Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS®

When collection of ejaculated sperm samples is not possible, as is the case with wild species, the epididymides of sacrificed wild males become the only possible source of spermatozoa. Mature cauda epididymal spermatozoa display characteristics similar to those of ejaculated sperm cells. The present work proposes a sperm staining technique suitable for the morphometric evaluation of red deer epididymal sperm using a new computerized system. Epididymides from wild animals were extracted no later than 2 h post mortem. After epididymal sectioning, sperm samples were collected, cooled to and equilibrated at 5 °C, and frozen in liquid nitrogen. Before staining, sperm samples were thawed for 20 s at 37 °C, and used for the preparation of slides. Three different sperm stains were tested: Hemacolor, Diff-Quik, and Harris’ Hematoxylin. Morphometric analyses of sperm samples were performed using the morphologic module of the ISAS®. Two hundred spermatozoa per sample and stain were captured at random and analyzed. Sperm morphometric values were significantly affected by the staining technique used. Moreover, significant differences were observed between animals. In our study, Diff-Quik could be considered to be the best sperm staining method, as it provided the highest percentage of well automatically analyzed cells by the ISAS®, and discriminates better between animals. This sperm staining technique also proved to be a useful method for characterizing and discriminating between sperm samples of different animals.This study was partially supported by the Ministerio de Ciencia y Tecnología, INIA (RZ01-008).Peer reviewe

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

Introduction and objectiveCryopreservation 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.MethodsTestes 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 discussionOur 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.ConclusionsIn conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.This work was supported by the Education and Science Council (PBI-05-011), by the Spanish Ministry of Education and Science (RZ2006-00006-C3), and by the Agriculture Council (PREG-05-004) of Junta de Comunidades de Castilla-La Mancha (JCCM). Olga García-Álvarez and Alejandro Maroto Morales were recipients of scholarships from INIA and JCCM, respectively. Felipe Martínez-Pastor, María Rocío Fernández-Santos, and Milagros C. Esteso were supported by the Juan de la Cierva program from the Spanish Ministry of Education and Science.Peer reviewe

Use of native chicken breeds (Gallus gallus domesticus) for the development of suitable methods of Cantabrian capercaillie (Tetrao urogallus cantabricus) semen cryopreservation

8 Pág.The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals.Fundación Biodiversidad-Ministerio para la Transición Ecológica y el Reto Demográfico (Ref-DOC-2017458); Zoitechlab S.L.-INIA, Grant/Award Number: CON18-141Peer reviewe

Head dimensions of brahman and their crossbred bull spermatozoa are affected by cryopreservation

The objective of this study was to determine the effect of cryopreservation on morphometrics characteristic of Brahman and their crossbred bull sperm heads. Five ejaculates were collected from 4 bulls and diluted at 30°C in a skim milk-egg yolk extender. Two microscope slides were prepared from single extended sperm samples prior to freezing in nitrogen vapors, and another one after thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor®. Sperm-head dimensions for a minimum of 150 sperm heads/samples were analysed from each sample by means of the Sperm-Class Analyser ® (SCA), and the mean measurements recorded. A GLM procedure was performed to evaluate the effect of ryopreservation on sperm head morphometric dimensions. Bull sperm heads were significantly (P<0.001) smaller in frozenthawed spermatozoa than in the extended samples for length (9.00 μm vs. 9.43 μm), width (4.82 μm vs. 5.13 μm), perimeter (32.46 μm vs. 33.69 μm) and area (36.20 μm� vs. 39.97 μm�) for all bulls. Also, differences (P<0.001) were found within all bulls for whole morphometric parameters. The individual variability of sperm head measurements across all bulls ranged from 5.9% to 10.2% for fresh and thawed samples, respectively. In conclusion, the present study indicate that cryopreservation of bull semen did affect the morphometry to reduce the dimensions of Brahman and crossbred bull sperm heads. The differences among bulls may be indicative of the individual bull resistance to the cryopreservation process

Effectiveness of ultra-rapid cryopreservation of sperm from endangered species, examined by morphometric means

8 Pág.This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ± 0.049 μm2) and dama gazelle sperm heads the largest (43.746 ± 0.291 μm2), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ± 0.150 μm2) and mouflon sperm heads the largest (38.258 ± 0.104 μm2). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing.This work was funded by MINECO/AEI/FEDER, UE, Spain grants AGL2014-52081-R and AGL2017-85753-R, and by the Fundación Parques Reunidos - INIA, Spain agreement CC16-001. The authors thank DG Biodiversidad (Principado de Asturias), and the Consejería de Medio Ambiente (Junta de Andalucía), for their unfailing help in the provision of animals from the mentioned wildlife reserves and in implementing the projects proposed. The authors thank the Ayuntamiento de Sedella (Málaga) for its help in the provision of a field laboratory close to the Tejeda and Almijara mountains. Thanks are also due to the Parque Zoólogico del Ayuntamiento de Córdoba, the Parque Zoológico del Ayuntamiento de Guadalajara and the Zoo-Aquarium, Madrid, for their help in the execution of this project.Peer reviewe

Evaluación objetiva de la morfometría de los espermatozides de ciervo. Relaciones con congelabilidad y calidad del semen

103 páginas.-- Tesis inédita de la Universidad de Castilla-La Mancha (UCLM) e Instituto de Investigación en Recursos Cinegéticos (IREC), Unidad de Producciones cinegéticas.-- Fecha de lectura: 14-12-2006.En esta Tesis Doctoral se plantea la identificación de subpoblaciones espermáticas según su morfometría, su evolución y su distribución tras el proceso de criopreservación en muestras epididimarias de ciervo Ibérico. Esto se lleva a cabo a través de los siguientes objetivos particulares:Determinar los efectos de la criopreservación sobre la morfometría de la cabeza de los espermatozoides epididimarios de ciervo Ibérico, utilizando un sistema ASMA.Evaluar la criosupervivencia espermática analizando la motilidad individual y la integridad de membranas tras la descongelación a partir de muestras recogidas de epidídimo de ciervo Ibérico.Determinar si el área y el perfil de la cabeza espermática son medidas útiles para predecir la congelabilidad de las muestras espermáticas epididimarias de ciervo Ibérico.Identificar subpoblaciones espermáticas morfométricamente diferentes, analizar su comportamiento tras el proceso de criopreservación y su relación con la congelabilidad en muestras epididimarias de ciervo Ibérico.Evaluar los efectos de dos protocolos diferentes de descongelación sobre el tamaño de la cabeza de los espermatozoides epididimarios de ciervo Ibérico y su distribución en distintas subpoblaciones espermáticas en base a las referidas dimensiones de la cabeza espermática.Esta tesis ha sido realizada en el laboratorio de Biología de la Reproducción de la Universidad de Castilla-La Mancha. Los diversos experimentos que forman parte de la misma han sido parcialmente financiados por el proyecto de la Consejería de Educación y Ciencia de Castilla-La Mancha: "Evaluación objetiva de la morfometría de los espermatozoides de ciervo. Relaciones con otras pruebas de evaluación seminal, congelabilidad y fertilidad de la inseminación artificial".Peer reviewe

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5°C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 °C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P > 0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin–eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 °C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.This study was sponsored by Grant AGL2000-0671 from the Spanish Ministry of Science and Technology. Ana J. Soler and M.R. Fernández-Santos were recipients of scholarships from the Consejería de Ciencia y Tecnología de la Junta de Comunidades de Castilla-La Mancha (Spain).Peer reviewe

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 − 22ºC (ambient temperature) and 5ºC − 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 ~ EG ~ 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 22ºC (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.This work was sponsored by Spanish Ministry of Science and Technology (AGL2000-0671).Peer reviewe