Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Cryo-scanning Electron Microscopy Discloses Differences in Dehydration of Frozen Boar Semen Stored in Large Containers

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50°C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from −5°C to −100°C, namely 2°C/min, 50°C/min and 1200°C/min, the lattermost by plunging the samples into liquid nitrogen (LN₂). The samples were thereafter fractured into LN₂ and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2°C/min and 50°C/min) but not at 1200°C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.     

Deep-freezing of boar semen in plastic film 'cochettes'

The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, 'cochettes', and in 'maxi-straws', in total doses of 5 x 10(9) spermatozoa/5 ml with glycerol (3%) used as cryoprotectant, were assessed after thawing. A computer-based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C-FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler-ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post-thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5%. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT-spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi-straws than in cochettes, 46.8 vs. 43.0% (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi-straws and those frozen in cochettes. The results indicate that although the deep-freezing of AI-doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen.     

Cryopreservation of Black Bengal buck semen: Effects of diluents and freezing on sperm motility and morphology

Semen from Black Bengal bucks was collected to establish a cooling protocol (to −196°C) for buck semen preservation, and to study the effect of freezing on sperm motility and morphology. Semen was diluted with diluents (Triladyl & Tris) and cryoprotectants, filled into straws, sealed, cooled (to 5°C) and equilibrated. After dilution, motility ranged from 75.00% to 76.67% and from 73.33% to 80.00% in Triladyl and Tris diluents, respectively. Motility of sperm after cooling to 5°C in Triladyl and Tris diluents ranged from 65.00% to 66.67% and from 63.33% to 70.00%, respectively. After equilibration in straws, the semen was subjected to a freezing protocol in a computer-controlled biofreezer CL-3000 (cooling at 10°C per minute, from 5°C to −80°C) and plunged into liquid nitrogen. Sperm motility of re-thawed semen varied from 38.33% to 43.33% and from 6.00% to−6.67% in Triladyl and Tris diluents, respectively. Sperm morphology of re-thawed semen was studied and head damage or cryoinjury was found in 2–3% of sperm in Triladyl diluents and 3–6% in Tris diluents. Whether the differences of sperm motility and head damage reflect fertility after artificial insemination is yet unknown and needs to be studied further.     

Assessment of Fertility after Using Different Procedures to Thaw Ram Spermatozoa Frozen in Mini Straws

Contents
In this study, fertility rates were compared after using different procedures (50°C and 70°C) to thaw ram spermatozoa frozen in mini straws. Semen from three, 1.5–2.5-year-old rams of the same breed, selected for use in an AI programme, was collected using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in 0.25-ml mini straws and frozen in a programmable freezer. Post-thaw sperm motility was assessed subjectively using a phase contrast microscope. Sperm membrane integrity was assessed with fluorescent dyes (Calcein AM/EthD-1). Statistically significant variation in the incidence of membrane integrity was found, both between rams and between freezing operations. Significant differences between the different thawing procedures used in this study were seen for membrane integrity (p < 0.01), as assessed with the fluorescent dyes (Calcein AM/EthD-1), but not for the post-thaw motility. The average fertility in this study was 39.7%, with a wide variation between freezing operations (not significant), rams (p < 0.001; 30.4, 33.3 and 64.6%) and flocks (p < 0.001, range: 14.8–61.6%). No statistically significant differences were found for the different thawing procedures, in terms of the fertility (39.0 and 40.4%, respectively) and the litter size (1.32 and 1.41, respectively). Thawing at 50°C for 9 s, instead of 70°C for 5 s, does not seem to further affect either fertility or litter size. The use of this lower temperature would facilitate the practical use of frozen–thawed ram semen under farm conditions in Sweden.     

In vitro Fertilizing Capacity of Deep Frozen Boar Semen Packaged in 0.5 and 5 ml Straws

The aim of this study was to evaluate the straw size effect used for freezing on the in vitro fertilizing capacity. Twenty-one ejaculates from seven fertile boars were frozen under controlled conditions in 0.5 and 5 ml straws. Thawed semen was compared to fresh semen. For fresh and thawed semen in 0.5 and 5 ml straws, the results were: 92.18, 77.38 and 79.04% sperm penetration; 80.68, 66.89 and 69.33% monospermy; 11.51, 10.49 and 9.74% polyspermy; 86.19, 47.14 and 47.02% motility and 75.52, 48.19 and 46.81% normal apical ridge (NAR), respectively. Analysis of variance and test of multiple comparisons showed that under the conditions employed, penetration, monospermy, motility and NAR were significantly reduced by freezing–thawing, but polyspermy was much less affected. The results obtained suggest that frozen boar semen is adequate for in vitro fertilization. In addition freezing in 5 ml straws did not have any detrimental effect on either penetration, monospermy, polyspermy, motility and NAR, in comparison with freezing in 0.5 ml straws.     

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM^® to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM^®. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN₂) vapour approximately 3 cm above the level of LN₂ for 20 min and then were plunged into LN₂. Thawing was achieved in warm water at 50°C for 12 s and then was incubated in a 38°C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM^® was added to the freezing extender. There was no difference (p   =   0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM^® was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p   =   0.02, p   =   0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM^® (p   >   0.05). The results of these investigations suggested that Equex-STM^® exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.     

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.     

Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

Effects of Freezing–Thawing on DNA Integrity of Boar Spermatozoa Assessed by the Neutral Comet Assay

A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

Control of Campylobacter fetus in artificially contaminated bovine semen by incubation with antibiotics before freezing

Fresh, diluted semen containing 1.55 X 10(6) cfu/ml of Campylobacter fetus subsp. venerealis was incubated with 500 iu of penicillin, 500 micrograms of streptomycin, 160 micrograms of lincomycin and 300 micrograms of spectinomycin per ml at 35 degrees C for 0, 1, 2, 5, 10, 20 or 40 minutes. The semen was cooled to 5 degrees C, packaged in 0.25 ml French straws and then frozen in liquid nitrogen for 2 weeks. Immediately after thawing and removal of the antibiotics by centrifugation semen samples from each of the seven treatment groups were cultured as for C. fetus. Semen samples were also examined by in-vitro tests for sperm motility prior to and post-freezing. Incubation with the antibiotics for 5, 10, 20 or 40 min prior to freezing reduced the numbers of C. fetus in the semen to non-detectable levels in 38%, 69%, 88% and 100% of samples respectively. The incubated semen showed no significant reduction of sperm motility although fertility trials have not been done.     

On Deep Freezing of Boar Semen: Investigations on the Effects of Different Straw Volumes, Methods of Freezing and Thawing Extenders

Contents: At the Bundesanstalt Wels tests with frozen-thawed boar semen in plastic straws were conducted. The influences of straw volume, method of freezing and thawing extender were investigated. The straw volumes of 1, 0 and 0, 5 ml showed significantly better thawing results in a preliminary trial than the 5, 0 ml Macrotub. When semen was frozen in a computerized freezer with automatic seeding, all tested straw. volumes gave significantly better results than the straws frozen in static N₂-vapor. A ready to use commercial extender was used for thawing with good results to simplify the handling of deep frozen semen for on farm insemination. In the main trial 60 ejaculates from 10 boars were frozen in 1, 0 ml straws in the computerized freezer. Three in vitro parameters for fresh semen (motility, osmotic resistance and keeping quality under standard conditions) and two parameters for thawed semen (motility and percentage of sperm with normal apical ridge) were recorded and correlated. The osmotic resistance test proved to be well suited as means of predicting the fitness of an ejaculate for deep freezing, but the other two fresh semen parameters showed poor correlation with the parameters for thawed semen. From the parameters for thawed semen a freezing score was derived as a measure of the freezability of single ejaculates and boars. A preliminary insemination trial gave satisfying farrowing rates. Inhalt: Zur Tiefgefrierung von Ebersamen: Untersuchungen zum Einfluß von verschiedenen “Straw”-Volumina, Einfriewerfahren und Auftaubedingungen Ander Bundesanstalt Wels wurden Tiefgefrierversuche mit Eberspermain Kunststoffpailletten durchgeführt. Die jeweiligen Einflüsse von Paillettenvolumen, Gefrierverfahren und Auftauverdünner wurden untersucht. In Vorversuchen erwiesen sich die Paillettenvolumina 1, 00 und 0, 5 ml gegenüber den 5,0 ml Makrotüb hinsichtlich Kopfkappenintegrität und Auftaumotilität signifkant überlegen. Die Auftauergebnisse der im computergesteuerten Freezer mit automatischem Seeding eingefrorenen Proben waren für alle untersuchten Paillettengröβen signifikant bis hoch signifikant besser, als die Werte der im statischen Stickstoffdampf eingefrorenen Proben. Als Auftauverdünner wurde ein handelsüblicher Fertigverdünner gewählt um eine besonders für den Eigenbestandsbesamer wichtige einfache Handhabung der TG-Besamung zu gewährleisten. Im Hauptversuch wurden 60 Ejakulate von 10 Ebern verwendet und in 1, 0 ml Pailletten im programmierbaren Freezer eingefroren. Drei Frischsamenparameter (Motilität, osmotische Resistenz und Haltbarkeit unter Laborbedingungen) und zwei Auftausamenparameter (Motilität und Kopfkappenintegrität) wurden erhoben und korreliert. Dabei enwies sichder osmotische Resistemtest als für die Beurteilung von Frischsperma hinsichtlich TG-Eignung gut geeignet, während die beiden anderen Frischsamenparameter keine bzw. nur schwache Korrelation zu den Auftauergebnissen aufwiesen. Aus den Auftausamenparametern wurde eine “Einfrierbarkeitszahl” als Maβ für die Eignung des Spermas zur Tiefgefrierkonservierung der einzelnen Eber bzw. Ejakulate erstellt. Dabei enwies sich der osmotische Resistenztest als für die Beurteilung von Frischsperma hinsichtlich TG-Eignung gut geeignet. Bei einem orientierenden Besamungsversuch wurden zufriedenstellende Abferkelraten erzielt.     

Effects of liquid nitrogen vapor sensitization conditions on the quality of frozen-thawed dog spermatozoa

The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN2) vapor and the height from LN2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: -4 to -22 degrees C/min) and 10 cm/15 min groups (cooling rate: -6 to -10 degrees C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9 degrees C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN2 to reduce the temperature at a slow cooling rate of about -10 degrees C/min, followed by plunging into LN2 after sensitization for 10-15 min, provides good semen qualities after thawing.     

Cryopreservation of the semen collected by electroejaculation from the Hokkaido brown bear (Ursus arctos yesoensis)

Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4 degrees C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean +/- SD) of motile and live sperm were 96+/-2 and 86.5+/-7.2% before freezing, and 43+/-5 and 67.4+/-3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8+/-1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination.     

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration,freezing rate and thawing rate on post-thaw semen quality

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality.

2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN₂) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined.

3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found.

4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN₂ surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.     

Pregnancy rates of mares inseminated with semen cooled for 18 hours and then frozen

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.     

Preliminary Trial: Motility Comparisons of a Unique Freezing Technology (UFT) to Liquid Nitrogen Mist Methodology for Cryopreservation of Porcine Spermatozoa

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.     

提高奶牛细管冻精精子活力的试验研究

为了提高奶牛细管冻精精子的活力,试验探索了稀释液种类、最佳熏蒸距离、最佳冷冻温度、最佳熏蒸时间、不同解冻温度及时间对精子活力的影响。结果表明:精子在由柠檬酸钠和果糖组成的稀释液中存活时间长,在三羟甲基氨基甲烷稀释液中冷冻后精子活力高于其他稀释液;牛细管冻精最佳熏蒸距离为2.5 cm,时间影响不显著(5～10 min均可);用50℃温水解冻15 s的精子活力比其他解冻温度和时间时的精子活力要好。     

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post‐thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%–1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing–thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%–0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.