis based in viability, total motility and progressive motility with Botucrio extender (57.1 ± 11.0%; 56.6 ± 20.0%; and 22.6 ± 10.3%, respectively) were significantly higher (p &lt; 0.05, p &lt; 0.0001, and p &lt; 0.0001, respectively) than with HM-0 extender (42,6 ± 11.4%; 14.9 ± 5.1%; and 1.0 ± 2.5%, respectively). We report new information on Poitou donkey semen and cryopreservation in the Southern Hemisphere that could be useful in donkey breeding and conservation programs to develop strategies that improve the effectiveness of population management of this breed.

scholarly journals Beneficial Influence of Soybean Lecithin Nanoparticles on Rooster Frozen–Thawed Semen Quality and Fertility

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1769
Author(s):  
Lingwei Sun ◽  
Mengqian He ◽  
Caifeng Wu ◽  
Shushan Zhang ◽  
Jianjun Dai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Quality ◽  
Antioxidant Activities ◽  
Soybean Lecithin ◽  
Sperm Quality ◽  
The Other ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
The Impact

The present study aimed to investigate the impact of different concentrations (0%, 0.5%, 1.0%, 1.5%, and 2.0%) of nano-soybean lecithin (SL) in the extender on sperm quality, sperm motion characteristics, and fertility outcomes of post-thawed rooster semen. Adult Ross broiler breeder roosters (n = 20) were subjected to semen collections twice a week for three weeks. At each collection, semen samples were pooled and allocated into five treatments corresponding to different nano-SL concentrations (control, SL0.5, SL1.0, SL1.5, and SL2.0). Sperm parameters, including motility (collected using a computer-assisted sperm analysis system), plasma membrane and acrosome integrities, and mitochondrial activity were assessed. Sperm malondialdehyde (MDA) and antioxidant activities (total antioxidant capacity (TAC); superoxide dismutase (SOD); glutathione peroxidase (GPx)) were evaluated. The fertility and hatchability obtained with frozen–thawed rooster semen supplemented with the optimum nano-SL concentration were assessed after artificial insemination. The results showed that the addition of 1% nano-SL into the extender led to a higher semen motility in roosters, improved plasma membrane and acrosome integrities, and higher mitochondrial activity of post-thawed rooster semen in comparison to controls (p < 0.05). The MDA levels in the SL0.5 and SL1.0 groups were lower than the other groups (p < 0.05). TAC activities in SL0.5, SL1.0, and SL1.5 groups were significantly higher than those in the other groups (p < 0.05). It was observed that the concentration of SOD was higher in the SL1.0 group than in the other groups (p < 0.05). The activity of GPx was not influenced in any of the cases (p > 0.05). Moreover, the percentages of fertility and hatchability in the SL1.0 group were higher (56.36% and 58.06%) than those in the control group (42.72% and 40.43%). In summary, the addition of nano-SL to the extenders enhanced the post-thawed semen quality and fertility of roosters by reducing the level of oxidative stress. The optimum nano-SL concentration was 1.0%. These results may be beneficial for improving the efficacy of semen cryopreservation procedures in poultry breeding.

65 ARE “BAD FREEZER” STALLIONS ALSO “BAD COOLER” STALLIONS?

2015 ◽  
Vol 27 (1) ◽  
pp. 125
Author(s):  
C. Ramires Neto ◽  
M. M. B. Castro-Chaves ◽  
Y. F. R. Sancler-Silva ◽  
R. C. Uliani ◽  
P. V. L. Oliveira ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Cooling Process ◽  
Freezing Resistance ◽  
Plasma Membrane Integrity ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Fresh Semen

Several factors can interfere with sperm cryopreservation resistance, especially the genetic factors and those related to the plasma membrane composition of the sperm and seminal plasma. However, it is still unclear if the same factors that confer freezing resistance will perform the same role during the cooling process. Thus, the aim of this study was to determine the relation between the resistance to freezing and cooling processes in stallions. Two ejaculates from each of 75 stallions were used. All animals showed good quality of fresh semen (total motility higher than 60% and plasma membrane integrity higher than 50%). After collection, the semen was diluted 1 : 1 with commercial skim milk-based extender (Botu-SemenTM, Botupharma, Brazil) and then a part was designed to cooling and the another to freezing. The cooled semen was divided into 2 groups: Group PS, in which the semen was diluted with Botu-SemenTM at a concentration of 50 × 106 sperm mL–1, and Group SPS, which was subjected to a centrifugation at 600 × g for 10 min and resuspended with Botu-SemenTM at 50 × 106 sperm mL–1. Semen samples from both groups were placed in the same cooling passive system for a period of 24 h/5°C. To accomplish the freezing process, the semen sample was subjected to centrifugation at 600 × g for 10 min. The supernatant was discarded, and the pellet was re-suspended in a Botu-CrioTM. The straws were frozen according to the manufacture. The sperm parameters from fresh semen, cooled semen for 24 h with and without seminal plasma, and frozen semen were evaluated for kinetics by computer-assisted semen analysis and for plasma membrane integrity (IMP%) by epi-fluorescence microscopy. The animals were classified in relation to their resistance to cooling and freezing processes as follow: “bad coolers” – reduction in sperm total motility and in plasma membrane integrity higher than 35% after 24 h of cooling in samples with seminal plasma; “good coolers” – reduction in sperm total motility and in plasma membrane integrity lower than 35% after 24 h of cooling in samples with seminal plasma; “bad freezer” – sperm total motility lower than 40% and progressive motility lower than 20% in seminal sample after thawing; “good freezer” – sperm total motility higher than 60% and progressive motility higher than 30% in seminal sample after thawing. The comparison between the resistance to cooling and freezing processes was performed by Fisher's exact test. The level of significance was 5%. No difference (P < 0.05) between the resistance to cooling and freezing processes was observed. The percentage of stallions “good freezer” and “good cooler” was 54%, “good freezer” and “bad cooler” was 22.6%, “bad freezer” and “good cooler” was 12%, and “bad freezer” and “bad cooler” was 10.6%. Within stallions classified as “good freezer” and “bad cooler,” 52.9% also were “good cooler” when the seminal plasma was removed before the cooling process, and 47.1% remained as “bad cooler.” The result of this study demonstrates that there is a strong relation between the resistance to cooling and freezing processes in stallions. In stallions categorized as “bad cooler,” the seminal plasma presents a major influence on the quality and longevity of cooled semen.

14 MORPHOLOGICAL AND FUNCTIONAL PARAMETERS OF ENDANGERED BERMEYA GOAT BREED SEMEN

2008 ◽  
Vol 20 (1) ◽  
pp. 87
Author(s):  
C. O. Hidalgo ◽  
A. Rodríguez ◽  
C. Díez ◽  
D. Martín ◽  
M. Carbajo ◽  
...  
Keyword(s):  
Semen Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Analysis Data ◽  
Computer Assisted ◽  
Genetic Stock ◽  
Term Survival ◽  
Sperm Analysis ◽  
Functional Parameters ◽  
Fresh Semen

The Bermeya goats are an endangered autochthonous breed distributed in the north of Spain. To ensure their genetic diversity and long-term survival, morphological and functional parameters of the semen must be known in order to preserve the current genetic stock in a germplasm bank. The aim of this work was to establish basic characteristics and post-thaw survival of Bermeya goat's semen obtained by electro-ejaculation, that is not well described in the literature. The semen was collected by electro-ejaculation from 7 bucks, 1 to 7 years old, twice per week, for 9 weeks (n = 83). Fresh semen was evaluated for volume (V), concentration (C), motility, morphology, functional integrity of the sperm (spz) membranes (hypoosmotic swelling test; HOST), and acrosome integrity rate (NAR). Individual and progressive sperm motility were analyzed by means of a computer-assisted sperm analysis system (CASA: SCA 2002�, Microptic, Barcelona, Spain) immediately after dilution with the extender at 37�C, and after cooling to 4�C; five fields per sample (diluted to 204 � 106 spz mL–1) were evaluated under a phase contrast microscope (100�). The NAR and morphological abnormalities of sperm head, midpiece, tail, and cytoplasmic droplets were determined by counting 100 spz under 1000�. For freezing, ejaculates with at least 80% motile spz were diluted at 32�C with Krebs-Ringer solution containing 20% egg yolk and 14% glycerol to a final concentration of 400 � 106 spz mL–1, cooled to 4�C for 90 min, aspirated into 0.25-mL plastic straws (IMV�, L'Aigle, France), frozen at 7 cm above liquid nitrogen (LN2) phase for 10 min, and then plunged into the LN2. Straws were thawed in a water bath at 39�C for 30 s for post-thaw survival analysis. Data were analyzed by the GLM and FREQ procedures (SAS; SAS Institute, Inc., Cary, NC, USA) and expressed as means � standard error. Fresh semen characteristics were: V = 1.7 � 0.1 mL; C = 2619 � 106 � 153 spz mL–1; total and progressive motility were 89.0 � 2.1% and 66.9 � 2.1%, respectively. Percentages of head abnormalities were 4.8 � 0.5; midpiece: 3.8 � 0.7; tail: 4.7 � 1.0; cytoplasmic droplets: 8.3 � 0.7; intact acrosome: 91.8 � 0.6; and membrane integrity: 49.2 � 2.1. At 4�C, the % of total motile spz was 62.6 � 1.6, and the post-thaw survival rate was 46.3 � 1.5. There were only individual differences (P < 0.001) between bucks on sperm concentration, head abnormalities, and cytoplasmic droplets. In conclusion, our results indicate that semen quality is related to each individual animal and that electro-ejaculation allows collection of semen of satisfactory quality to use as fresh and for cryopreservation. However, the validity of our results for possible future sperm banking of endangered Bermeya goats semen must be confirmed by field trials.

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.

scholarly journals Progesterone decrease plasma membrane in human sperm with subnormal hypoosmotic swelling test scores

2019 ◽  
Vol 38 (1) ◽  
pp. 56
Author(s):  
Sisca Sisca ◽  
Luluk Yunaini ◽  
Dwi Ari Pujianto
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Human Sperm ◽  
Uterine Wall ◽  
Control Group ◽  
Computer Assisted ◽  
Sperm Cells ◽  
Sample Treatment ◽  
Hypoosmotic Swelling Test ◽  
Sperm Membrane

Background<br />Progesterone (P4) is known as a female hormone affecting oocyte maturation and developing uterine wall. A proteomic study identified several receptors including P4 receptors on human sperm. The role of P4 in human sperm cells remains unknown as to whether P4 has non-genomic effects on human sperm. The present study aims to determine the effect of progesterone (P4) on the hyperactivated motility and membrane integrity of human sperm cells.<br /><br />Methods<br />Semen from normal individuals was obtained from donors. The semen was washed by gradient density centrifugation. P4 was added to each semen sample to final concentrations of 0 (control), 250, 500, 750 and 1000 ng/mL. After the sample treatment was completed, the sperm membrane integrity was assessed with the hypoosmotic swelling test (sodium citrate dihydrate and D-fructose) and the hyperactivated sperm motility parameter was determined with the Computer Assisted Sperm Analyzer [CASA] (Hamilton Thorne, IVOS II, USA). The percentage was then compared between the treatment groups and the control group. The percentage differences were analyzed with the Sigmastat version 2.0 statistical program.<br /><br />Results<br />Administration of P4 increased sperm hyperactivated motility when compared with the control group at a concentration of 500 ng/mL, but the increase was statistically not signicant (p&gt;0.05). In contrast, P4 decreased sperm membrane integrity significantly (p=0.042). And the mean of plasma membrane integrity in all groups was subnormal hypoosmotic swelling test score. <br /><br />Conclusion<br />Progesterone administration tends to increase sperm hyperactivated motility. The integrity of plasma sperm membrane was affected by progesterone.

scholarly journals Sperm Quality Assessment in Honey Bee Drones

Biology ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 174
Author(s):  
Jesús L. Yániz ◽  
Miguel A. Silvestre ◽  
Pilar Santolaria
Keyword(s):  
Honey Bee ◽  
Multiple Testing ◽  
Semen Quality ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Domestic Animal ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Sperm Volume

The quality of honey bee drone semen is relevant in different contexts, ranging from colony productivity to pathology, toxicology and biodiversity preservation. Despite its importance, considerably less knowledge is available on this subject for the honey bee when compared to other domestic animal species. A proper assessment of sperm quality requires a multiple testing approach which discriminates between the different aspects of sperm integrity and functionality. Most studies on drone semen quality have only assessed a few parameters, such as sperm volume, sperm concentration and/or sperm plasma membrane integrity. Although more recent studies have focused on a broader variety of aspects of semen quality, some techniques currently used in vertebrates, such as computer-assisted sperm analysis (CASA) or multiparametric sperm quality testing, still remain to be developed in the honey bee. This may be attributed to the particular sperm morphology and physiology in this species, requiring the development of technologies specifically adapted to it. This article reviews the present knowledge of sperm quality in honey bee drones, highlighting its peculiarities and proposing future lines of research.

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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