Removal Of Seminal Plasma Research Articles

Effect of glutathione on the quality of frozen buck semen

Ejaculates (30) collected twice weekly from five Beetal bucks were used to study the effect of addition of 0 (control), 4 mM, 6 mM and 8 mM glutathione on the quality of frozen Beetal buck semen by split sample technique. After removal of seminal plasma, the semen was primarily extended with Tris extender (1: 5) considering the volume of semen prior to removal of seminal plasma and then split into 4 parts and finally extended with equal volume of Tris extender that rose the extension rate to 1: 10. The mean percentage of sperm motility, live sperm, live intact acrosome, HOST-reacted sperm was significantly higher in Tris extender containing 4 mM glutathione than that containing 6 mM, 8 mM glutathione and control. The release of ALT and AST from post-thaw spermatozoa was also the lowest in semen containing 4mM concentration of glutathione. Based on post-thaw sperm motility, live sperm, live intact acrosome, HOST-reacted sperm, and ALT and AST release, addition of 4 mM glutathione in Tris extender was superior to 6 mM, 8 mM and 0 mM (control). It was concluded that glutathione at 4 mM could be used as an antioxidant in Tris for cryopreservation of Beetal buck semen which could provide a better environment in protecting the functional capacity of spermatozoa.

Comparison of cushioned centrifugation and SpermFilter filtration on longevity and morphology of cooled-stored equine semen

This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and...

45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P < 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P > 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P < 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P < 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen. Acknowledgments are extended to CAPES, Brazil, for the financial support.

Coenzyme Q10 and α-Tocopherol Prevent the Lipid Peroxidation of Cooled Equine Semen.

Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α-tocopherol (α-TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with the following concentrations: α-TOH: 2 mm; CoQ10: 40 μg/ml; and CoQ10 + α-TOH: 40 μg/ml + 2 mm for control (C) without the addition of antioxidants and for vehicle control (EtOH) with 100 μl ethanol. The CoQ10 group had a higher percentage of total motility (69.1 ± 16.2%) compared to control (62.1 ± 16.2%) and EtOH (58.1 ± 18.6%). CoQ10 + α-TOH and α-TOH groups were most effective in preventing LPO compared to controls (1765.9 ± 695.9, 1890.8 ± 749.5, 2506.2 ± 769.4 ng malondialdehyde/10(8) sptz, respectively). In conclusion, CoQ10 and α-TOH were effective during the cooling process of equine semen at 5°C for 72 h, providing increased levels of total motility, as well as lower LPO.

Coatis (Nasua nasua) semen cryopreservation

<p>Os procedimentos de criopreservação do sêmen em carnívoros devem ser iniciados após a lavagem e centrifugação em meios de cultura para retirada do plasma seminal e eliminação de microrganismos. O objetivo deste estudo foi realizar a criopreservação do sêmen de quatis comparando os efeitos entre dois extensores Ham’s F-10 e M199 para lavagem e centrifugação do sêmen antes do congelamento com o meio Dilutris. Amostras de sêmen (n = 36) foram coletadas por eletroejaculação em seis quatis (<em>Nasua nasua</em>) machos adultos entre maio e outubro de 2008, no Zoológico da Universidade Federal de Mato Grosso. Motilidade total (%), motilidade espermática progressiva (0-5), integridade da membrana plasmática (%) e integridade do acrossoma (%) foram analisados. Amostras de sêmen a fresco foram divididas em duas frações, diluídas em 1 mL de Ham’s F-10 (Ham’s F-10, Nutricel S.A., Brasil) ou M199 (M199, Nutricel S.A., Brazil) e centrifugado a 300 g por 10 min. O sobrenadante foi descartado e pellets ressuspendidos em 1 mL de Dilutris (Dilutris, Minitube<em>®</em>, Brasil), armazenados a 5ºC durante 3 horas, transferidos para palhetas de 0,25 mL, colocadas em vapor de nitrogênio líquido por 20 min e imersos em nitrogênio líquido. Os resultados para sêmen a fresco e sêmen congelado utilizando Ham’s F-10/Dilutris e M199/Dilutris foram, respectivamente: 84,28 ± 11,57, 45,38 ± 27,26 e 44,61 ± 25,03 para motilidade total; 3,64 ± 1,44, 2,15 ± 1,14 e 2,07 ± 1,03 para a motilidade progressiva; 92,76 ± 3,46, 84,69 ± 15,77 e 89,76 ± 13,97 para integridade da membrana plasmática; e 94,76 ± 2,89, 92,35 ± 4,73 e 90,58 ± 7,17 para integridade do acrossoma. Não houve diferença significativa (P < 0,05) entre os valores obtidos nos tratamentos Ham’s F-10/Dilutris ou M199/Dilutris. Ambos os tratamentos demonstraram ser adequados para a congelação de sêmen desta espécie.</p>

Cryopreservation of Sambar deer semen in Thailand.

Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer in developing countries.

Effect of butylated hydroxy toluene on the quality of frozen goat semen

Ejaculates (40) collected from 5 Beetal bucks twice weekly were used to study the effect of 0 (control), 150, 200 and 300 μM butylated hydroxy toluene (BHT) on the quality of frozen semen by split sample technique. After removal of the seminal plasma, the semen was primarily extended with Tris extender (1: 5) considering the volume of semen prior to removal of seminal plasma and then split into four parts and finally extended with equal volume of Tris extender containing 0 (control), 300, 400 and 600 μM BHT. Consequent upon addition of equal volumes of Tris extender the rate of extension of semen rose to 1: 10 and eventual concentration of BHT in the extender was 0, 150, 200 and 300 μM. The extended semen was cooled gradually to 5°C @ 1°C/3 min and equilibrated in the cold handling cabinet for 4 h at 5°C. The extended semen was filled in French mini straws (0.25 ml) and polyvinyl alcohol powder of different colours were used for sealing the extended semen for identification of semen of different bucks processed with different concentrations of BHT. The freezing of semen was done by rapid horizontal vapour freezing technique using liquid nitrogen. The mean percent post thaw sperm motility, live sperm, live intact acrosome and HOST-reacted sperm were significantly higher in the extender with 200 μM BHT than with 150, 300 μM BHT and control. The release of ALT and AST from post thaw spermatozoa was also the lowest (33.75 ± 0.17 and 221.61 ± 0.73 U/L respectively) in the extender containing 200 μM BHT. Based on sperm motility, live sperm, live intact acrosome, HOST-reacted sperm, and ALT and AST release, 200 μM BHT in Tris extender was superior to 0, 150, and 300 μM BHT in freezing Beetal buck semen. It was concluded that BHT at 200 μM provided a better protection to the functional capacity of buck spermatozoa for cryopreservation.

Control Methods and Evaluation of Bacterial Growth on Fresh and Cooled Stallion Semen

The penis and prepuce of the stallion have a high bacterial load on its surface, forming a natural microbial flora that contaminates the semen during ejaculation. Bacterial growth in semen may cause a decline on sperm quality, viability, and fertility and predisposes the occurrence of endometritis in inseminated mares. Thus, the aim of this study was to evaluate the effect of penile wash before semen collection, the addition of different commercial skim milk–based extenders containing antibiotics (BotuSemen and INRA96), and the removal of seminal plasma by filtration on the quality, viability, and bacterial proliferation on fresh and cooled stallion semen. Animals that were never submitted to penile wash before semen collection tended to have lower bacterial contamination in the ejaculate. Semen samples extended in BotuSemen showed superiority in total motility, progressive motility, average path velocity, and rapid sperm and lower bacterial contamination in relation to semen samples extended in INRA96 after 24 hours of cooling. No difference was found in these parameters between the storage temperatures (5°C and 15°C). Furthermore, the removal of seminal plasma by filtration reduced the bacterial load in semen after cooling. In conclusion, the penile wash before semen collection tended to reduce the bacterial growth in fresh semen. The use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration were effective in reducing the bacterial contamination and preserved the quality of cooled stallion semen.

67 EFFECTS OF ANTIOXIDANTS LACTOFERRIN AND CATALASE ON STALLION FROZEN SEMEN

During cryopreservation, the sperm were submitted to an increased generation of reactive oxygen species. Furthermore, because of the large portion of seminal plasma removal, there is a decrease of sperm antioxidant protection. Addition of antioxidants proteins found in seminal plasma, such as lactoferrin (Lf) and catalase (Cat), to the freezing semen extenders could protect the sperm during cryopreservation. Lactoferrin is a transferrin, which prevents the hydroxyl radicals generation, and Cat plays an antioxidant role. The aim of this study was to determine the effects of Lf and Cat supplementation to the INRA 82 freezing extender (Battelier et al. 1997) on sperm motility parameters and membrane functionality of stallion frozen semen. Semen from 6 stallions was collected with an artificial vagina, diluted with Kenney extender (1 : 1), and centrifuged (500 × g, 10 min). The supernatant was discarded, and sperm number per milliliter was calculated. Semen was resuspended with 3 extenders to 100 × 106 sperm mL–1. The treatments were distributed in (F1) control, INRA 82 freezing extender (Battelier et al. 1997), (F2) F1+ 500 μg mL–1 lactoferrin, and F3) F1 + 200 IU mL–1 catalase. Semen samples were packaged in 0.5-mL straws and cooled to 5°C (0.27°C min–1). For semen freezing, the straws were laid over the LN vapor for 20 min and plunged into the LN. The straws were thawed at 37°C for 30 s. Motility parameters of frozen semen were determined using a computer sperm cell analysis, and sperm membrane functionality was assessed by the hyposmotic swelling test (Lagares et al. 1998). The data were analysed using Friedman test using stallion as a block. A probability of P < 0.05 was considered significant. There was no significant difference between the percentage of total sperm motility (median, minimum-maximum value; F1: 29.9, 11.0–82.7; F2: 49.8, 7.7–55.2; F3: 39.8, 5.7–92) and progressive sperm motility (F1: 7.1, 3.2–23.3; F2: 13.4, 2.6-22.4; F3: 15.6, 1.1–29.6), and functional sperm membrane (F1: 26.7, 14.7–56.2; F2: 50.5, 15.7–61.7; F3: 46.6, 13.8–50.9) with regard to the treatment. However, the velocity parameters: velocity average path (F1: 29.3, 22.1–33.80; F2: 34.6, 24.8–44; F3: 35.7, 18.2–42.6), velocity curvilinear (F1: 36.9, 30.5–45.1; F2: 42.5, 34.7–51; F3: 44.6, 25.5–50.9), and velocity straight line (F1: 23.4, 17–3.60; F2: 28.9, 18.8–38.2; F3: 26.6, 13.6–37.2) in the treatment with Lf (F2) were higher compared with the control (F1; P < 0.05). These results corroborate with studies reporting the lack of positive effect on equine sperm motility when antioxidants were added to skim milk-based extenders. Although the addition of Lf or Cat to skim milk-based extenders did not improve the motility sperm characteristics and sperm membrane functionality, more studies about the positive effect of Lf on the velocity parameters are necessary. Lactoferrin could then play an important role on the oxidative metabolism, which provides energy to the sperm movement.Acknowledgments to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, for the financial support.

Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n=12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300mM Tris, 28mM glucose, 95mM citric acid 5% glycerol to a concentration of 200×106sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100mg/mL skimmed milk powder and 27.75mM glucose (without glycerol) to a concentration of 400×106sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200×106sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25mL straws, held for 2h at 4°C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean±SEM) were significantly lower (P<0.05) in P1 as compared to P2 (47.50±1.23% vs. 55.63±1.72%; 80.04±1.29% vs. 84.04±1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P>0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.

Effect of holding time and removal of seminal plasma on preservation of boar semen at liquid state

Effects of 3 holding times and 3 levels of removal of seminal plasma on the quality of Hampshire boar semen during preservation in BTS extender for different periods were studied. The semen samples were held at 24°C prior to extension and preservation. The effect of removal of 0, 50, 75 and 100% of seminal plasma was studied after holding for 5 h by subsequently replacing and extending it with BTS before preservation. The mean sperm motility, live sperm count and intact acrosome were significantly higher at 5 h than at 6 and 7 h of holding. The corresponding parameters recorded at zero hour of preservation were 83.89±1.25, 89.22±1.29 and 88.78±1.35%, which decreased significantly with increase in hour of preservation and recorded to be 66.39±2.17, 70.78±1.83 and 75.83±1.73% at 96 h of preservation in semen held for 5 h. All the 3 sperm parameters were significantly higher in semen without removal of seminal plasma, i.e., non-removal than in 50, 75 and 100% removal of seminal plasma. The mean sperm motility, live sperm count and intact acrosome recorded were 66.39±2.17, 71.39±1.36 and 70.61±1.22% at 96 h of preservation without removal of seminal plasma.

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1mg CLC/120×106sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.

New seminal plasma removal method for freezing stallion semen

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600×g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

Methods of Concentrating Stallion Semen

Removal of excess seminal plasma is sometimes necessary to increase the quality and the longevity of cooled equine semen; moreover, this procedure is an indispensable step aiming to concentrate the sperm cells before freezing equine semen. Typically, the removal of seminal plasma is achieved by centrifugation; however, studies have shown that the force and duration of centrifugation can damage sperm cells and reduce the sperm recovery rate. Recently, new methodologies, such as cushion and filtration, have been described that aim to decrease the mechanical damage of centrifugation to sperm cells. This study aims to compare different methods for concentrating stallion semen.

Effect of two extenders on fertility of mares inseminated with jackass semen diluted and cooled at 5°C for 12 hours

concentration as the first half. Spermwas then stored at 4 C for 24hours. Immediately prior to cooling and after 24hours of storage at 4 C, spermwas analyzed for kinetic and quality parameters: total motility (MT) and average path velocity (VAP) were assessed by Computer Assisted Sperm Analysis IVOS, membrane integrity (IM) was evaluated using NucleoCounter SP-100, and DNA integrity (expressed as DNA fragmentation index, DFI) was evaluated by Sperm Chromatin Structure Assay (SCSA) [1]. The Wilcoxon matched pairs test was used to evaluate the effect of seminal plasma on MT, VAP, IM, and DFI. Prior to storage, 50% seminal plasma had no effect on total motility, DNA and membrane integrity, when compared with samples containing 0% plasma; however, the presence of 50% plasma significantly (P < 0.01) increased average path velocity. After 24 hours of storage at 4 C, spermwith 0% seminal plasma showedbetter DNA quality (lower % DFI, P < 0.001), higher values of membrane integrity (P < 0.05) and total motility (P < 0.01) as compared to sperm stored with 50% seminal plasma, but no differences were found for average path velocity. During cooled storage, samples with 0% seminal plasma showed no changes with respect to kinetic and quality parameters, while a significant (P < 0.01) worsening of DNA integrity, totalmotility andmembrane integritywas evidenced for the samples extended in 50% seminal plasma after 24 hours at 4 C. In conclusion, the complete removal of seminal plasma during cooled semen storage was protective to sperm quality, while the presence of seminal plasma was associated with an increasing of sperm DNA damage and alteration of membrane stability after 24 hours storage at 4 C.

Effects of seminal plasma removal on bacterial growth in stallion semen

Effects of seminal plasma removal on bacterial growth in stallion semen

Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation

Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090gmL−1 and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165gmL−1. Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation.

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.

Removal of Seminal Plasma Enhances Membrane Stability on Fresh and Cooled Stallion Spermatozoa

Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4 °C: motility, plasma membrane integrity as evaluated by hypo-osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty-six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.

Cryopreservation of Cooled Semen and Evaluation of Sperm Holding Media for Potential Use in Equine-Assisted Reproduction Procedures

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.