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Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws
Abstract
The objectives were to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa of native Malpura and Bharat Merino breeds maintained under a semi-arid tropical environment. Good quality semen samples of both breeds were diluted, packaged in medium-sized straws, and frozen under controlled conditions. Straws were thawed at 60 degrees C for 10s and thawed samples were incubated at 37 degrees C for 4h. Post-thaw motion characteristics and acrosomal integrity of incubated spermatozoa were assessed (by computer-aided semen analysis and Giemsa staining, respectively) just prior to incubation and at hourly intervals thereafter. There was a significant effect of incubation time on motility characteristics and the proportion of spermatozoa with normal acrosomes; 81.4% (arcsin transformed value, 65.2) of spermatozoa were motile at the start of incubation, with 47.9% (arcsin transformed value, 44.4) motile after 4h. At the corresponding times, there were normal acrosomes in 65.8 (arcsin transformed value, 54.8) and 55.7% (arcsin transformed value, 48.9) of spermatozoa, respectively. The percentage straightness of spermatozoa varied during incubation (P < 0.01). However, there was no significant change in percentage linearity, curvilinear velocity, average path velocity, straight line velocity, lateral head displacement, and beat cross frequency of spermatozoa during incubation. There were no breed variations in any motility parameters during incubation, except percentage straightness (P < 0.05), lateral head displacement (P < 0.05) and beat cross frequency (P < 0.01). That sperm motility and acrosomal morphology were very acceptable immediately post-thaw and after 4h of incubation indicated the efficacy of cryopreserving ram spermatozoa under controlled conditions in medium-sized straws.
... Tanto la capacitación como la reacción acrosomal pueden ser alcanzadas in vitro bajo diferentes condiciones, dentro de las cuales se incluye la incubación a temperatura corporal o incubación térmica. Estudios realizados en animales han mostrado que la incubación térmica puede modular los procesos de capacitación espermática y reacción acrosomal (Si, 1997;Si, 1999;Thurston et al., 2003;Bag et al., 2004), conllevando de esta forma a la adquisición de una movilidad espermática progresiva rápida y a un aumento de la capacidad de fertilización del espermatozoide. ...
... Dentro de los procesos de crioconservación, el mantenimiento de la movilidad y viabilidad espermática postdescongelación es esencial para alcanzar altas tasas de preñez; no obstante, la adquisición de una movilidad progresiva rápida que permita el desplazamiento y fertilización del oocito, es aún más importante (Bag et al., 2004). El semen es evaluado antes y después de pruebas de estrés térmico para estimar la capacidad del espermatozoide de sobrevivir en el tracto reproductivo de la hembra y mantener su capacidad fertilizante (Bag et al., 2004). ...
... Dentro de los procesos de crioconservación, el mantenimiento de la movilidad y viabilidad espermática postdescongelación es esencial para alcanzar altas tasas de preñez; no obstante, la adquisición de una movilidad progresiva rápida que permita el desplazamiento y fertilización del oocito, es aún más importante (Bag et al., 2004). El semen es evaluado antes y después de pruebas de estrés térmico para estimar la capacidad del espermatozoide de sobrevivir en el tracto reproductivo de la hembra y mantener su capacidad fertilizante (Bag et al., 2004). En el caso del semen crioconservado, estos ensayos son de vital importancia ya que permiten evaluar a través del tiempo su calidad en cuanto a la movilidad y los posibles cambios en la morfología individual espermática, permitiendo de esta forma realizar procesos de inseminación artificial con espermatozoides viables y de movilidad aceptable. ...
Titulo en ingles: Effect of post-thawing incubation on cryoconserved porcine semen qualityRESUMEN: La crioconservación seminal juega un papel importante en la introducción y preservación de características genéticas de tipo productivo y reproductivo las cuales son expresadas por un grupo particular de animales; sin embargo, en cerdos su eficiencia ha sido frecuentemente comprometida por la reducida capacidad fertilizante del espermatozoide después del proceso de crioconservación. Algunos autores han descrito el efecto positivo de la incubación seminal postdescongelación a diferentes temperaturas, permitiendo alcanzar porcentajes de movilidad mayores a los mostrados por muestras recién obtenidas, lo cual podría mejorar significativamente su capacidad de fertilización. Por lo anterior, el presente estudio evaluó la calidad del semen crioconservado de cerdo con base en la viabilidad y morfología espermática, así como en la movilidad y velocidad espermática individual después de su incubación postdescongelación. Para esto, pajillas de 0.25 mL de semen crioconservado de las razas Pietran y Landrace fueron utilizadas (n=13). Las pajillas fueron descongeladas en baño de agua a 55 ºC por 12 seg, posteriormente diluidas (1:40) con solución BTS (Beltsville Thawing Solution) y llevadas a incubación en baño de agua a 37 ºC por 4 h. La calidad del semen durante el periodo de incubación fue determinado cada hora con base en la movilidad y velocidad espermática individual utilizando un sistema de análisis espermático computarizado (CASA), así como en su morfología espermática. Durante el periodo de incubación, no se observaron cambios significativos en la morfología espermática; sin embargo, la viabilidad disminuyó significativamente (p<0.05) entre la hora 0 y 4 de incubación (70.6±1.9 y 57.8±3.2 %, respectivamente).La movilidad progresiva lineal rápida y la velocidad en línea recta fueron significativamente mayores (36.4±2.6 % y 26.8±2.0 µm.seg-1, respectivamente) después de una hora de incubación del semen postdescongelación cuando comparadas con la hora 0 (4.4±1.8 % y 5.3±1.70 µm.seg-1, respectivamente) y 4 de incubación (21.5±3.0 % y 14.7±2.2 µm.seg-1, respectivamente). La movilidad total mostró un comportamiento similar a las anteriores sin diferencias significativas entre la hora 1 y 2 de incubación (63.5±3.1 y 58.9±3 %, respectivamente). En conclusión, se observó que el semen crioconservado de cerdo presenta mejores características espermáticas, en cuanto a movilidad y velocidad después de 1 hora de incubación a 37 ºC, no obstante, este resultado necesita ser evaluado en términos de fertilidad para su recomendación.Palabras clave: calidad seminal, verraco, crioconservación, espermatozoide, incubación, movilidad espermáticaABSTRACT: Sperm cryopreservation plays an important role in introducing and preserving productive and reproductive genetic characteristics which are expressed by a particular group of animals; however, in pigs its efficiency has been frequently compromised by the semen’s reduced fertilizing ability following cryoconservation. Some au- thors have described the positive effect of post-thawing semen incubation at different temperatures, allowing achieve motility percentage higher than those showed by recently obtained samples, which could signifi- cantly improve their fertilization ability. The present study thus evaluated the boar cryopreserved sperm quality based on spermatic viability and morphology, as well as individual spermatic motility and velocity following its post-thawing incubation. 0.25 mL cryopreserved from Pietran and Landrace were used (n=13). Straws were thawed in a water bath at 55ºC for 12 sec, then diluted (1:40) with BTS (Beltsville Thawing Solution) and incubated in a water bath at 37ºC for 4 h. Computer Assisted Sperm Analysis (CASA) was used for determining semen quality every hour during the incubation period based on individual spermatic motility and velocity as well as spermatic morphology. No significant changes were observed in spermatic morphology during the incubation period; however, viability became significantly reduced (p<0.05) between incubation hour 0 and 4 (70.6±1.9 and 57.8±3.2 %, respectively). Rapid lineal progressive motility and straight-line velocity were significantly greater (36.4±2.6 % and 26.8±2.0 µm.seg-1, respectively) after one hour’s incubation of semen post-thawing when compared to incubation hour 0 (4.4±1.8 % and 5.3±1.70 µm.seg-1, respectively) and 4 (21.5±3.0 % and 14.7±2.2 µm.seg-1, respectively). Total motility presented a similar pattern to the foregoing, presenting no significant differences between incubation hours 1 and 2 (63.5±3.1 and 58.9±3 %, respectively). It was thus observed that pig’s cryoconserved semen presented better spermatic characteristics (i.e. motility and velocity) following 1 hour’s incubation at 37ºC; nevertheless, this result needs to be evaluated in terms of fertility for it to be fully recommended.Key words: seminal quality, boar, cryopreservtaion, sperm, incubation, spermatic motility
... The sub-lethal capacitation-like changes including cholesterol efflux, protein tyrosine phosphorylation and lack of energy are considered responsible for the shorter post-thaw sperm survival [36,39,46]. Conventionally, during ram semen cryopreservation semen straws are progressively cooled from 30 • C to 5 • C over a period of 2½ h by putting inside a cold cabinet maintained at 5 • C or by using a programmable cell freezer for more controlled cooling [2,4,6,21,24]. Following cooling, the semen straws are subjected to equilibration at 5 • C for 2-4 h. ...
... The maintenance of sperm functions during freezing-thawing and their post-thaw recovery depend on several interrelated factors such as cooling rate, equilibration time, freezing and thawing methods [3,5,6,21,27,38,39]. The equilibration period is considered one of the crucial steps in cryopreservation of semen since during this time sperm remain in contact with the cryoprotectants that penetrate in to cells and establish a balance between the intracellular and extracellular concentrations [38]. ...
... Cryodamage caused by ice crystals during freezing of sperm involves predominantly membrane structures. The freeze-thawing procedure is known to cause damage to acrosomal membrane of sperm [6,12,39,45]. Acrosome disintegration and partial removal of the outer acrosomal membrane with depletion of acrosomal content are common alterations attributed to physical freezing events [7]. These defects are probably caused by ice crystal formation during the freezing of extracellular fluids, which results in expansion of sub-acrosomal region [18]. ...
Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 10⁶ sperm mL⁻¹) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL⁻¹ catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at −25 °C min⁻¹ up to −125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.
... Cooling is the first temperature change known to alter the physical properties of cell membranes (Hammerstedt et al., 1990) and thus is one of the variables potentially affecting the success of artificial insemination with frozen-thawed semen. Freezing of ram spermatozoa in cell freezer has been commonly carried out from 5°C after precooling of straws up to 5°C in the cold chamber (Fiser and Fairfull, 1986, 1989; Fiser et al., 1986; Pontbriand et al., 1989; Soderquist et al., 1997; Byrne et al., 2000; Gil et al., 2000; Bag et al., 2004; Joshi et al., 2005). A protocol based on controlled-rate cooling and freezing of ram semen in straws has been reported to improve ram semen freezing technique but the post-thaw attributes of spermatozoa were evaluated by subjective assessment (Kumar et al., 2003). ...
... In this study, all the samples were diluted approximately to the recommended range of 25 x 10 6 sperm/ml with normal saline solution at 37°C and the settings of the analyzer were kept constant prior to CASA analysis for all the observations so as to ensure reliability and repeatability of the results. Moreover, the time lapse between sample dilution and the CASA analysis was kept short, thereby enabling the spermatozoa to survive until completion of analysis (Bag et al., 2004). The semen analyzer was set up as follows: Image type: Phase contrast; Frames at frame rate: 30 at 60/sec; Minimum contrast: 60; Low and high static size gates: 0.8 to 6.25; Low and high static intensity gates: 0.25 to 1.50; Low and high static elongation gates: 20 and 70; Default cell size: 5 pixels; Default cell intensity: 55; Magnification: 1.89 (Kumar et al., 2007). ...
... Prolonged incubation of sperm also causes deterioration and changes in the acrosome integrity of ram spermatozoa (Pontbriand et al., 1989). Similar changes in the acrosomal integrity of ram spermatozoa were observed in the present study after postthaw incubation, which was in agreement with our earlier findings (Bag et al., 2004; Joshi et al., 2005). The controlled-rate cooling protocol, besides providing complete automation in the cryopreservation process, might also protect spermatozoa against some adverse effect caused by minor fluctuation in temperature imposed by the transfer of cooled straws from cold cabinet to cell freezer as done in the uncontrolled cooling rate ram semen freezing protocol. ...
Exposure of frozen-thawed spermatozoa to a thermal resistance test reveals damages, which are not apparent immediately after thawing but are useful to assess the fertilizing ability of ram spermatozoa. Our earlier study has shown that cryopreservation of ram spermatozoa under controlled rate of cooling and freezing significantly improves the post-thaw motility and acrosomal integrity, compared to uncontrolled rate of cooling prior to controlled rate of freezing. The purpose of this study was to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa cryopreserved under controlled (Group 1) and uncontrolled rate of cooling (Group 2) followed by programmable freezing. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 x 10 9 spermatozoa per ml with Egg yolk-Test-glycerol extender and packaged in 0.25 ml straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25 to 5°C at the rate of 0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5 to –125°C at the rate of 25°C per minute. Thawing of straws was done at 50°C for 10 seconds and thawed spermatozoa were subjected to a thermal resistance test at 37°C for 4 h. Samples were assessed immediately after thawing and at hourly interval for sperm motion characteristics by computer-aided semen analysis technique. Post-thaw incubated spermatozoa were also evaluated at 0, 1, 2, 3, and 4 h for acrosomal integrity after staining the dried semen smears with Giemsa stain. The % motility, % rapid moving spermatozoa, % linearity and % sperm with normal acrosome were significantly (P < 0.05) higher in Group 1 compared to Group 2. The effect of incubation time was also significant (P < 0.05) on % motility, fraction of rapid motile spermatozoa, % linearity, curvilinear velocity, average path velocity, straight line velocity, area of sperm head, lateral head displacement and % spermatozoa with normal acrosome. The % motility, % rapid motile spermatozoa, sperm velocity, lateral head displacement and % spermatozoa with normal acrosome progressively declined during 4 h of incubation but the decline in all the traits was less in Group 1 compared to Group 2. The results showed that controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa for post-thaw thermoresistance test compared to uncontrolled rate of cooling prior to programmable freezing.
... There was no significant difference observed in the per cent of sperm cells with non-progressive motility during the incubation period. In accordance with the present study, Bag et al. (2004) reported a significant reduction in motile sperm cells during post thaw incubation of ram spermatozoa. The decrease in motility during incubation might be due to a gradual decline in the ability of spermatozoa to generate adenocine triphosphate (ATP) through mitochondrial respiration as a consequence of mitochondrial ageing (Viswanath et al. 1997) or the toxic effect of membrane-bound aromatic amino acid oxidase enzyme released from dead spermatozoa during long storage in ambient temperature (Shannon & Curson 1982). ...
... Linearity of the sperm cell motility varied between 40% and 35%, the straightness of the movement varied between 70% and 65% with the per cent of wobble around 60. Amplitude of lateral head displacement of sperm cells was around 3 µm and the beat cross frequency was 9 Hz, during the incubation period. Though Krzyzosiak et al. (2000) and Bag et al. (2004) reported that velocity parameters did not change during incubation, the reduction in the velocity parameters observed in the present study was well in accordance with the observations of Gil et al. (2000). Variations in the reports on sperm velocity parameters might be due to differences in the settings used in the CASA such as frame rate and frames per field, chamber and time of analysis, sample preparations including thawing temperature, sperm sample concentration and media used for dilution (Contri et al. 2010). ...
Proteins present in the seminal plasma and sperm influence sperm function and fertilization. The present study was carried out to screen breeding bull semen samples for the presence of fertility-associated 28–30 kDa heparin-binding protein (HPB) and its effect on in vitro sperm characters and fertility. Semen samples were collected from 22 breeding bulls and the sperm proteins were extracted by Triton X detergent extraction method. HBPs were eluted, and the molecular weight of the proteins was assessed by discontinuous sodium dodecyl sulphate polyacrylamide gel elecrophoresis. Based on the presence/absence 28 kDa HBPs, bulls were categorized into group I and group II. Frozen semen samples were evaluated for in vitro sperm characters at immediate post thaw, 60, 120 and 180 min post-thaw incubation. To assess the field fertility of the bulls, 50 frozen semen straws/bull were used for insemination. Results indicated that only 50% of the bulls screened had 28–30 kDa HBPs in their sperm. Bulls positive for fertility-associated protein had better in vitro sperm characters, better protection against oxidative stress, readily underwent capacitation induction by heparin and had 13% higher conception than the bulls lacking the protein. So, it can be concluded that the bulls positive for of 28–30 kDa HBPs in sperm had higher chance of fertility and screening for its presence can be included in the regular breeding soundness examination for selection of bulls.
... Sperm motility is important for transport from the site of ejaculation or insemination to the site of fertilisation (Suarez and Pacey 2006). Sperm motility in fresh, diluted and frozen-thawed semen declines during incubation at body temperature (Bedford et al. 1999;Schuffner et al. 2002;Bag et al. 2004;Batista et al. 2011). Reduction in sperm motility is mainly due to mitochondrial impairment (Bag et al. 2004) and generation of reactive oxygen species (ROS; Guthrie and Welch 2006). ...
... Sperm motility in fresh, diluted and frozen-thawed semen declines during incubation at body temperature (Bedford et al. 1999;Schuffner et al. 2002;Bag et al. 2004;Batista et al. 2011). Reduction in sperm motility is mainly due to mitochondrial impairment (Bag et al. 2004) and generation of reactive oxygen species (ROS; Guthrie and Welch 2006). According to the Society of Theriogenology, bull semen should possess .30% of spermatozoa with progressive motility to achieve acceptable fertility (Barth 2007). ...
The extent of changes in sperm structure and function affect the success of fertilization ultimately during the pathway to ovum in the female reproductive tract. The success of AI with frozen-thawed semen varies in dairy and beef breeds of bovine because of differed alterations in sperm during transport in female tract after insemination. To our knowledge, no report is available comparing the changes in dairy and beef sperm leading to death in female tract. Therefore, this study was aimed to investigate the changes in motility, generation of reactive oxygen species (superoxide and hydrogen peroxide), and their relation to sperm death [asymmetry (apoptosis) and rupture of plasma membrane] of dairy and beef frozen-thawed semen during incubation at 37°C for 24h. This incubation was aimed to mimic the environment of female reproductive tract. Frozen dairy semen (n=4 bulls) was procured from a Canadian breeding station, whereas beef semen was collected from breeding beef bulls (n=3; 5 replicates), diluted with Tris-based extender (composition was same as used in dairy semen), cooled to +4°C over 90min, and cryopreserved by programmable freezer using standard rate as used in dairy semen. Two straws per replicate were thawed at 37°C from both types of semen, pooled separately, and incubated at 37°C for 24h in capped tubes. Each pooled semen sample was evaluated for motility with CASA, superoxide (O2(-), and hydrogen peroxide (H2O2) radical using HE/YoPRO and H2DCFDA/PI assay, respectively, and asymmetry of plasma membrane using YoPRO/PI assay through flow cytometric analysis at 0, 2, 4, 6, 12, and 24h of incubation. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) was used to analyse the data as 2×6 factorial model for 2 types of semen (dairy and beef) and 6 time points using time as repeated measure. A threshold limit of 30% was considered for motility and live sperm to get optimum fertility. Sperm motility remained higher (P<0.05) than threshold limit till 6h in dairy (50.95±2.62%) and 2h in beef semen (30.28±6.95%). Dairy semen possessed more (P<0.05) nonapoptotic sperm without O2(-) (HE-/YoPRO-) till 6h of incubation than beef semen. The increase in apoptotic sperm containing superoxide radical (HE+/YoPRO+) over time was more (P<0.05) in beef semen till 6h of incubation. The rise in dead sperm containing H2O2 (H2DCFDA+/PI+) was recorded more in beef than in dairy semen until 6h of incubation. Live sperm without apoptosis (YoPRO-/PI-) were higher until 24h in dairy (49.36±4.56%) compared with beef semen (24.89±3.85%), whereas viable sperm with apoptosis (YoPRO+/PI-) were found similar in both types of semen over time. In conclusion, dairy frozen-thawed semen possessed more live sperm without reactive oxygen species (superoxide and hydrogen peroxide) until 6h of incubation than did beef semen. The decrease in superoxide radical was more in dairy than in beef semen. Dead and apoptotic sperm increased more in beef frozen-thawed semen over time during incubation. This inference suggests performing the insemination late near ovulation with beef frozen-thawed semen because of less viable life than dairy semen.
... Sperm motility is important for transport from the site of ejaculation or insemination to the site of fertilisation (Suarez and Pacey 2006). Sperm motility in fresh, diluted and frozen-thawed semen declines during incubation at body temperature (Bedford et al. 1999;Schuffner et al. 2002;Bag et al. 2004;Batista et al. 2011). Reduction in sperm motility is mainly due to mitochondrial impairment (Bag et al. 2004) and generation of reactive oxygen species (ROS; Guthrie and Welch 2006). ...
... Sperm motility in fresh, diluted and frozen-thawed semen declines during incubation at body temperature (Bedford et al. 1999;Schuffner et al. 2002;Bag et al. 2004;Batista et al. 2011). Reduction in sperm motility is mainly due to mitochondrial impairment (Bag et al. 2004) and generation of reactive oxygen species (ROS; Guthrie and Welch 2006). According to the Society of Theriogenology, bull semen should possess .30% of spermatozoa with progressive motility to achieve acceptable fertility (Barth 2007). ...
This study was designed to compare the kinetics of sperm survival in different types of bull semen. Fresh ejaculates from four bulls were pooled, diluted in Tris-citric acid-egg yolk-glycerol extender, cooled to 4°C, frozen in LN2 and thawed at 37°C. Fresh, diluted, cooled and frozen-thawed semen were incubated at 37°C, and evaluated at 0, 2, 4, 6, 12 and 24h after the beginning of incubation. In Experiment 1, progressive sperm motility, normal acrosomes and plasma membrane integrity and asymmetry were determined. In Experiment 2, generation of superoxide anion (O2•) along with plasma membrane permeability and generation of hydrogen peroxide (H2O2) along with plasma membrane integrity were assessed. In Experiment 1, frozen-thawed semen had shorter survival times for progressive sperm motility, and spermatozoa with intact plasma membranes and acrosomes (IPM-IACR) as compared with other types of semen (P<0.05). Fresh spermatozoa underwent a necrotic pathway, diluted and cooled spermatozoa underwent an apoptosis-like pathway and frozen-thawed spermatozoa underwent both necrotic and apoptosis-like pathways. In Experiment 2, spermatozoa in all four types of semen exhibited O2•-generation and increased plasma membrane permeability, and became necrotic without H2O2 generation during incubation (P<0.05). In conclusion, frozen-thawed semen had shorter sperm longevity, which has important implications relating to the timing of artificial insemination. Different types of semen followed different death pathways. During incubation, spermatozoa in all types of semen generated O2•-, which increased the permeability and compromised the integrity of the plasma membrane.
... Sperm motility is important for transport from the site of ejaculation or insemination to the site of fertilisation (Suarez and Pacey 2006). Sperm motility in fresh, diluted and frozen-thawed semen declines during incubation at body temperature (Bedford et al. 1999;Schuffner et al. 2002;Bag et al. 2004;Batista et al. 2011). Reduction in sperm motility is mainly due to mitochondrial impairment (Bag et al. 2004) and generation of reactive oxygen species (ROS; Guthrie and Welch 2006). ...
... Sperm motility in fresh, diluted and frozen-thawed semen declines during incubation at body temperature (Bedford et al. 1999;Schuffner et al. 2002;Bag et al. 2004;Batista et al. 2011). Reduction in sperm motility is mainly due to mitochondrial impairment (Bag et al. 2004) and generation of reactive oxygen species (ROS; Guthrie and Welch 2006). According to the Society of Theriogenology, bull semen should possess .30% of spermatozoa with progressive motility to achieve acceptable fertility (Barth 2007). ...
Extent and timing of alterations in structures and functions of sperm after its placement in the female reproductive tract are important for successful fertilization. To our knowledge, the few reports are available on the kinetics of alterations in bovine sperm structures and functions during pathway to their death. Therefore, the present study was conducted to determine the changes in motility, acrosome and plasma membrane asymmetry in fresh and frozen-thawed semen during incubation at 37°C over the period of 24h. Semen was collected from 3 breeding beef bulls, pooled, and considered as one replicate (total replicates=5). Each pooled semen sample was diluted in Tris-citric acid egg yolk glycerol extender (pH 6.8), cooled to +4°C over 90min, and then cryopreserved by a programmable cell freezer. Fresh (pooled semen) and frozen-thawed semen were incubated at 37°C for 24h. Each semen sample was evaluated for sperm motility with computer-assisted semen analysis and acrosomal integrity and plasma membrane asymmetry using fluorescein isothiocyanate-peanut agglutinin/propidium iodide and Annexin V/propidium iodide assays, respectively, at 0, 2, 4, 6, 12, and 24h of incubation at 37°C, with a flow cytometer. Statistical analysis was conducted using PROC MIXED model in statistical analysis system as 2 (semen types)×6 (times) factorial model, using time as repeated measure. Progressive motility was higher (P<0.05) in fresh than in frozen-thawed semen until 6h. Progressive motility declined (P<0.05) below the threshold level (i.e. 30%) much later (12h) in fresh as compared with frozen-thawed semen (2h). However, acrosomal integrity and plasma membrane asymmetry deteriorated (P<0.05) below threshold at the same time interval (2h) in both fresh and frozen-thawed semen. Viable sperm (AN-/PI-) remained higher (P<0.05) during the first 6h in fresh than in frozen-thawed semen and declined (P<0.05) below the threshold at 12h in fresh and at 6h in frozen-thawed semen. In fresh semen, the necrotic sperm (AN-/PI+) population increased (P<0.05) over time and reached maximum (97%) at 24h. In frozen-thawed semen, a mixed population of late apoptotic (53%) and necrotic (34%) sperm was found at 24h. In conclusion, the alterations in sperm motility, acrosomes, plasma membrane integrity, and asymmetry were slower in fresh than in frozen-thawed semen. Fresh sperm followed necrosis and frozen-thawed sperm underwent necrosis and apoptosis-like pathways, respectively.
... É sabido que dentro de um sistema aeróbico ou parcialmente aeróbico, a produção de espécies reativas de oxigênios (ROS) é inevitável. Os radicais livres ânion superóxido (O 2 -), peróxido (H 2 O 2 ) e hidroxil (OH) são os mais nocivos e as reações que geram estes radicais parecem mais ativas em temperaturas mais altas (Bag et al., 2004), sendo a temperatura utilizada no teste de exaustão bastante favorável. No presente estudo, a incubação do sêmen a 37ºC foi realizada após o descongelamento de uma palheta de 0,25ml em um tubo de vidro de 5ml, e possivelmente o espaço entre a tampa do tubo e a superfície do sêmen proporcionou condições para o estabelecimento de um sistema aeróbico, esse fato pode ter contribuído para baixos resultados no final da incubação. ...
... Quanto às percentagens de espermatozóides vivos com acrossomo reagido e mortos reagidos pós-descongelação não foram observadas diferenças significativas entre os tratamentos (Tabela 2). Estes resultados estão de acordo com os estudosde Pérez et al (1996) eGillan et al. (1997), os quais sugerem que as etapas de congelação e descongelação aumentam o número de espermatozóides capacitados, mas possuem menor efeito sobre o número de acrossomos reagidos.O grau de dano espermático que ocorre nos espermatozóides durante o processo de criopreservação torna-se aparente durante a sua incubação; quanto maior esse dano, menor é a longevidade dessa célula no trato genital da fêmea(Bag et al., 2004).Dos resultados do teste de exaustão não foram observadas diferenças significativas na percentagem da motilidade e no vigor entre os quatro tratamentos ao final das 4 horas de incubação (Tabela 3). No entanto, notaram-se valores muito baixos para a motilidade espermática dos tratamentos, isto já era esperado, devido ao fato das membranas espermáticas estarem muito lesadas no momento da descongelação. ...
Different types of cooling systems of ram semen were evaluated by the measurement and comparison of cooling rates and their effects on the post-thawing quality. Motility, vigor, morphology damages, viability and acrosomal status were tested at the end of 90 minutes of cooling and post-thawing. Regarding post-thawed semen, the same parameters were used, in addition to the assessment of the integrity of the plasmatic membrane, and the resistance test by the incubation of semen at +37ºC for 4 hours. Semen refrigeration was carried out in a domestic refrigerator and in a horizontal refrigerator. To control the temperature drop of semen in both equipments, the slats were disposed between plastic bags containing water at +32ºC, constituting four combinationsof cooling procedures: RS (refrigerator without bag), RC (refrigerator with bag), BS (horizontal refrigerator without bag) and BC (horizontal refrigerator with bag), resulting in four cooling rates: -1.4ºC/min, -0.4ºC/min, -2.9°C/min and -0.45ºC/min, for RS, RC, BS and BC, respectively. At the end of the cooling period, BS treatment showed the lowest percentage of sperm motility (P<0.05), the lowest mean of live spermatozoa with intact acrosome, and the highest mean of dead spermatozoa with intact acrosome (P<0.05). As for morphological defects, BS treatment had the highest mean whereas the systems RC and BC resulted in the lowest means. There was no significant difference among treatments in relation to frozen-thawed semen at the end of the resistant test. It was concluded that the different cooling rates affected ram semen at the end of cooling stage but not at post-thawing.
... Estimation of capacitation and survivability of the sperm cells in the female genital tract directly affects fertilization and, therefore, the success rates of pregnancy. 17 Ozone is an inorganic colorless gas that is composed of three oxygen atoms with a characteristic odor at room temperature. 18 Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. ...
Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.
... Semen manipulation, during incubation, refrigeration, and cryopreservation processes produces deterioration and changes in the acrosome and membrane of spermatozoa [65]. These changes, therefore, could produce changes in sperm motility and sperm survivability [66]. Thus, acrosome integrity during freezing can be highly correlated with most sperm-quality-related variables. ...
Simple Summary
For the conservation of genetic resources in avian species, semen freezing is very helpful. The disadvantage of this process is that spermatozoa suffer different types of damage. Exogenous antioxidants can be added to the cryopreservation extender to mitigate this damage. This study aimed to test whether the addition of different hydroxytyrosol (HT; an antioxidant derived from olive oil) concentrations produces beneficial effects in the sperm of a local avian breed (Utrerana roosters). For this purpose, different semen quality parameters, which fell under the following macro-areas were evaluated in both fresh and thawed semen: motility, morphology, membrane functionality, and flow-cytometry-related traits. At the statistical level, a descriptive analysis and a canonical discriminant analysis were performed, which allowed us to extract valuable information about the different studied variables. Lastly, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was carried out and the reactive oxygen species (ROS) variable was found to have the highest power to discriminate between the different treatments according to the HT concentration. Low or no HT concentrations resulted in higher ROS values, and therefore, possible mechanical damage unrelated to plasma membrane peroxidation can be produced in the frozen–thawed rooster spermatozoa.
Abstract
Sperm cryopreservation is effective in safeguarding genetic biodiversity in avian species. However, during this process, spermatozoa are very susceptible to plasma membrane peroxidation in the presence of high concentrations of reactive oxygen species (ROS). To mitigate this effect, the addition of exogenous antioxidants, such as hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), an antioxidant derived from olive oil, to the cryopreservation sperm diluent, could be useful. To verify this, a cryopreservation diluent was supplemented with different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL, and 150 μg/mL) of HT. For this, semen was collected in 10 replicates from 16 roosters of the Utrerana avian breed, and a pool was prepared with the optimum quality ejaculates in each replicate. After cryopreservation, spermatozoa were thawed and different in vitro semen quality parameters were evaluated. A discriminant canonical analysis (DCA) was carried out and revealed that total motility (TM; Lambda = 0.301, F = 26,173), hypo-osmotic swelling test (HOST; Lambda = 0.338, F = 22,065), and amplitude of lateral head displacement (ALH, Lambda = 0.442; F = 14,180) were the variables with the highest discriminant power. Finally, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was performed excluding fresh semen samples and ROS was found to be the most valuable variable to discriminate between the different established freezing groups. Samples in the absence of HT or with low concentrations of this antioxidant showed less desirable ROS values in cryopreserved rooster semen. The present study could lead to the improvement of cryopreservation techniques for the genetic material of local poultry breeds and optimize the conservation programs of endangered native avian breeds.
... Furthermore, unlike what was observed for the G5L8 extender, no significant reduction in total motility and VCL were observed after 3 h of incubation when HB was added. The better stability observed during incubation after adding HB is an important in vitro parameter of sperm quality since longer motility maintenance after thawing is related to a higher pregnancy rate after AI (Bag et al., 2004). ...
How to cite: Snoeck PPN, Câmara DR, Moura LCO, Silva MC, Machado-Neves M, Teixeira-Neto MR, Henry M. Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants. Anim Reprod. 2023;20(1):e20220068. https://doi. Abstract The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.
... Effect of incubation period at 37 o C for third hours on the sperm characteristics increased during the first hour while, with continuing incubation time up to 3 hours decreased (P<0.05). The apparent decline in incubated post-thawing sperm characteristics may be due to released lactic dehydrogenase (LDH) may reflect the breakdown cell membrane (Maxwell, 1978), production of free radical (Fridovich, 1981), increasing in lactic acid exerts from respiration motile spermatozoa ( Zeidan, 1995) and released aromatic amino acid oxidase (AAAO) as toxic enzyme from dead spermatozoa (Marti et al., 2003 andBag et al., 2004). ...
... The efficient process of semen cryopreservation results in longterm preservation of semen and later for its desired use [2,3]. Frozen-thawed sperm possessing intact motility apparatus, active mitochondria, integral plasma and acrosomal membranes [4], and unfragmented DNA [5] ensure optimum fertility through AI [6]. ...
... However, they did not detect a significant change in the rate of linearity, curvilinear velocity, mean passage, lateral displacement of the head, and incidence of tail sperm strokes during incubation. Different dilution media ( Bag et al., 2004;Gil et al., 2003;Gholami et al., 2012;Singh et al., 2013;Soltanpour and Moghaddam, 2013;Mostafapor and Ardebi- li, 2014;Jiménez-Rabadán et al., 2015) also affect the characteristics of the sperm. ...
The aim of the study was to determine the sustainability of the ejaculates of rams from Lacaune breed by examining the effect of low temperatures on sperm parameters before and after cryopreservation.The experiment was carried out with seven 2-year-old rams from Lacaune breed, placed under the same conditions of growing, feeding and breeding use. From each ram we received 2 consecutive ejaculates. The received ejaculates were examined by computer assisted sperm analyzer (CASA) in terms of changes in kinetic parameters such as VCL, VSL and VAP. After the sperm assessment, the ejaculates were frozen by Cassu’s pasay technology, and after thawing the samples were analyzed for the same parameters by CASA. After freezing and subsequent thawing, the amount of sperm with progressive movement is greatly reduced (from 323.36 to 35.5 mill/ml) on the contrary of the amount of non-progressive moving (from 2970.86 to 1443.57 mill/ml) and especially of the static sperm (from 6.71 to 1796.92 mill/ml). These processes also lead to a decrease in VCL (from 102.44 to 21.57 μm / s), VSL (from 27.83 to 10.48 μm / s), VAP (53.99 to 14.36 μm /s). Lacaune rams are not susceptible to long term freezing due to the damaging effect of low temperatures on their sperm cells.
... Continuous linear velocity, VAP, rectilinear velocity, and linearity are mutually related to migratory efficacy in the sheep's cervix. Seed fluid incubation may also have an effect on its kinematics and acrosomatic integrity in ram semen [ 4 ]. There is a significant effect of incubation on the mobility characteristics and the number of sperm with normal acrosome. ...
The spermatozoa of agricultural breeds of ram are susceptible to the action of low freezing temperatures. This process leads to changes in main sperm parameters such as motility and speed. Various factors such as the type of storage media, the addition of some biological substances in the diet and the freezing regimes are influenced by the cryopreservation of the sperm. The aim of our study is to investigate the effect of cryopreservation on the speed parameters of sperm from breeds of Lacaune and Ile de France sheep. The experiment was carried out with 10 sexually mature, pure bred rams during their breeding campaign. The animals were divided in two groups according to the breed – 5 rams of each breed (Ile de France and Lacaune). The assessment of sperm motility, concentration and various kinematic parameters of motile spermatozoa were carried out by Sperm Class Analyzer (SCA, Microptic, Spain). Cryopreservation of semen straws was done by the method of Cassou [ ¹³ ]. The results show that in the Lacaune breed, the amount of static spermatozoa after thawing increases significantly while the Ile de France breeds even slightly decreases. With respect to progressive and non-progressive mobile sperm, both breeds showed a decrease after thawing, but in the case of Ile de France it was much less. All speed indicators (VCL, VSL and VAP) and parameters – percentage of linearity (LIN, %), percentage of straightness (STR, %) and percentage of fluctuations (WOB, %) for Ile de France breed increase, albeit a little after thawing, while the other breed – Lacaune, significantly decreases. In conclusion in the Lacaune breed after cryopreservation there is a significant decrease in both the progressive spermatozoa and the speed parameters, as opposed to the ejaculates of the Ile de France breed.
... The more reduction in motility during incubation at 4°C in rapidly cooled group of present study has been augmented by initial decline from extension to end of cooling. This decrease could be due to mitochondrial aging [2], loss of mitochondrial membrane potential, decreased production of ATP and depletion of cAMP due to cooling and freezing [4,16]. Reduction in motility at lowered temperatures might be due to disorganization of axoneme which occurs due to depolymerisation of tubulin [11] and damage to mitochondrial sheath and matrix [16]. ...
Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8h) on survival before freezing at 4°C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates=6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (-2.2(o)C/min) or MC (-0.3(o)C/min) from 37(o)C to 4(o)C in separate aliquots and further equilibrated at 4(o)C for 8h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4(o)C, of motility and PMI was observed 5 and 6h respectively in RC group while >8h in MC group. Rate of decline (slope) in motility and viability was higher (P<0.05) in RC overtime during equilibration at 4(o)C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P<0.05) in MC when equilibrated for 2-8 h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2-8 h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.
Copyright © 2015. Published by Elsevier Inc.
... ), peróxido (H 2 O 2 ) e hidroxil (OH) são os mais nocivos e as reações que geram estes radicais parecem mais ativas em temperaturas mais altas (Bag et al., 2004), sendo a temperatura utilizada no teste de exaustão bastante favorável. No presente estudo, a incubação do sêmen a 37ºC foi realizada após o descongelamento de uma palheta de 0,25ml em um tubo de vidro de 5ml, e possivelmente o espaço entre a tampa do tubo e a superfície do sêmen proporcionou condições para o estabelecimento de um sistema aeróbico, esse fato pode ter contribuído para baixos resultados no final da incubação. ...
This study aimed to evaluate the interference of daily gynecological exam on reproductive and productive parameters in Murrah buffaloes. Twenty-four buffaloes, which have calved in the autumn season, were milked once a day and kept on pasture, were divided into two groups: Research (RG; n=13) and Control (CG; n=11). The animals in RG were daily taken to the corral and submitted to gynecological examination and blood collection immediately after milking, from the 7th day postpartum until the first estrus and breeding day. Animals in CG were released directly to pasture after milking without any manipulation. The RG had smaller calving-first estrus interval (40.4±9.0 days) than CG (59.2±24.4 days; P0.05), as well as in milk production. The number of mating per conception was higher in RG (2.1±0.9) than in CG (1.5±0.5; P
... Moreover, frozen-thawed motility decreases significantly during incubation because of the gradual decline in ability of spermatozoa to generate ATP through mitochondrial respiration. This mitochondrial impairment might be due to mitochondrial aging and the toxic effect of membrane bound aromatic amino acid oxidase enzyme resulted from dead spermatozoa ( Bag et al., 2004). On the other hand, the decreasing motility during incubation may be related to change in spermatozoa morphology, the spermatozoa has usually containing mitochondria in mid-piece and tail after freezing and thawing. ...
... Thus, results were grouped for two hESC lines for each time point and plotted as a mean of the percentages of HNupositive ([HNu [+]) human cells carrying Tuj1 or recoverin in grafts, with corresponding standard error of the mean (SEM). Comparison of the statistical significance between expression of Tuj1 and recoverin in the subretinal space versus the epiretinal (vitreous) space was calculated with an unpaired Student t test (with p<0.05 considered statistically significant) after converting the percentage values to arc sin values [52]. ...
Purpose: To examine the potential of NIH-maintained human embryonic stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-1/IGF-1/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression. Methods: hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs). Wnt (Integration 1 and wingless) blocking morphogens Dickkopf-1 (Dkk-1) and Insulin-like growth factor 1 (IGF-1) were used to direct NPs to a rostral neural fate, and fibroblast growth factor 9 (FGF9)/fibroblast growth factor-basic (bFGF) were added to bias the differentiation of developing anterior neuroectoderm cells to neural retina (NR) rather than retinal pigment epithelium (RPE). Cells were dissociated and grafted into the subretinal and epiretinal space of young adult (4–6-week-old) mice (C57BL/6J x129/Sv mixed background). Remaining cells were replated for (i) immunocytochemical analysis and (ii) used for quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis. Mice were sacrificed 3 weeks or 3 months after grafting, and the grafts were examined by histology and immunohistochemistry for survival of hESC-RPCs, presence of mature neuronal and retinal markers, and the dynamics of in vivo maturation and integration into the host retina. Results: At the time of grafting, hESC-RPCs exhibited immature neural/neuronal immunophenotypes represented by nestin and neuronal class III β-tubulin, with about half of the cells positive for cell proliferation marker Kiel University -raised antibody number 67 (Ki67), and no recoverin-positive (recoverin [+]) cells. The grafted cells expressed eye field markers paired box 6 (PAX6), retina and anterior neural fold homeobox (RAX), sine oculis homeobox homolog 6 (SIX6), LIM homeobox 2 (LHX2), early NR markers (Ceh-10 homeodomain containing homolog [CHX10], achaete-scute complex homolog 1 [MASH1], mouse atonal homolog 5 [MATH5], neurogenic differentiation 1 [NEUROD1]), and some retinal cell fate markers (brain-specific homeobox/POU domain transcription factor 3B [BRN3B], prospero homeobox 1 [PROX1], and recoverin). The cells in the subretinal grafts matured to predominantly recoverin [+] phenotype by 3 months and survived in a xenogenic environment without immunosuppression as long as the blood–retinal barrier was not breached by the transplantation procedure. The epiretinal grafts survived but did not express markers of mature retinal cells. Retinal integration into the retinal ganglion cell (RGC) layer and the inner nuclear layer (INL) was efficient from the epiretinal but not subretinal grafts. The subretinal grafts showed limited ability to structurally integrate into the host retina and only in cases when NR was damaged during grafting. Only limited synaptogenesis and no tumorigenicity was observed in grafts. Conclusions: Our studies show that (i) immunosuppression is not mandatory to xenogenic graft survival in the retina, (ii) the subretinal but not the epiretinal niche can promote maturation of hESC-RPCs to photoreceptors, and (iii) the hESC-RPCs from epiretinal but not subretinal grafts can efficiently integrate into the RGC layer and INL. The latter could be of value for long-lasting neuroprotection of retina in some degenerative conditions and glaucoma. Overall, our results provide new insights into the technical aspects associated with cell-based therapy in the retina. Photoreceptor death in retinal and macular degenerative diseases is a leading cause of inherited vision loss in developed countries. Novel therapeutic strategies have recently emerged, from mechanical to cell based, to repair Correspondence to: Igor O. neural circuits affected by photoreceptor (PR) cell loss [1]. Trophic factor delivery to extend the life of dying PRs has been pursued in experimental animals [2-5] and in some instances in the clinic [6]. Gene therapy approaches have been applied successfully in one type of Leber congenital amaurosis and remain viable when etiology of disease is understood and the size of a gene is not prohibitive for packaging capacity of the viral vector. Retinal implants [7,8] utilize a high-tech mechanical device placed on the retina to
... Freezing of ram spermatozoa in cell freezer has been commonly carried out from 5°C after precooling of straws up to 5°C in the cold chamber Pontbriand et al., 1989;Soderquist et al., 1997;Byrne et al., 2000;Gil et al., 2000;Bag et al., 2004). ...
Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The
aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolledrate)
on pre-freezing and post-thaw sperm motility parameters. Ejaculates were collected using the
artificial vagina from four Chal rams and diluted with a Tris-based extender and packed in 0.25 ml
straws. Then, the sample was processed according to the two methods; method 1: straws were cooled
from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and
equilibrated at 5°C for 80 min and then were frozen at the rate of -0.3°C/min from 5 to -10 and -25°C from
-10 to -150°C and plunged into liquid nitrogen for storage; method 2: straws were transferred to a
refrigerator and maintained at 5°C for 3 h, then, the straws were frozen in liquid nitrogen vapor 4 cm
above the liquid nitrogen, for 15 min and plunged into liquid nitrogen. A computer-assisted sperm
motility analysis was used to analyze sperm motion characteristics. The controlled rate of freezing
(method 1) significantly improved the pre-freezing and post-thaw total and progressive motility
compared with the uncontrolled rate (method 2). In specific kinetic parameters, method 1 gave
significantly higher value for straight linear velocity (VSL) and curvilinear velocity (VCL) in comparison
with method 2. There were no significant differences between the two methods for average path
velocity (VAP) and linearity (LIN). In conclusion, the controlled rate of cooling conferred better
cryopreserving ability to ram spermatozoa compared with the uncontrolled rate of cooling prior to
programmable freezing.
... Thus, results were grouped for two hESC lines for each time point and plotted as a mean of the percentages of HNu – positive ([HNu [+]) human cells carrying Tuj1 or recoverin in grafts, with corresponding standard error of the mean (SEM). Comparison of the statistical significance between expression of Tuj1 and recoverin in the subretinal space versus the epiretinal (vitreous) space was calculated with an unpaired Student t test (with p<0.05 considered statistically significant) after converting the percentage values to arc sin values [52]. ...
To examine the potential of NIH-maintained human embryonic stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-1/IGF-1/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression.
hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs). Wnt (Integration 1 and wingless) blocking morphogens Dickkopf-1 (Dkk-1) and Insulin-like growth factor 1 (IGF-1) were used to direct NPs to a rostral neural fate, and fibroblast growth factor 9 (FGF9)/fibroblast growth factor-basic (bFGF) were added to bias the differentiation of developing anterior neuroectoderm cells to neural retina (NR) rather than retinal pigment epithelium (RPE). Cells were dissociated and grafted into the subretinal and epiretinal space of young adult (4-6-week-old) mice (C57BL/6J x129/Sv mixed background). Remaining cells were replated for (i) immunocytochemical analysis and (ii) used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Mice were sacrificed 3 weeks or 3 months after grafting, and the grafts were examined by histology and immunohistochemistry for survival of hESC-RPCs, presence of mature neuronal and retinal markers, and the dynamics of in vivo maturation and integration into the host retina.
At the time of grafting, hESC-RPCs exhibited immature neural/neuronal immunophenotypes represented by nestin and neuronal class III β-tubulin, with about half of the cells positive for cell proliferation marker Kiel University -raised antibody number 67 (Ki67), and no recoverin-positive (recoverin [+]) cells. The grafted cells expressed eye field markers paired box 6 (PAX6), retina and anterior neural fold homeobox (RAX), sine oculis homeobox homolog 6 (SIX6), LIM homeobox 2 (LHX2), early NR markers (Ceh-10 homeodomain containing homolog [CHX10], achaete-scute complex homolog 1 [MASH1], mouse atonal homolog 5 [MATH5], neurogenic differentiation 1 [NEUROD1]), and some retinal cell fate markers (brain-specific homeobox/POU domain transcription factor 3B [BRN3B], prospero homeobox 1 [PROX1], and recoverin). The cells in the subretinal grafts matured to predominantly recoverin [+] phenotype by 3 months and survived in a xenogenic environment without immunosuppression as long as the blood-retinal barrier was not breached by the transplantation procedure. The epiretinal grafts survived but did not express markers of mature retinal cells. Retinal integration into the retinal ganglion cell (RGC) layer and the inner nuclear layer (INL) was efficient from the epiretinal but not subretinal grafts. The subretinal grafts showed limited ability to structurally integrate into the host retina and only in cases when NR was damaged during grafting. Only limited synaptogenesis and no tumorigenicity was observed in grafts.
Our studies show that (i) immunosuppression is not mandatory to xenogenic graft survival in the retina, (ii) the subretinal but not the epiretinal niche can promote maturation of hESC-RPCs to photoreceptors, and (iii) the hESC-RPCs from epiretinal but not subretinal grafts can efficiently integrate into the RGC layer and INL. The latter could be of value for long-lasting neuroprotection of retina in some degenerative conditions and glaucoma. Overall, our results provide new insights into the technical aspects associated with cell-based therapy in the retina.
... The cooling rate determines whether the cells remain in equilibrium with their extracellular environment or become progressively super cooled with the increasing possibility of intracellular ice formation [10] . Freezing of ram spermatozoa in cell freezer has been commonly carried out from 5°C after precooling of straws up to 5°C in the cold chamber11121314151617. Computer-controlled cooling machine was built in the laboratory to achieve rapid cooling of samples in straws under controlled conditions. ...
Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters.
Ejaculates were collected using the artificial vagina from four Chal rams and three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen for 15 min and plunged into liquid nitrogen. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics.
Controlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing.
... The percentage of cells with iAC and H⌬⌿m throughout refrigeration and freezing remained similar to the values found for fresh/diluted semen, which corroborated a previous report [38] regarding iAC in ovine sperm. However, latent damage may have occurred; in that regard, perhaps the degree of injury during the freezing/thawing process is manifested during the post-thaw incubation, with a greater degree of latent damage decreasing sperm longevity in the female genital tract [40]. ...
The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.
Wider implementation of AI in sheep in the field condition has not been possible till date due to very poor conception rate after cervical insemination with cryopreserved semen. Poor cervical penetrability in ewe and diminished sperm functions in cryopreserved semen are considered responsible for it. In the present study, effect of carboxymethyl cellulose (CMC) on post-thaw qualities of ram semen was investigated. Ejaculates from eight adult Malpura rams were pooled and diluted (800 × 10⁶ sperm mL⁻¹) with TES-Tris-fructose-egg yolk extender having either 5 or 6% glycerol and supplemented with 0, 0.25, 0.5, 0.75 and 1.0% (w/v) CMC and packaged into 0.25 mL French mini straws. The straws were progressively cooled to 5 °C inside a cold cabinet (5 °C) and then equilibrated for 22 h inside a refrigerator (2-5 °C). Straws were frozen at -25 °C min⁻¹ up to -125 °C using a programmable cell freezer (Planer Biomed R-204, UK) and finally plunged into liquid nitrogen. The post-thaw progressive motility was higher (P<0.05) in 0.75% CMC-treated group compared to control. Overall, both pre-freeze and post-thaw sperm kinetics was comparable between CMC-treated and control groups. The post-thaw sperm viability, acrosomal integrity and sperm with high mitochondrial membrane potential (hMMP) were relatively higher while sperm with high membrane cholesterol was significantly (P<0.05) higher in presence of 0.25% CMC compared to the control. Both sperm having hMMP and non-capacitated sperm were significantly (P<0.05) higher in presence of 5% glycerol than 6% glycerol. Similarly, functional membrane integrity (FMI) was higher in presence of 5% glycerol than 6% glycerol when CMC was added at 0.5% to extender. In conclusion, both 0.25% CMC and 5% glycerol resulted in improvement in several post-thaw sperm functions in cryopreserved ram semen. Thus CMC demonstrated cryoprotective effect on ram sperm in a synergistic manner with glycerol.
This study aims to examine the quality of frozen semen in Tris egg yolk (TEY) extender and skimmed milk extender with or witout omega-3. A total of 18 Simmental bulls belong to Lembang Artificial Insemination Centre were divided into three groups. Each was fed with standard feed (R1), standard feeds supplemented with minimal Se and Zn (R2) and standard feed supplemented with maximal of Se and Zn concentration. Semen were collected using an artificial vagina and were evaluated macro- and microscopically. The semen then were divided into four tubes and each diluted with Skimmed, SkimmedOmega 3, TEY or TEY-O. The semen was then packed into a 0.25 ml straw and equilibrated at 5 oC for 4 hours, then frozen above liquid nitrogen vapor, and stored in liquid nitrogen container (-196 oC). The qualities of frozen semen were evaluated on the motility, individual score, viability and integrity of the plasma membrane of sperms. Sperm motility of bulls fed with standard feed (R1) in TEY extender and R3 in TEY and TEY-Omega-3 extender were higher (p <0.05) compared to the other combinations. No difference was found on the individual score. The viability of sperms in bulls fed with standard feed in SkimmedOmega-3 extender was higher than the other treatments and the highest sperm plasma membrane integrity was demonstrated by sperm in bull feeding with R2 in TEY extender.
Bu çalışmanın amacı, Honamlı teke spermasının kısa süreli saklanmasında farklı büyüklükteki payet ve deney tüplerinin spermatolojik parametrelere etkisini araştırmaktır. Araştırmada beş baş Honamlı tekesi kullanıldı. Tekelerden haftada iki kez suni vagina yardımıyla üreme sezonu içinde sperma alındı. Her bir tekeden alınan nativ ejakülatlar birleştirilerek tris yumurta sarısı sulandırıcısı ile sulandırıldı ve payet (0.25; 0.5 ml), Ependorf tüpü (0.5; 1.5 ml), Falkon tüpü (15; 50 ml) olarak 6 farklı grup oluşturuldu ve +4 ⁰C’de saklandı. Gruplar oluşturulduktan sonra 24 saat aralıklarla spermatolojik parametreler 96. saate kadar sperma motilitesi (%), morfolojik bütünlük (%), membran bütünlüğü (HOS test, %) yönünden incelendi. Morfolojik bütünlük yönünden gruplar arasında istatistiki olarak farklılık bulunmazken (P>0.05); 96. saatin sonunda en yüksek motilite (%57.50±2.50) ve membran bütünlüğü (%56.00±3.73) 0.5 ml’lik payet grubunda tespit edildi (P<0.05). Sonuç olarak +4 ⁰C’deki saklama koşullarında motilite ve membran bütünlüğü açısından en iyi sonuç 0.5 ml’lik payet grubunda tespit edildi.
A trial was conducted to assess the semen production potential of FecB gene carrier Garole x Malpura rams during the non-breeding season and to evaluate objectively their semen quality by computer-aided semen analysis (CASA) technique. Semen was collected from 4 homozygous (FecB(BB)) and 11 heterozygous (FecB(B+)) adult Garole x Malpura rams on 8 occasions twice in a week. The data derived from CASA estimates were subjected to repeated measures analysis. The overall means of traits which did not differ significantly with age or FecB genotyping were volume (0.6 ml), mass motility (4.52), sperm concentration (3714.6 x 10(6)/ml), motility (91.4%), rapid motile (83.0%), linearity (61.1%), curvilinear velocity (234.9 mu m/s), average path velocity (168.7 mu m/s), straight-line velocity (147.5 mu m/s), amplitude of lateral head displacement (6.93 mu m), sperm head elongation (48.8%) and sperm head area (6.96 mu m(2)). The age of rams had significant effect (P < 0.05) on the percent straightness of spermatozoa, which was higher in young rams (< 4 yr) compared to old ones (> 4 yr). However, straightness was not significantly affected by the FecB status of semen donor rams. The results indicate that FecB(BB) and FecB(B+) Garole x Malpura rams are capable of producing good quality semen during the non-breeding season in a hot semi-arid climate.
The present study was conducted to observe the effect of osmolality of glycerolated TEST-yolk glycerol extenders on post-thawing sperm kinematics of ram spermatozoa of the native Malpura breed maintained in a semi-arid tropical environment. Good quality semen obtained from adult rams was pooled, split and diluted to 1,000 million spermatozoa per ml in complete TEST-yolk-glycerol extenders of 900, 1,200, 1,500 and 1,800 mOsm/kg osmolality. Diluted semen samples were loaded in 0.25 ml straws and cooled down to -125??C freezing temperature at the rate of -25??C per minute under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50??C in a water bath for 10 seconds and sperm kinematics of the frozen-thawed spermatozoa were assessed by a computer-assisted sperm analysis technique. Osmolality of diluent had no significant effect on post-thawing % motility, % rapid, % medium and % slow moving frozen-thawed spermatozoa but significantly (p< 0.05) affected the % linearity and % straightness.The post-thawing % motility and % rapid motile spermatozoa were highest in samples extended in diluent of 1,500 mOsm/kg osmolality and lowest in 900 mOsm/kg. The curvilinear velocity of spermatozoa was significantly (p
A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.
The present study was conducted to compare sperm motion characteristics of adult Malpura and Bharat Merino rams by the computer-aided sperm analysis (CASA) technique. Malpura is a hardy native sheep breed of the semi-arid tropical environment and Bharat Merino is a crossbred evolved in the same environment by crossing native sheep with exotic rams. Semen was collected from 8 donor rams of each breed at the onset of autumn season on 5 occasions at three days interval. The data were analyzed by analysis of variance using the general linear model repeated measures procedure. The CASA parameters which differed significantly (p<0.05) between the breeds were rapid motile sperm, medium motile sperm, slow motile sperm, linearity, straightness, curvilinear velocity, average path velocity, straight-line velocity, amplitude of lateral head displacement, beat frequency, sperm head area and sperm head elongation with higher values in all traits in Malpura breed. The semen volume and sperm concentration were higher in Bharat Merino breed but the differences were not significant. The body weight of rams had significant (p<0.05) effect on mass motility, curvilinear velocity and amplitude of lateral head displacement. The m ass motility was higher in rams of more than 50 kg body weight while curvilinear velocity and amplitude of lateral head displacement was higher in rams of less than 50 kg body weight. A significant (p<0.05) influence of age of rams was observed on linearity and amplitude of lateral head displacement. The linearity was higher in rams of less than 3.5 years of age whereas amplitude of lateral head displacem ent was higher in rams of more than 3.5 years of age. In conclusion, CASA derived sperm motion characteristics revealed that the semen quality of native Malpura rams was better compared to crossbred Bharat M erino rams during major breeding season in a sem i-arid tropical climate.
A study was conducted to evaluate the semen production and sperm motion characteristics of ram lambs by computer-aided semen analysis technique. Eight Malpura rams were raised under intensive management system and were trained for semen collection at a weekly interval from the age of 6 months. Rams were scheduled for semen collection at a weekly interval up to 1 year of age to assess their potential for semen production and objective evaluation of semen quality. The average age of ram lambs at the time of first ejaculation was 219 days ranging from 186 to 245 days. The age of ram lambs significantly (p < 0.05) influenced sperm concentration, sperm velocities, and beat frequency of spermatozoa, which were higher in 9-12-month-old compared to 6-9-month-old ram lambs. However, the effect of age was not significant on semen volume, percent motility, percent rapid, medium or slow motile spermatozoa, percent linearity, percent straightness, amplitude of lateral head displacement, percent elongation, and area of sperm head. The body weight of ram lambs was significantly (p < 0.01) and positively correlated (r = 0.46) with age. The results indicate that Malpura ram lambs of 9-12 months of age raised under the intensive management system in a semiarid tropical environment can produce good quality of semen.
The arid and semi-arid tropical climates of India are endowed with vast diversity of non-prolific sheep breeds. The GarolexMalpura sheep has been evolved in a semi-arid tropical environment by introgression of FecB gene via artificial insemination of Malpura ewes using diluted semen of prolific microsheep Garole and subsequently multiplied by inter se mating among GarolexMalpura halfbreds. The aim of the present study was to identify FecB mutation in sexually mature GarolexMalpura rams by forced RFLP-PCR of BMPR-1B gene and evaluate: (i) semen production and sperm motion characteristics of GM rams and (ii) influence of age and FecB genotype on their semen attributes. Semen was collected during autumn season from 12 donor rams by artificial vagina on 8 occasions at weekly interval. The overall means of traits which did not differed significantly with age or FecB genotyping were volume (0.72 ml), mass motility (4.44), sperm concentration (2721.56 x 10(6)ml(-1)), curvilinear velocity (134.51 microm/s), motility (81.3%), amplitude of lateral head displacement (6.24 microm), beat frequency (44.43 Hz), sperm head elongation (48.9%) and sperm head area (10.01 microm(2)). The FecB genotyping had a significant effect (P<0.05) on percent linearity and rapid motile sperms, which did not vary significantly with age. Although sperm concentration was higher in FecB(BB) and FecB(B+), compared to FecB(++) genotypes but the effect was non-significant. The age and FecB genotyping had significant effects (P<0.05) on straightness, average path velocity, straight-line velocity and percentage of medium or slow motile sperms. It is concluded that GarolexMalpura rams with introgressed FecB gene are capable of producing good quality semen in a semi-arid tropical climate.
Garole is a prolific, rare, less known and small size Indian sheep breed found in low and humid Sunderban region of West Bengal. Although information on stored Garole ram liquid semen upto 24 h is available, but there is a need to further investigate the short-term and long-term preservability of Garole ram semen for extensive utilization of this valuable germplasm by artificial insemination. The aim of the present study was to apply computer-assisted sperm analysis technique for assessing the motion characteristics of Garole ram semen stored (i) in liquid state at refrigeration temperature for short-term preservation upto 48 h and (ii) in frozen state at -196°C for long-term preservation after packaging in mini straws. Short-term preservation had a significant effect on motility (p<0.01) as the motility progressively decreased from 90.1% at 0 h to 85.5% and 73.2% after 24 and 48 h of storage, respectively. Although the decline in rapid moving sperms was also significant (p<0.01) on storage but the decrease was more pronounced at 48 h as compared to 24 h of storage period. Storage of chilled semen had also a significant effect on % linearity (p<0.05), % straightness (p<0.01), sperm velocities (p<0.01), amplitude of lateral head displacement (p<0.01) and beat frequency (p<0.01) of spermatozoa. The replication had a significant effect for all the variables except average path and straight line velocity. However, the interactions of short-term storage and replication were non-significant for most of the variables except % of medium moving sperms, sperm velocities and beat frequency. On long-term preservation of Garole ram spermatozoa under controlled conditions the mean post-thaw recovery of 70.4 and 71.4% motile spermatozoa was achieved having 48.8 and 48.9% of rapidly motile spermatozoa, respectively in both the replicates. The effect of replication on cryopreservation was significant (p<0.05) on amplitude of lateral head displacement and beat frequency, but there was no significant effect on motility, rapidly motile spermatozoa, linearity, straightness and sperm velocities of frozen-thawed spermatozoa. It can be concluded from these results that an average 70% motility can be achieved on storage of Garole ram semen in chilled liquid state upto 48 h or in liquid nitrogen after freezing under controlled conditions in straws. However, further studies are required to evaluate the fertility of short-term and long-term preserved Garole ram semen for extensive use of this prolific sheep breed.
To obtain better ram semen extenders for artificial insemination (AI), we developed effective trehalose-containing hypertonic diluents. The cryoprotective action of trehalose has been explained by its dehydrating activity and interaction with cell membranes. Accordingly, we tested the cryopreserving capacity of different combinations of a Salamon's modified plus trehalose extender with EDTA. Evaluations were based on the percentage of motile spermatozoa and acrosome integrity, measured after thawing and after a 4-h post-thaw resistance test at 37 degrees C. We conclude that the combination of trehalose plus EDTA confers the highest cryopreserving activity tested, not only for freeze-thawing but also for post-thawing resistance, possibly by removing calcium from the medium thereby preventing cation competition with trehalose for membrane-binding sites.
Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).
Semen of adult Malpura rams was processed for freezing after initial dilution @ 1000 million spermatozoa/ml. Extended semen samples were aspirated into 0.5 ml straws and frozen in a programmable cell freezer. Straws were thawed at 50°C or 60°C for 10 sec and the motion characteristics of spermatozoa were assessed by computer-assisted sperm analysis technique. The mean post-thaw recovery of motile spermatozoa at 60°C thawing was significantly higher (P<0.01) ranging from 72.1 to 72.9% in 2 replicates as compared to 53.8 to 55.4% at 50°C thawing. The effect of replication was significant (P<0.01 ) only for beat frequency of spermatozoa. The results indicate that thawing at 60°C has also a significant effect on % rapid, %linearity, % curvilinear velocity, average path velocity, straight line velocity (P<0.01) and beat frequency of spermatozoa (P<0.05) as compared to thawing at 50°C. However, there was no significant effect of thawing temperature on % medium, % slow, % straightness and amplitude of lateral head displacement of spermatozoa.
Research up to 1993 is reviewed. Freezing and thawing of ram semen causes ultrastructural, biochemical and functional damage to a significant proportion of spermatozoa. These changes are accompanied by a reduction in motility, impaired transport and decreased viability of spermatozoa in the female genital tract, and reduced fertility after cervical insemination. Methods used to improve the fertility after cervical insemination include increased concentration of spermatozoa in the inseminate, treatment of ewes with or addition to the semen of hormones, double and deep cervical inseminations. The most effective method is to increase the depth of deposition of frozen-thawed semen into the cervical canal. Recently developed transcervical insemination techniques achieve deep cervical insemination or even uterine deposition of semen. However, at present satisfactory and reliable lambing results are only obtainable by using intrauterine insemination by laparoscopy.
Laparoscopic insemination with frozen-thawed semen is currently used for planned matings in the Sarda breeding programme. In order to find a fast and less intrusive artificial insemination (AI) method that could replace laparoscopic insemination, a field comparison of laparoscopic and transcervical techniques was carried out on 200 mature Sarda ewes. After AI, ewes were assigned to teaser and fertile rams for 2 months. Return rates and cumulative (AI + natural mating) lambing rates were recorded over three subsequent 23-day periods. Lambing rates to AI were significantly different (P < 0·01), and were 62% and 7% respectively for laparoscopic and transcervical AI. Cumulative lambing rates after two further 23-day periods of natural mating were no longer significantly different (P > 0·05) and reached 82% and 74% respectively. Ewes with body condition scores at AI higher than 2·75 showed better overall reproductive performance, but not higher pregnancy rate to AI. Plasma cortisol concentrations, sampled twice, before and after AI, were higher (P < 0·01) in the last sample, suggesting a stress response to insemination. Cortisol levels after AI were lower (P < 0·01) for ewes submitted to transcervical rather than laparoscopic insemination (P < 0·01). However, cortisol levels after AI were no greater than those recorded when ewes were restrained in a milking yoke different from that usually employed. Laparoscopic AI was confirmed as the most suitable technique for insemination offrozen semen in the Sarda breeding scheme.
Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25ml straws and cooled to −25, −75 or −125°C freezing temperature at the rate of −25°C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50°C in a water bath for 10s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125°C and lower at −25 or −75°C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P
This study comprising of two trials was undertaken in ewes exhibiting natural oestrus to evaluate a simple transcervical artificial insemination technique suitably modified for use in tropical sheep. Twenty four adult multiparous Malpura ewes which had lambed 6 to 12 months prior to the experiment were utilized for two oestrous cycles during the autumn breeding season. The cervical penetration (n=47) was conducted in both first (n=24) and second (n=23) trial ≤6 to 18 h following onset of oestrus, while lambing rate was assessed in the second trial (n=23) following deposition of frozen-thawed semen having >70% mean post-thaw motility at os, mid cervix or uterus. The overall success achieved in cervical penetration was 44.7%. There was no difference in cervical penetration rate at ≤6 and 18 h after detection of oestrus (46.1% vs. 42.8%, respectively). A mean lambing rate of 22.7% was recorded in ewes inseminated using this technique. Mid cervical and transcervical insemination with frozen semen resulted in similar lambing rate (28.5 vs. 22.7%, respectively). However, no lambing was achieved on os-cervical insemination. Further efforts are needed to determine factors controlling the cervical penetration and conception rate following artificial insemination with frozen semen.
Research up to 1993 is reviewed. Various methods for processing, freezing and thawing of semen have been elaborated and their effect on survival and fertility of spermatozoa have been examined in vitro or in fertility tests. Diluents initially used for freezing bull semen had their limitations and were modified or replaced by new media. The freezing diluents investigated included citrate-sugar-, milk-, lactose-, saccharose-, raffinose-, trisbased and other media containing protective agents (mainly glycerol and egg yolk), and other substances including antioxidants. Satisfactory sperm survival rates were obtained with a number of diluents and different combinations of the factors studied.Lambing results after cervical insemination varied depending on the parameters examined in the freezing technology or at insemination, but were low in comparison with those obtainable with fresh diluted semen. As a consequence, cervical insemination with frozen-thawed semen had a relatively limited application in sheep.
In this study, three groups of ewes were inseminated with a transcervical intrauterine technique in order to evaluate the suitability of the technique for a commercial artificial insemination (AI) program using frozen semen. The ewes were synchronized into estrus with vaginal pessaries and pregnant mare serum gonadotrophin (PMSG) and were inseminated between 48 and 55 h following pessary removal. In Flock I, maiden and multiparous ewes (n = 12) received fresh semen containing 400 × 106 spermatozoa. Flock II was divided accordingly: 40 multiparous ewes were inseminated with frozen semen using the transcervical technique and 40 ewes were inseminated laparoscopically with frozen semen. In Flock III, 38 multiparous ewes were inseminated transcervically with frozen semen containing 150 × 106 spermatozoa. Pregnancy diagnosis was based on blood progesterone analysis 18 d following breeding, and real time ultrasound examination at 40 d. Pregnancy rates were 50, 50 and 68%, respectively, for the three groups inseminated transcervically and 70% for the ewes inseminated laparoscopically. The lambing rates were 50, 55 and 40%, respectively, for the ewes inseminated transcervically and 65% for the ewes inseminated laparoscopically. Lambing results were confirmed by matching the breeding dates to the lambing dates. These results demonstrate that the described technique is suitable for further evaluation using frozen semen in a commercial sheep AI program.
A series of five experiments was conducted to examine ewe fertility following insemination with ram spermatozoa frozen by the pellet method. With thawed semen of low concentration and relatively poor quality, fertility was very poor with an overall non-return rate of 5 -6% (269 ewes). Following insemination with thawed semen of low and high sperm concentration (0-5 versus 1·5 109 motile cells/ml) the proportions of ewes lambing were 23-7 and 43-8% respectively. There was no difference in the proportion of ewes lambing when the inseminate volume was reduced from 0-3 to 0-1 ml (Exp. 2, dilute semen) or from 0-15 to 0 5 ml (Exp. 3, concentrated semen). Two inseminations gave higher fertility than a single insemination (Exp. 2, 38-8% versus 22-6%; Exp. 3, 53-0% versus 39-7%) but the magnitude of the response varied according to both the time of insemination and the concentration and volume of the inseminate. High doses of oxytocin (5 or 10 i.u., i.m.) depressed the proportion of ewes lambing and relaxin (100 to 12,500 guinea-pig units/ewe) did not affect the depth to which the inseminating pipette could be inserted into the cervix. Overall lambing results (percentage of ewes lambing) for Exps 2, 3, 4 and 5 were 30-5% (266 ewes), 46-3% (231), 49-3% (69) and 27-5% (291) respectively, whereas results for the best treatment combinations in these experiments were 61-9%, 56-7%, 64-7% and 40-4%.
Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to −10°C retained fluorescence into the post-thaw temperature range of 9−24°C (mean ± SEM; 13.25 ± 0.91°C), whereas cells cooled to −20°C lost fluorescence shortly after thawing (mean ± SEM; 2.62 ± 0.91°C). Sperm cooled to 5°C, but not frozen, retained fluorescence during rewarming up to 20−30°C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing.
Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.
Acrosomal structures of ram spermatozoa were prominently stained when air dried smears of diluted semen were fixed for 15 minutes in buffered formal saline and stained for 90 minutes in a 6 per cent (v/v) buffered solution of Giemsa stain. Progressive disruption of the acrosomes was demonstrated during chilling and deep-freezing of the spermatozoa, and the degree of damage was systematically scored. A rapid and repeatable estimate of the state of the acrosomes in a sample could be made from the mean score of 20 spermatozoa examined per slide.
The reactive superoxide radical, O2-, formerly of concern only to radiation chemists and radiobiologists, is now understood
to be a normal product of the biological reduction of molecular oxygen. An unusual family of enzymes, the superoxide dismutases,
protect against the deleterious actions of this radical by catalyzing its dismutation to hydrogen peroxide plus oxygen.
Thirty human semen specimens were analyzed using a standard manual method, then videotaped and reanalyzed using two different computer-aided sperm analysis (CASA) instruments (the HTM system, Hamilton-Thorn Research, Danvers, MA, and the CTS system, Motion Analysis Corp., Santa Rosa, CA). Videotaped specimens were analyzed by CASA for 5 frames for sperm concentration (CON) and percent motility (MOT), and for 15 frames for kinematic variables (straight-line velocity, VSL; curvilinear velocity, VCL; linearity, LIN; and amplitude of lateral head displacement, ALH). Machine parameter settings for the two instruments were matched as closely as possible. CASA values were compared with each other for all measures and with manual results for sperm count and the percentage of motile sperm. Results show: 1) HTM and CTS average values for CON are not different from manual measures for the 5- or 15-frame analysis, but slight differences are seen between CTS and HTM; 2) average values for MOT for the 5-frame analysis are higher than the 15-frame analysis for both instruments, but the average manual measurement for percent motility is much higher than any CASA value; 3) average VSL and LIN are slightly higher for HTM than CTS, but pair-wise comparison shows a high degree of concordance between the instruments; and 4) the mean values for VCL and ALH are equal for the two instruments, and there is a close concordance for the pair-wise comparison for VCL; however, pair-wise comparison of ALH reveals significant differences between the instruments. Overall, the differences seen between these instruments are slight, and are probably not biologically or clinically significant.
A new, rapid, thermal stress test for assessing the viability of boar semen, requiring only 45 min of incubation at 42.5 degrees C, was developed and compared with a widely used stress test of 180 min incubation at 37 degrees C. The shorter procedure was found to have the same discriminatory ability as the standard test in assessing the effects of freezing conditions on the percentage of spermatozoa remaining motile. Neither test was able to show differences in the kinetic rating of motile sperm after freezing in relation to the glycerol concentration present during freezing. However, the new test had a greater ability to distinguish the effects of different concentrations of glycerol, over the range of 0 to 6%, and to reveal different degrees of acrosomal damage sustained during freezing. The longer procedure was unable to distinguish among glycerol concentrations from 0 to 4% with respect to acrosomal damage and produced an overall lower proportion of sperm having a normal apical ridge. The new thermal stress test thus has the advantages of greater sensitivity and more rapid execution over the test hitherto in widespread use.
A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.
Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.
A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.
The effect of dialysis on extended ram semen prior to cryopreservation was studied. Techniques were developed to improve post-thaw recovery of dialyzed semen and a fertility trial was used to evaluate the viability of dialyzed and frozen semen. Dialysis prior to freezing was shown to increase post-thaw recovery of motile cells and percentage of cells passing through a Sephadex filter. Freezing semen in pellets on dry ice was superior to freezing in French straws. Pellets were thawed in an aluminum thaw block at 42 to 45 degrees C before insemination of progestagen-PMSG synchronized ewes. Double inseminations were made at 12-hr intervals. Natural service of synchronized ewes was also made at 12-hr intervals as a control. There was no significant difference (P greater than 0.05) in fertility between naturally serviced ewes (44.4%) and ewes inseminated with frozen semen (44.7%).
A successful method for low temperature preservation of bull semen was modified for use with boar semen and resulted in recovery of twenty to fifty per cent motile cells immediately after thawing. Recovered cells did not survive five hours incubation at 37 degrees C. and no pregnancies resulted following insemination of twenty-four sows and gilts with frozen semen.
The toxic effect of dead sperm on diluted bovine semen and the modifying effect of certain compounds on this toxicity were investigated. Toxicity is associated with an amino acid oxidase which becomes active only after death of sperm. A metabolic product of this enzyme, peroxide, is responsible for the toxic effect of dead sperm. The enzyme is active against aromatic amino acids only. Its toxic effect is enhanced by increasing egg yolk, inhibited by ethylenediaminetetra- acetate (EDTA), and eliminated by cat- alase. Inhibition of toxicity by EDTA is by protection of sperm against deleterious effects of peroxide and not by inhibition of the enzyme. The enzyme is inhibited by ethyl maleimide indicating active sulph- ydryl groups. It is labile to heat being inactivated by heating to 90 C for 2 minutes. It is also labile to pH 3 and pit 9. An active fraction was obtained from dead sperm by precipitation with 30% acetone. Activity of ejaculated dead sperm is only slightly less than freshly killed sperm. Practical implications are discussed.
Excerpt
The results from the insemination of frozen-thawed bull semen approach or equal those expected from natural mating, and contrast with the poor results obtained using frozen-thawed ram semen (Emmens & Robinson, 1962). Healey (1969), describing the ultrastructural changes in spermatozoa resulting from freezing, noted differences between species in the degree of acrosomal deterioration.
This report summarizes the results of experiments on the cooling and deep-freezing of ram and bull spermatozoa. The degree of change in the acrosomes was estimated by means of light microscopy after the specimens had been stained with Giemsa, using the following scoring system: 0, normal acrosome; 1 and 2, stages of acrosomal damage; 3, acrosome entirely lost. A typical spermatozoon in each of the categories is shown in Text-fig. 1.
Ejaculates from fifteen bulls were examined in one experiment and from twelve rams in a second experiment. Semen was diluted tenfold at 30° C and
A factorial experiment was conducted to examine the effects in Merino ewes of method of insemination and semen type on embryonic loss (fertilization rate versus lambing rate).
The results showed that:
• frozen semen (pellet method) was of lower fertility than fresh semen (49% versus 70% respectively).
• there was little embryonic mortality following either the cervical or cervical traction methods of insemination (13% and 6% respectively), but substantial loss occurred following uterine insemination (47%). Results for both fresh and frozen semen were similar in this respect.
Normal cervical insemination (two inseminations at a 12-hr interval within one oestrus) with frozen semen of high concentration (1·6 × 10⁹ motile spermatozoa/ml, 0·1 ml dose) resulted in ewe fertilization and lambing rates of 58% and 50%, respectively.
Most (94%) of the aromatic L-amino acid oxidase activity in dead bovine spermatozoa was recovered in tail preparations. The enzyme was released from the cell in sodium citrate but not in sodium phosphate or sodium chloride solutions but oxidase activity was not significantly different in sodium phosphate or sodium citrate buffers (9.6 microliters O2/h and 11.2 microliters O2/h). The activity in ejaculated spermatozoa was correlated with the percentage of dead spermatozoa (r = 0.954, P less than 0.01) but could not be detected in freshly collected epididymal spermatozoa. Killed epididymal spermatozoa showed oxidase activity (10.1 microliters O2/h) similar to that of killed ejaculated spermatozoa (9.2 microliters O2/h). It is concluded that death of spermatozoa occurs in the ampulla and/or at ejaculation and that between-bull differences in the percentage of dead spermatozoa are a consequence of differences between bulls in conditions in the ampulla and/or at ejaculation.
The objective of this investigation was to examine the nature of freeze/thaw-induced plasma membrane damage in an effort to validate hypotheses about cryoinjury in ram spermatozoa. Spermatozoa were loaded with fluorescein diacetate (FDA), a marker for plasma membrane integrity, and cooled (15 degrees C/min) to temperatures between -10 degrees C and -30 degrees C on a cryomicroscope stage. Post-thaw fluorescence intensity measurements of individual cells indicated that freezing to temperatures between -10 degrees C and -15 degrees C did not induce significant membrane permeabilization. However, freezing below -15 degrees C was followed by membrane permeabilization immediately after thawing. A majority (> 60%) of flagellar plasma membranes of cells frozen to -10 degrees C remained ultrastructurally intact during thawing; principal-piece membranes were more robust than middle piece membranes (p = 0.001). Significant middle-piece membrane breakage was, however, induced as the post-thaw temperature increased from +10 degrees C to +30 degrees C (10 degrees C, 64 +/- 12.3% intact membranes [mean +/- SEM]; 30 degrees C, 43 +/- 12.5% intact membranes [mean +/- SEM]; p = 0.0085). Cells frozen to -30 degrees C did not exhibit this thawing effect, although the distinction between middle-piece and principal-piece plasma membranes was evident (p = 0.002). All sperm head plasma membranes were damaged by freezing and thawing to any combination of temperatures. Although acrosomes became swollen after freezing and thawing, the incidence of outer acrosomal membrane vesiculation remained at control (unfrozen) levels with all treatments used. Experimental exposure to the hyperosmotic conditions generated during freezing induced little flagellar membrane permeabilization, but significant damage was caused by restoration of osmotic equilibrium. It is suggested that membranes are initially destabilized during the freezing process, both by low temperature effects and by exposure to high salt concentrations. The resultant post-thaw degeneration of the plasma membrane is caused by a combination of temperature and osmotic effects.
Computer-aided sperm analysis (CASA) technology is 7 years old. Over 120 papers have been written that verify the technology or apply it in basic and clinical studies. Most of the technical problems with CASA, such as the dependence of velocity on video frame rate, inaccuracy of count and percent motility for low- and high-concentration specimens, parameter dependence on the number of frames analyzed, sensitivity of the subjective threshold setting, confusion over the presence of debris, and different implementations of algorithms across instruments, still persist. A critical review of the literature reveals that no standard practices are followed within or across instruments. Moreover, no standards have been embraced or recommended by professional societies. Despite its potential to provide objective measurements of specimen and individual sperm parameters, and to automate the laboratory semen analysis, the promise of CASA has not been fulfilled. Unless laboratory medicine defines instrument performance and laboratory standards and co-operates with industry to achieve these goals, CASA technology may remain a research curiosity. This outcome is especially worrisome in the context of increasing requirements for laboratory accuracy, precision, standardization, and accreditation under the Clinical Laboratory Improvement Act of 1988.
New research on the cooling and cryopreservation of mammalian spermatozoa is reviewed in the context of the older literature. Cryoinjury to a variety of cell organelles is regarded as being due to the two major stresses of cryopreservation, i.e., the change in temperature, and the formation and dissolution of ice and its consequences. Since the cryopreservation process involves departure of the cells from and return to body temperature, both cold shock and warm shock are included as potential stresses to be considered, as well as the stages involving cooling below the freezing point of the medium. The causes of cryoinjury are reconsidered and new concepts concerning the influence of osmotic stress are presented. Heterogeneity of the sperm population is discussed in the context of the success with which spermatozoa can be cryopreserved between and within ejaculates and individuals. The functional state of frozen and thawed spermatozoa is examined on the basis of published results of structural and functional tests of sperm competence. The hypothesis is advanced that cryopreserved mammalian spermatozoa are in a state resembling capacitation, which accounts for their relatively reduced longevity and their readiness to undergo egg penetration without incubation. The importance of this to the utilization of cryopreserved spermatozoa is examined, and proposals are made for new avenues of research to overcome these problems.
The antioxidants superoxide dismutase (SOD), catalase (CAT), cytochrome c (CHc) and glutathione peroxidase (GP) were added at various concentrations to Tris-glucose-yolk diluent (TGY), and their effects on motility, acrosome integrity and fertility of ram spermatozoa were assessed after extension and liquid storage. All the antioxidants improved the motility and acrosome integrity of spermatozoa, and a combination of SOD and CAT had an additive effect on the survival of spermatozoa stored at 5 degrees C but not at 25 degrees C. There was a linear improvement in survival of spermatozoa with increasing dose of antioxidants except for CAT for which doses higher than 200 U mL-1 were toxic. The proportion of oocytes fertilized in vitro declined with time of semen storage (P < 0.001), and was better for semen diluted with TGY containing SOD or CAT than TGY without antioxidants when stored for 7 days (116/246, 47% v. 25/79, 32%; P < 0.05) but not for 14 days (23/174, 13% v. 8/66, 12%). Fertilization rates were unaffected by the presence or absence in the diluent of CHc or GP. The proportions of ewes with fertilized ova and of recovered ova fertilized were better after insemination with semen diluted in TGY containing SOD and CAT than TGY without antioxidants when stored for 14 days (9/18, 50% and 20/40, 50% v. 2/13, 15% and 5/32, 16%; P < 0.05) but not for 7 days (9/20, 45% and 16/48, 33% v. 8/16, 50% and 24/41, 59%). Pregnancy rates were better after intrauterine insemination of ewes with fresh semen than stored semen (11/18, 61% v. 21/75, 28%; P < 0.01), and with semen stored in TGY containing SOD and CAT than in TGY without antioxidants (15/37, 41% v. 6/38, 16%; P < 0.05).
In a liquid environment, at high dilutions, fertility of bull sperm is maintained for 3-5 days when stored at ambient temperatures (10-21 degrees C), after which time it steadily declines at a rate of 3-6% per day. This decline in fertility occurs irrespective of whether the sperm are stored at 5 degrees C or at 15 degrees C, but the rate is greater once storage temperatures exceed 25 degrees C. Sperm motility can be maintained for extended periods in an environment where the extracellular oxidative stress is minimized by reducing the oxygen tension, by addition of antioxidants and chelating agents; however, this will not prevent a significant drop in fertility after five days of storage at ambient temperature. The requirement of energy by the sperm-motility apparatus demands a high level of respiratory activity. This system is very active and the free radicals produced in vivo during this process could lead to chromatin damage. As no internal repair mechanism exists in sperm, an extraneous supply of protectants, or an environment where damage is minimized, is essential to maintain its fertilizing potential. The lack of extended storage potential of sperm, even in the presence of antioxidants, seems to suggest that although oocyte-penetrating ability of the sperm could still be intact, the high rate of intracellular metabolic activity could lead to mitochondrial DNA damage and chromosomal abnormalities that would compromise the viability of the resulting conceptus.
The epididymal compounds taurine, hypotaurine and inositol, and the antioxidants carnosine and ascorbic acid, were added to Tris-based diluents containing varying concentrations of glycerol, and their effect on the post-thaw motility characteristics and fertility of ram spermatozoa was examined. Overall, the post-thaw motility characteristics of spermatozoa were better when semen was frozen in the presence rather than in the absence of glycerol. Only taurine protected spermatozoa during cryopreservation; the presence of 25 mM or 50 mM taurine significantly improved the post-thaw percentage of motile spermatozoa but this had no effect on fertility after cervical or laparoscopic insemination of ewes. Increasing the concentration of taurine to more than 100 mM significantly reduced the percentage of motile spermatozoa, compared with the lower concentrations of the amino acid. The presence of more than 50 mM carnosine or ascorbic acid significantly reduced all motility characteristics compared with the control diluent. Given that hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility, the cryoprotective effect of taurine may be attributable to its osmoregulation rather than to its antioxidant properties.
The fertility of ewes after artificial insemination and the relationship between fertility and motility characteristics assessed by a computerized motility analysis system were examined with ram semen frozen in diluents reported to improve postthaw motility. The percentages of motile and progressive spermatozoa were better when frozen in proline- or glycine betaine-containing or HEPES-based, rather than Tris-based, diluents (P < 0.01). The fertility of spermatozoa frozen in diluents containing proline or glycine betaine was slightly reduced, whereas when both compatible solutes were present, the reduction was more pronounced, in comparison with semen frozen in Tris- or HEPES-based diluents (9.5 versus 71.1 and 66.6%; P < 0.01). Fertility of frozen-thawed spermatozoa was higher after laparoscopic insemination than after cervical or transcervical insemination (P < 0.01). Similarly, higher fertility was obtained after cervical insemination with fresh than with frozen-thawed semen (32.4 versus 11.3%; P < 0.01). Furthermore, loss of embryos was lower after laparoscopic insemination of ewes with semen frozen in a Tris diluent than with semen frozen in proline diluents, in glycine betaine diluents, or in proline-plus-glycine betaine diluents (0.0 versus 26.0, 38.5, and 60.0%; P < 0.001). A wide variation in the postthaw percentage of motile (31.6-59.7%) and progressive (22.6-43.1%) spermatozoa and in the fertility of spermatozoa from individual rams was also observed after laparoscopic (29.2-59.7%) or cervical insemination (8.7-30.5%). Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure. The correlation analysis showed relationships between some of the motility characteristics (P < 0.01), but there were no relationships between the motility characteristics and fertility.
Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.
An experiment was conducted to determine whether factors affecting pregnancy rate out-of-season are associated more with transcervical artificial insemination (T-AI) procedures or with the reproductive state of the ewe. Twenty Finncross ewes were treated with progesterone sponges, and at sponge removal (0 h) 10 ewes were treated with eCG. Blood samples were collected for LH and progesterone analyses, and follicular development was monitored using ultrasonography. Ewes were inseminated from 48 to 52 h with 200 million motile frozen-thawed spermatozoa. The incidence of estrus, LH surges and ovulation was greater (P < 0.01) and intervals to these responses were shorter (P < 0.01) in the eCG-treated ewes. The number of follicles > 5 mm was higher (P < 0.05) in eCG-treated than control ewes. Progesterone concentrations increased and remained elevated through Day 19 in 7 eCG-treated and in 1 control ewe, and these ewes were pregnant based upon ultrasonographic examination. The results demonstrate that the T-AI technique using frozen-thawed semen produces a relatively high (70%) pregnancy rate out-of-season. The pregnancy rate was found to reflect primarily the reproductive condition of the ewe.
Storage of ram semen in liquid and frozen state, the diluents used for both methods, processing, cooling, freezing and thawing of semen are reviewed. Factors influencing the fertility of stored semen and methods used for improvement are discussed, and fertility results of long-term frozen stored ram semen are also given.
Sheep play an important role in the Indian economy by providing employment to a large population of marginal and landless farmers. The production from native breeds is relatively low due to their poor reproductive efficiency. Embryo transfer technology can be utilised for faster multiplication of elite animal to increase the genetic gain. A great deal of research is involved to overcome the constraints in the technology, i.e. expensive and complicated nature of the technology and low success rate. In order to avoid surgical involvement, procedures of laparoscope aided embryo collection and transfer have been developed. Although the use of FSH of ovine origin has given more consistent superovulatory response, but it is still too low to get sufficient numbers of progeny from a donor ewe. The progress made in cryopreservation of ram semen has opened the possibility for conservation and utilisation of frozen semen of elite rams in sheep improvement programme. The lambing rate obtained after laparoscope aided intrauterine artificial insemination with frozen semen is encouraging but the impetus is now to develop the non-invasive transcervical insemination technique.
The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.
The interaction of glycerol concentration from 0 to 16% and cooling velocity from 1 to 100 degrees C/min on freeze-thaw survival of ram spermatozoa was studied using a diluent based on 15% skim milk (450 mOs/kg water). Optimal spermatozoa survival (percentage motility and rating) was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min. Similar results were obtained with 8% glycerol at freezing rates of 5 to 30 degrees C/min. Although the ram spermatozoa tolerated several cooling velocities at each glycerol concentration, increasing the concentration of glycerol resulted in a downshift in the range of optimal cooling velocities. Glycerol concentrations above 8% were toxic and contributed greatly to the progressive decrease in spermatozoa survival. Comparison of the 15% skim milk diluent (450 mOs/kg water) with a 19% skim milk diluent (600 mOs/kg water) showed that optimal cryosurvival was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min with both diluents.
In commercial artificial insemination (AI) of sheep, fresh extended semen is deposited into the vagina or cervical os, or fresh extended or frozen semen is placed laparoscopically into the uterus. Transcervical intrauterine insemination of the ewe is not used commercially. In this study, methods of restraint and instrumentation for AI were evaluated and modified to produce a transcervical intrauterine technique suitable for commercial application. Four methods of restraint, four vaginal specula, three forceps and four instruments suitable for transcervical passage were compared. From these comparisons a technique was developed in which the ewes were positioned in dorsal recumbency with their hindquarters elevated. The vagina was dilated using a duck-billed speculum, the cervix was grasped and retracted using forceps, and an inseminating instrument was introduced into the cervical opening and manipulated through the cervical canal. The technique was repeated on 89 mature, multiparous ewes: the difficulty in locating the cervical opening, the force required to retract the cervix and the time required to penetrate into the uterus were recorded. Uterine penetration was achieved in 82% of the ewes. This technique has the potential to be applied in commercial artificial insemination programs of sheep.
The cervical canal of the ewe does not allow for the consistent transcervical passage of insemination instruments. To define the factors affecting transcervical passage, the gross anatomy of the cervix and canal were studied in 100 estrous ewes and then in their reproductive organs following slaughter. In each ewe, the vagina and cervical opening was examined and the external os was classified into one of four types. Insemination instruments were introduced into the cervical opening and manipulation through the canal was attempted. Fluoroscopy was used to record the flow of contrast material through the canal. Ultrasound, xeroradiography and computed axial tomography were used to image the canal of the recovered reproductive tracts. Following imaging, each cervical canal was filled with silicone to create a mold which was used to measure and describe the canal. The average length (+/-SD) of the cervical canal was 6.7 +/- 1.1 cm and contained 4.9 +/- 1.0 funnel-shaped rings (n = 79). Successful passage of insemination instruments was limited by failure to identify the cervical opening and the small openings in the rings, 2.7 +/- 1.1 mm on average (+/-SD) which were not concentrically aligned. The eccentric rings were most consistently the second or third rings from the external os. The design of effective instrumentation and technique for transcervical passage must take these factors into account.
A transcervical technique (the Guelph System for transcervical AI) was used to inseminate 2060 ewes on 65 farms (average 31 ewes, range 5 to 107) in Ontario, Canada, from October 1990 to September 1992, using previously frozen semen. Estrus was synchronized using progestagen pessaries and PMSG with median inseminations done at 54 h from pessary removal. Maiden ewes were not included. Only ewes in which the cervix could be penetrated were inseminated with 150 million spermatozoa per insemination. A total of 1809 were penetrated and inseminated (penetration rate 87.8%). Success of penetration increased from 76.3% in the first 500 ewes to 97.9% in the last 500 (P=0.01). Cervical penetration was more successful in ewes in the accelerated lambing program (92.3%, average 3.1 mo since the previous lambing) than those in the annual lambing program (82.4%, average 7.0 mo since the previous lambing; P=0.06). The lambing rate for ewes bred during the combined traditional breeding seasons (Fall of 1990, 1991, 1992) was 50.7% compared to 24.4% for ewes bred at other periods (P=0.00001). The average time required for handling and insemination decreased from 8.62 min in the first 500 ewes to 3.62 min in the last 500 ewes. The Guelph System for Transcervical AI was found to be successful for cervical penetration in most ewes. Penetration success was affected by period since the last lambing and by inseminator experience. The lambing rate was higher for ewes bred during the traditional Fall breeding seasons than during other times of the year.
Motility parameters and fertility Control of sperm motility: biological and clinical aspects
- Rj Aitken
Aitken RJ. Motility parameters and fertility. In: Gagnon C, editor. Control of sperm motility: biological and clinical aspects. Boca Raton: CRS Press; 1990. p. 285–302.
Semen quality tests and their relationship to fertility Columbia: National Association of Animal Breeders
- Saacke Rg
- White
Saacke RG, White JM. Semen quality tests and their relationship to fertility. In: Proceedings of the 4th NAAB Technological Conference on Artificial Insemination, Reproduction, Madison, WI, 18–20 April. Columbia: National Association of Animal Breeders; 1972. p. 22–7.