Slow freezing versus vitrification for the cryopreservation of zebrafish (Danio rerio) ovarian tissue

Abstract The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.

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Omega-3 Polyunsaturated Fatty Acids Counteract Inflammatory and Oxidative Damage of Non-Transformed Porcine Enterocytes

Animals ◽

10.3390/ani10060956 ◽

2020 ◽

Vol 10 (6) ◽

pp. 956 ◽

Cited By ~ 2

Author(s):

Tamil Selvi Sundaram ◽

Carlotta Giromini ◽

Raffaella Rebucci ◽

Antonella Baldi

Keyword(s):

Fatty Acids ◽

Cell Membrane ◽

Polyunsaturated Fatty Acids ◽

Membrane Integrity ◽

Sodium Sulphate ◽

Epithelial Cell Line ◽

Mitochondrial Activity ◽

Omega 3 ◽

Cell Membrane Integrity ◽

Enterocyte Apoptosis

Marine and plant-based omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are widely added to animal diets to promote growth and immunity. We tested the hypothesis that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and their 1:2 combination could counteract acute or long-term damage of lipopolysaccharides (LPS), dextran sodium sulphate (DSS) and hydrogen peroxide (H2O2) in Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2). The results showed that 24 h treatment with EPA or DHA exhibited proliferative effects in IPEC-J2 cells at low to moderate concentrations (6.25–50 μM) (p < 0.05). Further, 24 h pretreatment with individual DHA (3.3 µM), EPA (6.7 µM) or as DHA:EPA (1:2; 10 µM) combination increased the mitochondrial activity or cell membrane integrity post-LPS (24 h), DSS (24 h) and H2O2 (1 h) challenge (p < 0.05). Additionally, DHA:EPA (1:2, 10 µM) combination decreased the apoptotic caspase-3/7 activity around twofold after 24 h LPS and DSS challenge (p < 0.05). Our study confirms the proliferative and cytoprotective properties of EPA and DHA in IPEC-J2 cells. Increased intracellular mitochondrial activity and cell membrane integrity by ω-3 PUFAs can play a role in preventing enterocyte apoptosis during acute or chronic inflammatory and oxidative stress.

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

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.116285 ◽

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.

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The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽

10.3390/ani11123452 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3452

Author(s):

Uchechi Linda Ohaneje ◽

Uchebuchi Ike Osuagwuh ◽

Manuel Alvarez-Rodríguez ◽

Iván Yánez-Ortiz ◽

Abigail Tabarez ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Membrane Integrity ◽

Egg Yolk ◽

Quality Parameters ◽

Mitochondrial Activity ◽

Dna Integrity ◽

Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

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101CRYOPRESERVATION OF RAT EPIDIDYMAL SPERM: COMPARISON OF TWO COOLING PROTOCOLS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab101 ◽

2004 ◽

Vol 16 (2) ◽

pp. 172

Author(s):

N. Kashiwazaki ◽

Y. Okuda ◽

A. Takizawa ◽

N. Nakagata ◽

M. Shino

Keyword(s):

Cooling Rate ◽

Plasma Membranes ◽

Membrane Integrity ◽

Egg Yolk ◽

Molecular Probes ◽

Mature Male ◽

Epididymal Sperm ◽

Slow Cooling ◽

Freezing Medium ◽

Sperm Membrane

The present study examined post-thaw motility, plasma membrane integrity and fertility of rat epididymal sperm cooled by two procedures to +5°C, and then cryopreserved by controlled-rate freezing. Wistar rats were used in the present study. In protocol-I (2001 Reproduction 122, 463), epididymides were collected from a mature male and placed in a plastic dish containing 2mL of freezing medium I [23% (v/v) egg yolk, 8% (w/v) lactose monohydrate and antibiotics]. The epididymides were dissected with scissors to release epididymal sperm. The semen was kept at 15°C for 30min and then held at 5°C for 30min. The cooling rate from 15°C to 5°C was 0.3°Cmin−1. The cooled semen was diluted with 2mL of freezing medium II [freezing medium I with 1.4% (v/v) Equex Stm (ES, Nova Chemical Sales, Inc., Scituate, MA, USA)]. Mixed semen was aspirated into 0.25-mL straws and exposed to liquid nitrogen (LN) vapor for 10min. The straws were then plunged into LN. In protocol-II, epididymides were collected from a mature male and placed in 4mL of freezing medium III [freezing medium I and 0.7% (v/v) ES]. The epididymides were dissected with scissors and held for 10min at room temperature to release epididymal sperm. The semen was loaded into 0.25-mL straws and kept at 15°C for 15min and then held at 5°C for 15min. The cooling rate from 15°C to 5°C was 0.7°Cmin−1. The cooled straws were then exposed to LN vapor for 10min and plunged into LN. Straws were thawed in a 37°C water bath for 10s. Thawed semen in a straw was diluted with 1mL of KRB medium with 0.4% (w/v) bovine serum albumin (BSA, fraction V, Sigma, Tokyo, Japan) at 37°C and then incubated at 37°C in 5% CO2 in humidified air. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37°C under a light microscopy at 100×. The sperm membrane integrity was determined using a commercial Live/Dead sperm viability kit (Molecular Probes, Inc., Eugene, OR, USA) which differentiates between cells with intact plasma membranes and those with damaged membranes by fluorescent staining patterns observed with a fluorescence microscope (Olympus, IX-71, Tokyo, Japan). Similar levels of sperm motility were observed immediately after thawing of sperm from both protocols. However, after 2h of incubation, the post-thaw motility of sperm frozen by protocol-II was significantly (P<0.01) higher than that of protocol-I. Sperm membrane integrity immediately after thawing was also higher for sperm frozen by protocol-II (22.1% v. 9.3%, P<0.01). Sperm frozen/thawed by protocol-II was inseminated into the top of the uterine horns of recipient females to confirm fertility. Two of three inseminated females became pregnant and gave birth to 5 pups. These results suggest that loading sperm into straws before cooling and subsequent slow cooling at 5°C to 0.7°Cmin−1 increases post-thaw survival of rat epididymal sperm.

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

Zygote ◽

10.1017/s0967199418000576 ◽

2018 ◽

Vol 27 (1) ◽

pp. 17-24 ◽

Cited By ~ 2

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.

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Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2016000800015 ◽

2016 ◽

Vol 36 (8) ◽

pp. 767-774 ◽

Cited By ~ 4

Author(s):

Patrícia C. Sousa ◽

Erika A.A. Santos ◽

Andréia M. Silva ◽

José A.B. Bezerra ◽

Ana L.P. Souza ◽

...

Keyword(s):

Membrane Integrity ◽

Fluorescence Analysis ◽

Egg Yolk ◽

Ultrastructural Analysis ◽

Semen Parameters ◽

Mitochondrial Activity ◽

Morphological Alterations ◽

Fluorescent Markers ◽

Euphractus Sexcinctus ◽

Nuclear Modifications

Abstract: The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol.

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Use of phosphatidylcholine in Tris-based extender with or without egg yolk to freeze Bapedi ram semen

South African Journal of Animal Science ◽

10.4314/sajas.v50i3.5 ◽

2020 ◽

Vol 50 (3) ◽

pp. 389-396

Author(s):

K.S. Mafolo ◽

C.M. Pilane ◽

T. Chitura ◽

T.L. Nedambale

Keyword(s):

Sperm Cell ◽

Agricultural Research ◽

Membrane Integrity ◽

Germplasm Conservation ◽

Egg Yolk ◽

Protective Agent ◽

Liposome Membrane ◽

Cell Membrane Integrity ◽

Agricultural Research Council ◽

Acrosome Integrity

Traditionally, egg yolk is a protective agent that is used to freeze semen in various species. However, the addition of egg yolk in extender risks the introduction of disease. Therefore, an alternative cryoprotective agent should be found to preserve ram semen. The aim of this study was to evaluate the effect of phosphatidylcholine (PC) as a protective agent in extender with or without egg yolk on semen characteristics and acrosome integrity of frozen then thawed Bapedi ram semen. Semen was collected from four mature Bapedi rams, in the Agricultural Research Council (ARC) Germplasm Conservation Programme, using an artificial vagina. Following collection, semen samples were randomly diluted into Tris-based extender (1: 2), with and without egg yolk, and supplemented with four concentrations of PC liposome (0 mg/ml), 0.25 mg/ml, 0.5 mg/ml and 0.75 mg/ml). Supplementation of PC liposome in extender with or without egg yolk did not improve the semen total motility (TM), progressive motility (PM) and rapid motility (RM) rate. The sperm cell membrane integrity in extender with or without egg yolk was not influenced by the supplementation of PC liposome after thawing (P >0.05). The addition of PC liposome to Tris-based extender with egg yolk had a similar result to control (Tris-based extender with egg yolk) on sperm cell acrosome integrity. In conclusion, supplementation of PC liposome to Tris-based extender without egg yolk had lower sperm cell viability and motility rates compared with the extender with egg yolk, regardless of concentration.Keywords: acrosome, cryoprotectant, liposome, membrane, motility

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82 EVALUATION OF CELL DEATH IN CRYOPRESERVED MOUSE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab82 ◽

2005 ◽

Vol 17 (2) ◽

pp. 191

Author(s):

A.R.S. Coutinho ◽

A.B. Nascimeto ◽

C.M. Mendes ◽

R. Simoes ◽

C.F. Lucio ◽

...

Keyword(s):

Cell Death ◽

Liquid Nitrogen ◽

Chromatin Condensation ◽

Membrane Integrity ◽

Embryonic Cell ◽

Slow Freezing ◽

Cell Membrane Integrity ◽

Frozen Embryos ◽

Mammalian Embryos ◽

Cryopreserved Embryos

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis may be important in determining the viability of cryopreserved embryos. Our goal was to detect apoptosis and characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were collected, selected, and separated into three groups: fresh, slow-freezing, and vitrification. In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. After loading, the straws were placed into methanol at −7°C for 5 min, seeded and after 5 min cooled at 0.5°C/minute. After 10 minutes at −31°C, straws were plunged into and stored in liquid nitrogen. Slow-frozen straws were thawed in air for 10 s, and then immersed in a 25°C water bath for 20 s. Embryos were vitrified by exposing them to 10% and 20% EG for 5 min followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and the 0.25-mL straws then plunged into and stored in liquid nitrogen. The vitrified straws were warmed by immersing them in 25°C water for 20 s. Cell membrane integrity was assessed by Hoechst and propidium iodide double staining (H/PI). Fresh and thawed embryos were scored (following IETS recommendations) and then fixed after 30 min in PBS + 10% FCS. Morphology and apoptosis were assessed with Haematoxylin-Eosin staining (HE) and by electron microscopy (MET). The number of Grade I embryos recovered after thawing was higher for slow-frozen embryos (61.5%) than vitrified embryos (29.5%). H/PI detected more membrane permeability in the vitrified embryos (69.7%), than in the slow-frozen (48.4%) or non-frozen (13.8%) groups (P < 0,05, Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow-freezing induced pyknosis and chromatin condensation. Mitotic pattern was observed in the fresh and slow-frozen group, but not in vitrification group suggesting that the embryos were either not randomly allocated to the groups or not-treated and fixed at the same age, or that vitrification changed the nuclear status of the embryos. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), while in the slow-frozen group the presence of cytoplasmic condensation and eosinophilic structures indicated apoptosis. The ultrastructure examination confirmed the HE observations. In conclusion, the results demonstrated that staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. According to these data, vitrification caused more cellular injuries than slow-freezing, and oncosis was the predominant injury. It is important to point that specific molecular apoptosis tests must be performed to confirm these results. This work was supported by FAPESP 04/01252-4.

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EXPOSURE TO FLUORIDE SIGNIFICANTLY DECREASE THE CELL MEMBRANE INTEGRITY AND MITOCHONDRIAL ACTIVITY IN MURINE RENAL CELLS

International Journal of Advanced Research ◽

10.21474/ijar01/10356 ◽

2020 ◽

Vol 8 (1) ◽

pp. 735-743

Author(s):

Thanusha Perera ◽

◽

Kasun Godakumara ◽

Shirani Ranasinghe ◽

Neil Alles ◽

...

Keyword(s):

Cell Membrane ◽

Membrane Integrity ◽

Mitochondrial Activity ◽

Renal Cells ◽

Cell Membrane Integrity

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Influence of Coconut Powder Water-Based Conservation Medium (APC-102c) for Maintaining Mitochondrial Activity of Cryopreserved Ram Sperm

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.98684 ◽

2019 ◽

Vol 47 (1) ◽

Author(s):

Bruna Farias Brito ◽

Bárbara Mara Bandeira Santos ◽

Leonardo Alves Rodrigues Cabral ◽

David Baruc Cruvinel Lima ◽

Cristiane Clemente De Melo Salgueiro ◽

...

Keyword(s):

Plasma Membrane ◽

Liquid Nitrogen ◽

Sperm Motility ◽

Artificial Insemination ◽

Membrane Integrity ◽

Egg Yolk ◽

Mitochondrial Activity ◽

Plasma Membrane Integrity ◽

Significant Difference ◽

Motility Parameters

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders is key for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is through microscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and may not provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to add reliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a more accurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10). Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS + 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through the refrigeration curve up to 4°C (0.35° C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cm above liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Both fresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x), and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with the Eosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained). Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3'-diaminobenzidine (DAB); 200 sperms were counted, and classified into four classes (I, II, III, and IV) according to the degree of coloration of the intermediate part. Fresh semen showed no significant difference (P > 0.05) between treatments with respect to motility parameters; however, T2 showed significantly inferior results regarding plasma membrane integrity. After thawing, T2 was significantly higher in sperm motility parameters compared to T1. The mitochondrial activity and plasma membrane integrity parameters did not show any significant difference between the treatments.Discussion: The TRIS-based diluent showed higher motility values than ACP-102c; however, motility rates in ACP-102c diluent, although lower, are considered satisfactory for insemination, which requires semen with minimal progressive motility of 30%. Notably, the cryopreservation protocol used in this study is the standard for TRIS-based diluent, and it is known that the optimal rate of refrigeration and cryopreservation may differ according to the composition of the storage medium; therefore, we may assume that the protocol used is not yet appropriate for the ACP-102c diluent, and further studies are required. IMP is an essential attribute for fertilization, and cryopreservation can affect the plasma membrane as observed in this study. Cryopreserved semen reduced the percentage of class I mitochondrial reaction sperms in both treatments, demonstrating that cryopreservation affects the mitochondrial activity of the intermediate portion of the sperm; however, there was no difference between treatments in thawed semen. Thus, we concluded that the ACP-102c conservation medium maintains seminal quality after thawing, and it can be used in artificial insemination processes.

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