Relative content of Niemann-Pick C2 protein (NPC2) in seminal plasma, but not that of spermadhesin AQN-1, is related to boar sperm cryotolerance.

Variation between and within boar ejaculates in terms of their ability to withstand freeze-thawing is a limitation for sperm cryopreservation. Consequently, searching for freezability markers not only in sperm but also in seminal plasma (SP) is imperative. The present study aimed to evaluate the relationship between cholesterol content, relative levels of NPC2 and AQN-1 at two different holding times (0 h: HT0 and 24 h: HT24) at 17 °C, and boar sperm freezability. Forty-five ejaculates were cryopreserved and subsequently classified as of good (GFE) or poor (PFE) freezability according to their post-thaw sperm viability and total motility. Prior to cryopreservation, relative abundances of two SP proteins (NPC2 and AQN-1) and cholesterol content in sperm and SP were determined through immunoblotting and colorimetric methods, respectively. These determinations were made after ejaculation (HT0) and after 24 h of storage at 17 °C (HT24). Two bands for NPC2 protein (16 kDa and 19 kDa) were identified. Relative amounts of the 16 kDa-band were significantly (P < 0.05) higher in poor (PFE) than in good (GFE) freezability ejaculates both at HT0 and HT24, whereas those of the 19 kDa-band were significantly (P < 0.05) higher in PFE than in GFE at HT24 only. In the case of AQN-1, no significant differences between GFE and PFE were observed. In addition, no variations in the cholesterol content of sperm and SP were observed either between HT0 and HT24 or between GFE and PFE. We can conclude that the content of two NPC2 isoforms in SP, but not of that of spermadhesin AQN-1, may be involved in the sperm resilience to withstand freeze-thawing procedures and may predict ejaculate freezability. While a possible mechanism through which NPC2 during HT could affect boar sperm cryotolerance is suggested to be related to its ability to bind the plasma membrane cholesterol, further research is warranted.

A new test based on the hypotonic resistance and functional competence to evaluate the sperm quality, cryotolerance and in vitro fertilizing ability in pigs.

The present work aimed at setting a new test of boar sperm functional competence (SFCt) to determine the association with sperm in vitro fertilizing ability and cryotolerance. Three independent experiments were conducted. In the first experiment, a gage repeatability and reproducibility (R&R) study was carried out to determine the reliability of the SFCt. Following this, 1565 ejaculates were clustered on the basis of sperm membrane and acrosome integrity, total motility, morphology and membrane functional integrity. Two groups were obtained and their SFCt values were compared. In the second experiment, 45 ejaculates were classified into two groups based on cleavage rates after in vitro fertilization and the SFCt outcomes of the two groups were examined. In the third experiment, 45 ejaculates were cryopreserved and classified as with good (GFE) or poor freezability (PFE) based on their post-thaw sperm membrane integrity and total motility; the SFCt outcomes in liquid-stored semen were also compared. ROC curves were used to address the accuracy of the SFCt in each experiment. In the R&R study, the greater variability of the study was attributed to the differences between ejaculates (97.61%); SFCt values were significantly (P < 0.01) higher in the good than in the poor sperm quality group. Ejaculates with high cleavage rates showed significantly (P < 0.05) higher SFCt values than ejaculates with low cleavage rates. SFCt values of GFE before cryopreservation were significantly (P < 0.05) higher than those of PFE. The SFCt had a fair discriminatory value in all experiments. In conclusion, the SFCt is a useful and reliable test to evaluate the sperm quality and is also related to the sperm resilience to withstand freeze-thawing procedures. However, further studies evaluating blastocyst formation and AI trials with a large number of boars are needed to confirm the accuracy of this test.

Relationship among damaged chromatin, motility and viability in cryopreserved spermatozoa from Brahman bulls / Relación entre la alteración de la cromatina, movilidad y viabilidad en espermatozoides criopreservados de toros Brahman / Relação entre a alteração da cromatina, motilidade e viabilidade dos espermaotozoides criopreservados de touros Brahman

The aim of this study was to determine the percentage of sperm with damaged chromatic measure with toluidine blue stain and it´s relationship with motility and viability in criopreserverd semen from Brahman bulls. Three ejaculates from six Brahman bulls were used. Immediately after thawing, sperms were stained with toluidine blue to establish chromatin integrity (sperms with normal chromatin were light blue or green while sperms with damaged chromatin were dark blue or violet). Sperms were also stained with eosin-nigrosin to determine viability (live sperms were unstained while dead sperms were pink). Motility was measured under light microscope. Effects of bull, ejaculate, and the interaction between variables were assessed. The percentage of live sperms was 50.02 ( ± 14.13%). The mean motility was 33.88 (± 12.43%), while the percentage of sperms with damaged chromatin was 4.17 ( ± 2.96%). Viability was positively correlated with motility (r=0.77217, p=0.0002), and negatively correlated with damaged chromatin sperms (r= -0.43104, p=0.0087). Motility percentage was negatively correlated with the percentage of sperms with damaged chromatin (r=-0.48337, p=0.0421). In conclusion, cryopreserved semen of Brahman bulls presented a low level of chromatin damage, and this trait was negatively correlated with sperm motility and viability.

El objetivo del presente trabajo fue determinar el porcentaje de espermatozoides con cromatina dañada medida con la tinción de azul de toluidina, y su relación con la motilidad y la vitalidad del semen criopreservado de toros Brahma. Para ello, se utilizó semen de tres eyaculados de seis toros Brahman, el cual una vez descongelado se procedió a teñir con azul de toluidina para determinar la integridad de la cromatina (espermatozoides con cromatina normal teñidos de azul o verde claro; espermatozoides con cromatina anormal teñidos de azul oscuro o violeta), también se tiñeron con eosinanigrosina para determinar la viabilidad (espermatozoides vivos permanecen blancos; espermatozoides muertos se tiñen de rosado) y se estimó la motilidad espermática mediante microscopía óptica. Se evidenciaron las diferencias en todos los parámetros evaluados debidas al efecto toro y al eyaculado, así como a la interacción entre estas dos variables. El porcentaje de espermatozoides vivos fue de 50.02 ± 14.13% y la motilidad espermática promedió un 33.88 ± 12.43%, mientras que el porcentaje de espermatozoides con cromatina dañada fue de 4.17 ± 2.96%. El porcentaje de espermatozoides vivos se correlacionó positivamente con la motilidad (r=0.77217, p=0.0002), y negativamente con el porcentaje de espermatozoides con cromatina dañada (r= -0.43104, p=0.0087), mientras que el porcentaje de motilidad se correlacionó negativamente con el porcentaje de espermatozoides con cromatina anormal (r= -0.48337, p=0.0421). En conclusión, el semen criopreservado de toros Brahman presenta un bajo nivel de espermatozoides con daño en la cromatina, lo cual se correlaciona negativamente con la motilidad y la vitalidad espermática.

O objectivo deste estudo foi determinar a percentagem de espermatozóides com cromatina danificada, determinada pela coloração com azul de toluidina e sua relação com a viabilidade e a mobilidade do esperma cripreservado de touros Brahman. Para isso, foram utilizados três ejaculados de sêmen de seis touros Brahman, que uma vez descongelado foram coradas com azul de toluidina para determinar a integridade da cromatina (espermatozóides com cromatina normal coloream de azul ou verde; cromatina de espermatozóides con cromatina danificada, coloream de azul escuro ou violeta). Também foram corados com eosina nigrosina para determinar a viabilidade (espermatozóides vivos permanecem brancos e os mortos de cor rosa) e a motilidade espermática foi estimada por microscopia de luz. Foram encontradas diferenças significativas em todos os parâmetros, devido ao efeito de touro e o ejaculado, bem como a interacção entre essas duas variáveis. A percentagem de espermatozóides vivos foi de 50.02 ± 14.13% e motilidade espermática média de 33.88 ± 12.43%, enquanto a percentagem de espermatozóides com cromatina danificada foi de 4.17 ± 2.96%. A percentagem de espermatozóides vivos foi positivamente correlacionada com a motilidade (r=0.77217, p=0.0002) e negativamente com a porcentagem de espermatozóides com cromatina danificada (r = -0.43104, p= 0.0087), enquanto que a percentagem de motilidade correlacionou negativamente com a percentagem de espermatozóides com cromatina danificada (r = -0.48337, p=0.0421). Em conclusão, o sêmen de touros Brahman criopreservados tem um baixo nível de dano da cromatina, que está correlacionada negativamente com a motilidade e a vitalidade do esperma.

Efecto de la criopreservación sobre la integridad de la membrana plasmática y acrosomal de espermatozoides de toros / Effect of Cryopreservation on Integrity of Plasmatic and Acrosomal Membrane of Bulls Sperm

La integridad de la membrana plasmática (MP) y acrosomal (MA) han sido dos de los parámetros de valoración seminal más estudiados por su rol preponderante como límite celular y por ser responsable de hacer efectivas las interacciones entre células, tanto en términos de integridad morfológica, como funcional. El objetivo de este estudio fue determinar el efecto de la criopreservación sobre el porcentaje de espermatozoides vivos (VIT), acrosomas dañados (DAR), y la integridad estructural y funcional de la MP y MA de espermatozoides provenientes de 5 eyaculados frescos y 5 pajuelas descongeladas de 4 toros mediante frotis teñidos con eosina-nigrosina. La integridad funcional de estas membranas fue evaluada mediante los tests osmóticos (HOST y ORT). Los datos obtenidos fueron analizados con el procedimiento GLM (SAS®) y cuando se observaron diferencias se cuantificaron los efectos mediante el LSMEANS. Todos los valores de calidad espermática estudiados fueron afectados significativamente por la criopreservación (VIT, DAR, ORT y HOST). El proceso de congelación-descongelación causó un marcado efecto detrimental sobre la integridad estructural y funcional de la MP y MA de los espermatozoides evaluados (P<0,05). Sin embargo, se pudo demostrar como los espermatozoides resisten de manera diferente los efectos detrimentales de la criopreservación. Así mismo, el estudio confirma que el daño criogénico puede ocurrir indistintamente sobre la integridad estructural y funcional MP y MA, lo que afectaría la capacidad fecundante de las muestras seminales destinadas a Inseminación Artificial.

Plasmatic and acrosomal membrane integrity has been used as valuable information for determining sperm quality, because is important to know the morphological and functional role as cellular delimitation and in effective cell interactions. The aim of this study was to determine cryopreservation effects on sperm percentage of vitality, number of damaged acrosomes (DAR), the structural and functional sperm plasma membrane integrity in 5 fresh ejaculates and 5 thawed strauws of 5 bulls by using the eosin-nigrosin stain. The functional integrity of these membranes were evaluated by osmotic tests (HOST and ORT). The data was analysed by GLM procedure, and when differences were detected, LSMEANS was used to quantify the effects. Significant differences were found on seminal quiality parameters (VIT, DAR, ORT and HOST) between fresh and thawed sperm. Freezing-thawing procedure had detrimental effect on the integrity structural and functional of MP and MA in the spermatozoa evaluated (P<0.05). However, it was possible to demonstrate that the spermatozoa have different pattern of resistence to the detrimental effects of cryopreservation. Then, this study confirms that cryodamage could happen indistinctly on the structural and functional MP and MA integrity, which would affect the fertilizing capacity of the seminal samples destined for Artificial Insemination.

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.

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.

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

El objetivo de este estudio fue determinar el efecto de la criopreservación sobre las características morfométricas de las cabezas de espermatozoides de toros Brahman y sus mestizos. Cinco eyaculados fueron colectados de 4 toros y diluidos a 30°C en una solución de leche descremada-yema de huevo. Por cada muestra se hicieron dos frotis: uno del semen diluido, antes de su congelación en vapores de nitrógeno líquido, y otro de semen descongelado una semana después de la congelación. Todos los frotis fueron secados al aire y coloreados con Hemacolor®. Se analizaron las dimensiones de la cabeza espermática para un mínimo de 150 espermatozoides por muestra mediante el Sperm Class Analyser® (SCA). El procedimiento GLM se realizó para evaluar el efecto de la criopreservación sobre las dimensiones morfométricas de las cabezas espermáticas. Las cabezas espermáticas de los toros fueron significativamente (P<0,001) menores en los espermatozoides criopreservados que en las muestras frescas para la longitud (9,00 µm vs. 9,43 µm), el ancho (4,82 µm vs. 5,13 µm), el perímetro (32,46 µm vs. 33,69 µm) y el área (36,20 µm²vs. 39,97 µm²) para todos los toros. Así mismo, se encontraron diferencias (P<0,001) de todos los parámetros morfométricos de los toros evaluados, encontrándose dimensiones de cabeza menores en las muestras descongeladas. La variabilidad individual (CV) de las medidas de cabeza espermática de los toros osciló entre el 5,9 y el 10,2 por ciento para las muestras frescas y descongeladas, respectivamente. En conclusión, este estudio indica que el proceso de criopreservación de semen de toro afecta la morfometría, al reducir las dimensiones de la cabeza espermática de toros Brahman y sus cruces. Las diferencias entre los toros evaluados puede ser indicativo de diferencias individuales al proceso de criopreservación.

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 cryopreservation on sperm head morphometric dimensions. Bull sperm heads were significantly (P<0.001) smaller in frozen-thawed 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 µm2 vs. 39.97 µm2) 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.

Análisis de tres procedimientos estadísticos para la evaluación del crecimiento de mautas mestizas bajo diferentes regímenes nutricionales / Analysis of Three Statistical Procedures to Evaluate Growth in Crossbred Heifer Under Different Nutritional Levels.

Existen investigaciones donde se miden variables en varios períodos de tiempo sobre el mismo animal. Este tipo de información puede analizarse estadísticamente mediante tres opciones: Análisis univariados con la instrucción RANDOM del GLM; Análisis univariados o multivariados a través de transformaciones lineales mediante la instrucción REPEATED del GLM; y con modelos mixtos de covarianza con el procedimiento MIXED. Con el objetivo de evaluar estos tres métodos estadísticos y conocer cual es más preciso, se analizaron durante 7 meses los pesos corporales quincenales de una finca ubicada en el estado Táchira, Venezuela, (bosque húmedo tropical), donde 30 mautas mestizas con un peso y edad promedio de 176,9 ± 24,6 Kg y 17,22 ± 2,23 meses respectivamente, fueron distribuidas aleatoriamente dentro tres grupos de suplementación: (1) Control, (2) Alimento balanceado comercial, y (3) Harina de Gliricidia sepium con harina de maíz y melaza. Se obtuvieron estructuras de covarianzas, comparándose el procedimiento GLM con sus instrucciones RANDOM y REPEATED vs. el procedimiento MIXED en sus opciones CS, UN y AR1, todas del paquete estadístico SAS. Como variable respuesta se evaluó el peso de las mautas durante el período del ensayo y como variable independiente el grupo de suplementación, el período y la interacción lineal entre ambas. Así mismo, al realizar el análisis de la varianza utilizando la estructura de errores más indicada, se pudo corroborar que existe una interacción significativa entre el tratamiento y el período (P<0,01), es decir, que las curvas de crecimiento tienden a no ser paralelas. Los resultados indican que el análisis más ajustado es el Procedimiento MIXED con la opción AR1, ya que permite ajustar la matriz de covarianza.

There are investigations where variables are measured in periods of time on the same animal. This type of information should be analyzed statistically trough three ways: univariate analyses with the RANDOM statement of the GLM procedure; univariate or multivariate analysis with the method of lineal transformations with the REPEATED statement of the GLM; and with mixed models of covariance with the MIXED procedure. With the objective of evaluating these three statistical methods and to know the most precise, biweekly live weight coming from a rehearsal carried out located in the Tachira State, Venezuela (topical damp woods) was analyzed during 7 weeks, where 30 crossbred heifers with an average weight and age of 176.9 ± 24.6 Kg and 17.22 ± 2.23 months respectively, were randomly distributed between three groups: (1) control, (2) balanced commercial feed, and (3) Flour of Gliricidia sepium with flour of corn and molasses. It was modeled covariance structures, comparing the GLM procedure with its RANDOM and REPEATED statements vs. the MIXED procedure in its CS, UN and AR1 options, of the statistical package SAS. As dependent variable it was studied the weight of the heifers during the assay period and as independent variables the supplementation group, the period and the linear interaction among both. When carrying out the analysis of variance using the most suitable structure of errors, it can be conclude that there was a significant interaction between treatment and period (P<0.01), and that is to say that the curves of growth spread unless parallel. Results indicate that the best fitting analysis is the Proc MIXED with the AR1 option, since it allows to adjust the covariance womb.

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.

Natural Mediterranean photoperiod does not affect the main parameters of boar-semen quality analysis.

Photoperiod is an important factor in the modulation of male reproduction in mammals. In boars, however, it is a controversial factor. The main aim of this work is to determine the precise effect of the natural, Mediterranean photoperiod on boar-semen quality. To do this, boars were housekept in strictly controlled temperature and humidity conditions, whereas light periods were also strictly adjusted to obtain a light-cycle in the farm. The work was performed over a period of one year, thus allowing for the determination of the putative yearly oscillations of boar-semen quality. Variations of the natural Mediterranean photoperiod do not induce substantial changes in overall semen-quality parameters like the percentages of viability, morphological abnormalities and total motility, the response to the osmotic resistance test and sperm motion characteristics. Only the motile-sperm subpopulation structure was significantly (P<0.05) changed depending on the variations of the natural photoperiod. Furthermore, the boar-semen ability for storage at 15-17 degrees C in a commercial extender was not modified by photoperiod changes. Our results indicate that the natural Mediterranean photoperiod does not induce strong changes in boar-semen characteristics, probably due to boar sperm having a strong capability of adaptation to the light variations of this photoperiod.

Prediction of the day of ovulation in mares through physiological parameters measured during estrous

Con el fin de predecir el día de ovulación, fueron estudiados 120 períodos preovulatorios en 80 yeguas. Para tal fin, analizaron parámetros como el comportamiento sexual, tamaño del folículo, textura del folículo, apariencia ecográfica del útero. Entre el cuarto y segundo día previo a la ovulación, el tamaño folicular incrementó, la textura folicular a la palpación presentó un ablandamiento folicular progresivo y la apariencia ecográfica demostró una triangulización del folículo. Los otros parámetros estudiados no presentaron variación estadísticamente significativa, sin embargo, algunos obtuvieron propensión numérica favorable. El procedimiento Stepwise seleccionó la textura folicular a la palpación y el tamaño folicular como las variables que presentan mayor significancia estadística y que predicen la probabilidad de ovulación en los días que la preceden. La función logit con sus 3 interceptos estimados (-4,35; -3,06 y -1,59; 1, 2 y 3 días para la ovulación) y las pendientes, 0,495 para tamaño folicular y 1,05 para la textura del folículo, permite calcular las probabilidades acumuladas de ovulación en los días posteriores. Si encontramos un folículo muy blando y mayor de 45 mm de diámetro se obtiene un 79 por ciento de probabilidad de que la yegua ovule en 24 horas. Sin embargo, varias combinaciones de tamaño folicular y textura del folículo ofrecen probabilidades mayores del 80 por ciento de que la yegua ovule dentro de las próximas 48 horas

Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis.

A precise estimation of the fertilizing ability of a boar ejaculate would be very useful to improve pig assisted reproduction results. For this purpose, we tested the mathematical combination of several parameters of the boar semen quality analysis, including the computer-assisted semen motility analysis (CASA), as a predictive fertility tool. The utilized mathematical relations among parameters were logistic and linear regressions. Two mathematical models obtained by logistic regression involving Osmotic Resistance Test (ORT Test), Hyperosmotic Resistance Test (HRT Test) and viability of fresh samples, showed a significant (P<0.05) correlation between semen characteristics and conception rate. However, none of the obtained models produced a significant correlation model between semen characteristics and prolificacy. The CASA analyses show that three separate subpopulations of spermatozoa with different motility characteristics coexist in boar ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among boars, but no clear relationship between motile subpopulation structure and fertility was obtained. Our results support the belief that the predictive use of the results obtained in a standard boar semen quality analysis can reasonably be achieved by applying logistic correlation analyses among several function parameters of boar semen quality analysis and in vivo conception rates obtained after artificial insemination (AI).

Uso de la gliricidia sepium en la suplementación de mautas mestizas y su efecto sobre el crecimiento y la aparición de la pubertad / Use of gliricidia sepium in feed supplementation of crossbred heifers and its effect on growth and the onset of puberty

Con el objetivo de determinar el efecto del uso de la leguminosa forrajera Gliricidia sepium (GS) en la suplementación de mautas mestizas sobre el crecimiento e inicio de la pubertad, se realizó un ensayo durante 10 meses, en una finca comercial ubicada en el sector "Río Grande", Municipio Panamericano, Estado Táchira, Venezuela. Treinta mautas mestizas con peso y edad promedio de 176,9 ± 24,6 kg y 17,22 ± 2,23 meses respectivamente, alimentadas a base de pastoreo en potreros de tanner (Brachiaria arrecta), fueron distribuidas aleatoriamente dentro de 3 grupos de suplementación: (T1) sin suplementación, (T2) alimento balanceado comercial y (T3) 50 por ciento de harina de maíz, 30 por ciento de harina de hojas de GS y 20 por ciento de melaza. Se registraron los pesos corporales cada 15 días y se tomaron muestras semanales de sangre para la determinación de progesterona en suero a través de radioinmunoanálisis. Los datos fueron analizados mediante un análisis de varianza-covarianza del sistema de análisis estadístico (SAS). Las variables evaluadas fueron peso corporal a la pubertad (PP), ganancia diaria de peso corporal (GDP) y edad a la pubertad (EP). Los resultados muestran que la suplementación en los grupos T2 y T3 hace que duperen significativamente (P<0,05) al grupo T1 en cuanto a la GDP (570 y 556 vs 457 g/día) y EP (670 y 720 vs 783 días). El PP no arrojó diferencias significativas. la suplementción con los tratamientos 2 y 3 logra reducir la edad de la aparición de la pubertad en hembras mestizas a pastoreo en condiciones tropicales; por lo tanto, la suplementación con GS mejora el rendimiento animal