DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification.

Introduction and objective: Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species. Methods: Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope. Results and discussion: Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls. Conclusions: In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.

Cryopreservation of ferret (Mustela putorius furo) sperm collected by rectal massage and electroejaculation: Comparison of a decelerating and an accelerating freezing rate protocol.

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.

Seminal plasma amino acid profile in different breeds of chicken: Role of seminal plasma on sperm cryoresistance.

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

Effect of two cooling protocols on the post-thaw characteristics of Iberian ibex sperms.

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).

Sperm cell population dynamics in ram semen during the cryopreservation process.

BACKGROUND: Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. METHODOLOGY/PRINCIPAL FINDINGS: We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. CONCLUSIONS/SIGNIFICANCE: Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.

Valoración morfométrica de la cabeza del espermatozoide del cerdo doméstico según su edad / Morphometric evaluation of sperm head of domestic pig according their age

Se utilizó el Análisis Automatizado de la Morfometría Espermática (ASMA) con el fin de determinar las dimensiones de la cabeza del espermatozoide (DCE) en semen de cerdos domésticos según la edad, además de agrupar las medidas obtenidas en subpoblaciones espermáticas (SP). Se evaluaron 36 muestras de semen fresco y diluido de 20 cerdos los cuales se clasificaron en dos categorías. A: menores de 18 meses de edad y B: mayores de 18 meses de edad. Las DCE (Largo, µm/ Ancho, µm/ Área, µm 2 y Perímetro, µm) se analizaron en frotis teñidos con Hemacolor ® mediante Sperm-Class Analyser ® (SCA) y los valores obtenidos guardados en una base de datos. El procedimiento GLM fue utilizado para evaluar el efecto de la edad del cerdo sobre las DCE y el análisis de agrupamiento (FASTCLUS) para identificar las SP. Los espermatozoides provenientes de cerdos mayores de 18 meses de edad presentaron mayor longitud (8,84 vs. 8,95 µm) que los cerdos menores de 18 meses de edad, sin embargo, las medias correspondientes al ancho (4,44 vs. 4,32 µm), área (33,33 vs. 32,39µm 2) y perímetro (27,65 vs. 26,3 µm) fueron más pequeñas en los cerdos de mayor edad. Dos SP fueron obtenidas con el fin de ratificar las diferencias observadas entre las 2 categorías de edades evaluadas (P<0,001). La población que incluyó los espermatozoides con las mayores dimensiones disminuyó de 41,61 por ciento en cerdos menores de 18 meses a 20,78 por ciento en cerdos mayores de 18 meses. Contrariamente, la SP que contenía los espermatozoides de menor tamaño incrementó de un 58,39 por ciento en cerdos menores de 18 meses a 79,22 por ciento en cerdos mayores de 18 meses. En conclusión, la edad de los cerdos influye significativamente sobre las DCE. Los cerdos de mayor edad tienen 20 por ciento más de espermatozoides de menor tamaño que los cerdos más jóvenes.

Assisted Sperm Morphometry Analysis (ASMA) was used to determine the sperm head dimensions (DCE) of boar by age, and then the data set clustered in sperm subpopulations (SP). To this purpose were evaluated 36 fresh and diluted semen samples of 20 Dalland domestic pigs, which were classified in 2 categories: under 18 months old and over 18 months old. The DCE (Length, µm/ Width, µm/, Area, µm 2 / and Perimeter, µm) were analyzed in slides stained by Hemacolor ® by the Sperm-Class Analyser ® (SCA), and the mean measurements recorded. A GLM procedure was performed to evaluate the effects of boar age on sperm head dimensions and clustering analysis (FAST-CLUS procedure) to separate in SP. Spermatozoa collected from older boar (over 18 months old) had head length larger (8.84 vs. 8.95 µm) than younger boar (under 18 months old), however, the width (4.44 vs. 4.32 µm), area (33.33 vs. 32.39 µm 2) and perimeter (27.65 to 26.3 µm) were smaller in older boar than younger boar. Two SP were clustered in this trial toratify the differences between younger and older pigs. The mean values of each DCE among the SP were significantly dif-ferent (P<0.001). Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the largest decreased from 41.61 percent in pigs under 18 months old to 20.78 percent in pigs over 18 months old. Whereas, the percent of representation of the SP containing the smallest sper-matozoa increased from 58.39 percent in pigs under 18 months old to 153 79.22 percent in pigs over 18 months old. In conclusion, the age of sexually mature domestic male pig had a significant effect on the morphometric traits of their spermatozoa. Older boar had 20 percent more of smaller spermatozoa than younger boar.

Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa.

Fe(2)(+)/ascorbate, hydrogen peroxide (H(2)O(2)), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 microM Fe(2)(+) (hydroxyl radical generator); 1 mM, 100, and 10 microM H(2)O(2); and 100, 10, and 1 mU/ml XOD (superoxide and H(2)O(2) generator), incubated at 37 degrees C for 180 min. Intracellular reactive oxygen species (ROS; H(2)DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Deltapsi(m)) were considerably decreased by H(2)O(2) (1 mM and 100 microM) and XOD (100 and 10 mU/ml). Only 1 mM H(2)O(2) reduced viability. The antioxidant Trolox (10 microM) reduced intracellular ROS, but could not prevent the H(2)O(2) or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H(2)O(2) increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Deltapsi(m) loss, confirming that H(2)O(2) was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H(2)O(2) increased their proportion after 60 min. There were important differences between ROS generators, H(2)O(2) being the most cytotoxic. Although H(2)O(2) and XOD caused Deltapsi(m) dissipation, this was not reflected in increasing apoptotic markers.

Caracterización morfométrica de la cabeza del espermatozoide porcino mediante análisis computarizado (resultados preliminares) / Morphometry characterization of boar sperm head with computer assited analysis (preliminary results)

Para determinar los parámetros morfométricos de la cabeza espermática en semen porcino, así como evidenciar la presencia de subpoblaciones espermáticas fueron evaluadas 20 muestras seminales de 10 verracos Dalland. Sobre semen fresco y refrigerado fue evaluada la motilidad, vitalidad, acrosomas alterados y/o ausentes y anormalidades espermáticas. Mediante el análisis automatizado de la morfología espermática (ASMA), en frotis teñidos con Hemacolor®, se realizaron las mediciones de la cabeza espermática: Longitud (µm), Ancho (µm), Área (µm2), Perímetro (µm) y función Largo/Ancho. El efecto del proceso de refrigeración sobre las variables de calidad seminal y morfometría, se analizaron utilizando el GLM (SAS®) y para identificar las subpoblaciones espermáticas, se utilizó el procedimiento FASTCLUS (SAS®). La refrigeración a 16°C por 24 horas no afectó las características de calidad seminal de los eyaculados, pero si afectó las características morfométricas. La longitud de la cabeza disminuyó de 8,82 a 8,71 mm, así como el perímetro de 30,08 a 29,05 µm, mientras que aumentaron los valores de ancho (4,36 a 4,45 µm) y área (33,13 a 33,14 µm2). Se identificaron tres subpoblaciones espermáticas, con valores de distribución de 28,45 por ciento para la subpoblación 1 (espermatozoides grandes), 51,20 por ciento para la subpoblación 2 (medianos) y 20,35 por ciento para la subpoblación 3 (pequeños), las cuales se ven alteradas significativamente durante el proceso de refrigeración a 16°C.

To determine the morphometric parameters of the sperm head, and identify the presence of separate sperm subpopulations in boar semen were evaluated 20 ejaculate samples of 10 boars. Sperm motility, viability, acrosome integrity and morphological abnormalities were evaluated on fresh and cooling semen samples. By means Assisted Sperm Morphometry Analysis (ASMA), in slides stained by Hemacolor®, were determined the morphometric dimensions: Length (µm), Width (µm), Area (µm2), Perimeter (µm), and function Length/Width. Effect of cooling procedure on variables of semen quality and morphometric parameters were analyzed using GLM (SAS®). For identify the sperm subpopulations was used FASTCLUS procedure (SAS®). Cooling at 16°C for 24 hours did not affect the parameters of semen quality, but affected morphometric characteristics. Sperm head length decreased of 8.82 to 8.71 µm, and the sperm head perimeter of 30.08 to 29.05 µm, however, the width (from 4.36 to 4.45 mm) and area sperm head increased (33.13 to 33.14 µm2). Our results demonstrated that three separate sperm subpopulations coexist in boar ejaculates, 28.45% in the subpopulation 1 (larges), 51.20% in the subpopulation 2 (average), and 20.35% in the subpopulation 3 (small). These sperm subpopulation changed their distribution during cooling process.

Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants.

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.

Male fertility and sex ratio at birth in red deer.

Efforts to test sex ratio theory have focused mostly on females. However, when males possess traits that could enhance the reproductive success of sons, males would also benefit from the manipulation of the offspring sex ratio. We tested the prediction that more-fertile red deer males produce more sons. Our findings reveal that male fertility is positively related to the proportion of male offspring. We also show that there is a positive correlation between the percentage of morphologically normal spermatozoa (a main determinant of male fertility) and the proportion of male offspring. Thus, males may contribute significantly to biases in sex ratio at birth among mammals, creating the potential for conflicts of interest between males and females.

Influence of various permeating cryoprotectants on freezability of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of concentration and temperature of addition.

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of approximately 400 x 10(6) spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 degrees C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition -22 degrees C (ambient temperature) and 5 degrees C- on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22 degrees C (81.94 +/- 2.4% vs 72.38 +/- 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22 degrees C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22 degrees C, as an alternative to the more common 3%-4% of GLY and addition at 5 degrees C, in red deer semen freezing protocols.

Functional significance of the sperm head morphometric size and shape for determining freezability in iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples.

In the present study, computer-automated sperm head morphometry of epididymal samples was used to determine if sperm head area and shape are useful measurements for separating "good" and "bad" Iberian red deer freezers. A microscope slide was prepared from single diluted sperm fresh samples collected from 38 mature stags. Slides were air-dried and stained with Hemacolor. The sperm head area and shape (length/width) for a minimum of 145 sperm heads were determined for each male by means of the Sperm-Class Analyser. The remainder of each sample was frozen. After thawing, sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. All sperm parameters evaluated at thawing were placed in a statistical database and a multivariate cluster analysis performed. Mean sperm parameters of the 2 clusters generated ("bad" and "good" freezers) were compared by ANOVA. Our results show that sperm quality at thawing for all sperm parameters evaluated was significantly higher (P < .01) for "good" freezers than for the "bad" ones (sperm motility index: 67.4 +/- 2.0 vs 57.1 +/- 2.8; NAR: 67.1 +/- 2.5 vs 54.5 +/- 3.5; viability: 68.8 +/- 2.0 vs 60.1 +/- 2.8; HOST: 71.3 +/- 2.2 vs 63.1 +/- 3.1). Additionally, differences (P < .01) in epididymal sperm head area and shape were found between "good" and "bad" freezers before freezing, with the smallest overall sperm head dimensions found in the "good" freezers group (area: 32.04 microm2 vs 34.42 microm2). Thus, the lower the sperm head area in the fresh samples, the greater the sperm cryoresistance. Our results show that the 2 groups of males also differ in sperm head shape in fresh samples (good: 1.96 vs poor: 1.72; P < .01). It is possible that sperm head area and shape influence total sperm volume, thus causing differences in heat exchange as well as in movements of water, ions, and cryoprotectants and, in turn, on sperm freezability.

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality.

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa.

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C.

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.

Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure.

The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 degree celsius for 20 s vs. II = 70 degree celsius for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl-20 percent egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility. The thawing procedure had an effect on sperm motility percentage (P = 0.05), with the best overall recovery rates found with the use of protocol I (76.8 + or - 1.8 vs. 70.6 + or - 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P = 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 square micrometers vs. 30.32 square micrometers; width = 4.47 micrometers vs. 4.51 micrometers; length = 8.05 micrometers vs. 8.11 micrometers), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P = 0.001). In addition, differences were found within all stags for whole morphometric parameters (P = 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.