Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep[TM], or Cushion Fluid[TM]. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep™, or Cushion Fluid™. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥ 90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

Somatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.

Onset of puberty was observed in 12 bison bulls by the collection of semen at monthly intervals for 12 months beginning at 13 months of age. Onset of puberty was defined as the time in development when an ejaculate contained a minimum of 50x106 sperm showing at least 10% progressive motility. At each collection, data were recorded on body weight, semen quality, fecal testosterone concentration, and physical development. Semen was evaluated for gross motility, individual progressive motility, sperm morphology, sperm concentration and volume. From these data bison bulls attained onset of puberty at an average age of 16.5 months and an average body weight of 353 ± 52.8 kg. Age was the greatest determining factor for onset of puberty in this group of bulls. <p> Monthly abattoir collections of epididymal sperm (n=288) and testicular tissue (n=120) were evaluated to determine if bison bulls undergo seasonal changes in sperm production. Although epididymal sperm morphology did not give any indication of seasonal variation, the histological study of testicular tissue showed greater seminiferous tubule diameter (27.0 ± 4.3 ìm) during the breeding months (July, August and September) than during any other seasons. Semen collected at 4 different occasions during the year (June, November, January, and April) from live mature breeding bulls (n=21) was used to verify data collected from abattoir samples. Semen from mature bulls showed a significantly greater proportion of normal sperm in June than in November (73.8 ± 9.1%; 44.1 ± 24.3%), respectively. There was little improvement in sperm morphology at the January sampling but in April morphology improved to a level close to that observed in June. Fecal testosterone concentrations were highest in June (128.6 ± 67.4 ng/g) and lowest in April (48.5 ± 33.3 ng/g). Although there was no clear seasonal trend in sperm morphology from bulls sampled at the abattoir, mature bulls showed slight seasonal variations in semen quality.

El primer objectiu d'aquest estudi va ser determinar la presència de subpoblacions espermàtiques amb pautes específiques de motilitat en semen de cavall, porc i conill. Es va utilitzat per aquest fi l'anàlisi computaritzat de la motilitat espermàtica (CASA). L'optimització de les variables que millor expliquen el moviment espermàtic es va realitzar mitjançant una anàlisi d'agrupament de variables basat en l'estudi de la seva matriu de covariança. La investigació va demostrar que tres subpoblacions espermàtiques en semen de porc i quatre en semen de cavall i conill coexisteixen en els ejaculats. Es varen trobar diferències significatives (P <0.01) en la distribució d'aquestes subpoblacions en totes les espècies, sobretot en cavalls i conills. No obstant, no va existir una relació clara entre les subpoblacions espermàtiques i la fertilitat "in vivo" del semen. P'altra banda, l'estimació precisa sobre la capacitat fecundant de l'ejaculat de mamífers seria molt útil per l'optimizació de les tècniques de reproducció assistida. Amb aquest propòsit, es va plantejar el segon objectiu d'aquest estudi, que va ser determinar la possibilitat d'utilitzar la combinació matemàtica de varis paràmetres de qualitat seminal en semen de porc i conill, incloent el CASA. En porc, dos models matemàtics obtinguts per regressió logística varen seleccionar el test de resistència osmòtica, el test de resistència hiperosmòtica i la viabilitat espermàtica com els paràmetres que millor prediuen la taxa de concepció (P<0.05). No obstant, cap dels models fets per regressió lineal es va relacionar amb la prolificitat. En conill, les regressions logística i lineal van proporcionar dos models matemàtics significatius (P<0.05) que van seleccionar la viabilitat i les anormalitats espermàtiques amb els paràmetres de major predicció de la fertilitat i la prolificitat. En cavalls, els ejaculats amb almenys una fertilitat confirmada van presentar espermatozoides amb gran linealitat i progressivitat. A més, la totalitat de les mostres fèrtils presentaven un número total d'espermatozoides per ejaculat superior a 20 x 109. Les nostres observacions recolzen l'opinió de que la utilització predictiva dels resultats obtinguts en l'anàlisi de semen d'aquests mamífers pot aconseguir-se de forma raonable mitjançant l'aplicació dels anàlisis de regressió que permetin relacionar tots els paràmetres de qualitat avaluats en cada espècie. Així, la metodologia utilitzada explica sistemàticament la qualitat del semen de mamífers, a més de relacionar-la amb la taxa de concepció obtinguda després de la inseminació artificial i la prolificitat en els mamífers avaluats.Paraules claus: Anàlisi seminal en mamífers, Subpoblacions espermàtiques, Taxa de concepció, Tamany de la camada, Proves funcionals. / El primer objetivo de este estudio fue determinar la presencia de subpoblaciones espermáticas con pautas específicas de motilidad en semen de caballo, cerdo y conejo. Se utilizó para este fin el análisis computarizado de la motilidad espermática (CASA). La optimización de las variables que mejor explican el movimiento espermático se realizó mediante un análisis de agrupamiento de variables basado en el estudio de su matriz de covarianza. La investigación demostró que tres subpoblaciones espermáticas en semen de cerdos y cuatro en semen de caballo y conejo coexisten en los eyaculados. Se encontraron diferencias significativas (P<0.01) en la distribución de estas subpoblaciones en todas las especies, sobretodo en caballos y en conejos. Sin embargo, no existió una relación clara entre las subpoblaciones espermáticas y la fertilidad "in vivo" del semen. Por otra parte, la estimación precisa de la capacidad fecundante del eyaculado de mamíferos sería muy útil para la optimización de las técnicas de reproducción asistida. Con este propósito, se planteó el segundo objetivo de este estudio, que fue determinar la posibilidad de usar la combinación matemática de varios parámetros de calidad seminal en semen de cerdo y conejo, incluyendo el CASA. En cerdo, dos modelos matemáticos obtenidos por regresión logística seleccionaron el test de resistencia osmótica, el test de resistencia hiperosmótica y la viabilidad espermática como los parámetros que mejor predicen la tasa de concepción (P<0.05). Sin embargo, ninguno de los modelos hechos por regresión lineal se relacionó con la prolificidad. En conejo, las regresiones logística y lineal proporcionaron dos modelos matemáticos significativos (P<0.05) que seleccionaron la viabilidad y las anormalidades espermáticas como los parámetros de mayor predicción de la fertilidad y la prolificidad. En caballos, los eyaculados con al menos una fertilidad confirmada presentaron espermatozoides con gran linealidad y progresividad. Además, la totalidad de las muestras fértiles presentaban un número total de espermatozoides por eyaculado superior a 20 x109. Nuestras observaciones respaldan la opinión de que la utilización predictiva de los resultados obtenidos en el análisis de semen de estos mamíferos puede conseguirse en forma razonable mediante la aplicación de análisis de regresión que permitan relacionar todos los parámetros de calidad seminal evaluados en cada especie. Así, la metodología empleada explica sistemáticamente la calidad del semen de mamíferos, además de relacionarla con la tasa de concepción obtenida después de la inseminación artificial y la prolificidad en los mamíferos evaluados. Palabras claves: Análisis seminal en mamíferos, Subpoblaciones espermáticas, Tasa de concepción, Tamaño de la camada, Pruebas funcionales. / The first aim of this study was to test the presence of separate sperm subpopulations with specific motility characteristics in stallion, boar and rabbit ejaculates by using a computer-assisted semen analysis system (CASA). Sperm motility descriptors were analyzed thorough the clustering of variables based on a covariance matrix. This matrix selects the descriptors of sperm motility that better explain the spermatozoon kinetics. Sperm subpopulations were obtained by disjointing cluster analysis where the observations are divided into clusters by which each observation belongs to one specific cluster. This test showed that three separate sperm subpopulations with different motility characteristics in boar, and four in stallion and rabbit, coexist in the ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among individuals in all of the studied species, but no clear relationship between motile subpopulation structure and fertility was obtained. A second aim of the study was to test the possibility for a precise estimation of the fertilizing ability of mammalian ejaculate based upon the results of semen analysis. For this purpose, we tested the mathematical combination of several parameters of the boar and rabbit semen quality analysis as predictive "in vivo" fertility tools The main mathematical relations utilized among parameters were logistic and linear regressions. In boar, two mathematical models obtained by logistic regression involving osmotic resistance test, hyperosmotic resistance test and viability of fresh samples, showed a significant (P<0.05) relationships between semen characteristics and conception rate. However, none of the obtained models produced a significant relation between semen characteristics and litter size. In rabbits, logistic and linear regression analysis rendered two mathematic, significant (P<0.05) models, with related some semen characteristic (sperm viability and abnormalities) with "in vivo" fertility and litter size. In stallion, the study of subpopulations in ejaculates which showed confirmed fertilizing capacity showed that these ejaculates had the majority of their motile spermatozoa included in a subpopulation with high progressiveness and low linear velocity. Moreover, all the ejaculates with proven fertility which have a total sperm count &#8805;20x109 spermatozoa/ ejaculate showed all of their motile sperm included in this subpopulation. Our results support that the use of the values obtained in a standard boar, rabbit and stallion semen quality analysis to predict life fertilizing ability of a single ejaculate can reasonably be achieved by applying logistic and linear regression analyses to the parameters included in this analysis. Thus, our methodology can explain in a systematic manner mammal semen quality, relating it to conception rate and litter size.Key words: Mammalian semen analysis, Sperm subpopulations, Conception rate, Litter size, Functionality tests.

En este trabajo se presenta el estudio clínico de 335 hombres seropositivos al VIH-1 que acuden a un centro de reproducción con deseos de tener hijos. Se estudia también cómo influyen los diferentes par metros cl'nicos e inmunológicos relacionados con el SIDA en la calidad seminal de estos hombres. Se describe la técnica de lavado seminal utilizada para eliminar el virus del semen y aislar la fracción de espermatozoides móviles libres de virus para su utilización en TRA. Tras los lavados se aplica la técnica de PCR para detectar la presencia del VIH-1. Sólo las muestras de semen lavadas que han sido negativas al virus se utilizan clínicamente para la aplicación de TRA.Estudio del hombre seropositivo al VIH-1Las vías de contagio fueron: uso de drogas inyectadas, 233 (69,5%); sexual, 69 (20,6%); sangre o hemoderivados contaminados, 23 (6,9%), otras, 10 (3%). La mayoría (63%) eran seropositivos pero no afectos de SIDA. El 80,9% estaban bajo tratamiento antirretroviral. Se realizaron controles de carga viral el 66,9% de los pacientes, de ellos, el 58,5% mostró niveles indetectables de copias de RNA.Calidad seminal del hombre seropositivo al VIH-1Los pacientes seropositivos al VIH-1 presentaron una disminución significativa del volumen seminal (P=0,04), del porcentaje de movilidad traslativa (P<0,001) y del porcentaje de espermatozoides normales (P<0,001) respecto a un grupo de candidatos a donantes de semen. Se observó una disminución significativa del volumen seminal (P=0,04) y del porcentaje de espermatozoides normales (P=0,03) en el grupo de pacientes que habóan recibido terapia antirretroviral respecto a los que no.Lavado seminal y detección del VIH-1 por PCRLa técnica de doble lavado empleada en este estudio presenta una baja tasa de recuperabilidad espermática (6,8%). El 96,4% de las muestras son negativas al VIH-1 tras los lavados. Técnicas de inseminación artificial (IA) y fecundación in vitro con microinyección (FIV-ICSI)La IA consigue altas tasas de gestación por ciclo (26,3%) y reduce la probabilidad de contagio de la pareja respecto a la práctica de coito no protegido. Aplicando la técnica de IA, los hombres en un estadio más avanzado de la enfermedad (grupos A3, B3, C1, C2 y C3 del CDC) presentan una probabilidad menor de gestación estadísticamente significativa respecto a los pacientes de los grupos A1, A2, B1 y B2 (30,2% vs 18,5, P <0,01). La técnica de FIV-ICSI con semen lavado, congelado y testado es una técnica útil que permite solucionar los problemas de esterilidad masculina a los pacientes seropositivos al VIH-1; puede aplicarse como recurso en los pacientes en los que la IA haya fallado y a mujeres que presenten obstrucción tubárica. Las tasa de embarazo aplicando FIV-ICSI en estos pacientes es alta (49,2%/ciclo). / This paper presents a clinical study of 335 HIV-1-seropositive men who visited the reproduction centre because they wanted to father children. The effect of the different AIDS-related clinical and immunological parameters on the menÕs semen quality was also studied. We describe the semen-washing technique used to eliminate the virus from the semen and isolate the fraction of motile spermatozoa free of virus for use in assisted-reproduction techniques. After washing, the PCR technique was applied to detect the presence of HIV-1. Only those samples of washed semen that tested negative for the virus were used clinically for the application of assisted-reproduction techniques. Study of HIV-1-seropositive menThe routes of infection were: injection-drug use, 233 (69.5%); sexual, 69 (20.6%); contaminated blood or blood products, 23 (6.9%); other, 10 (3%). Most of the men (63%) were seropositive but not affected with AIDS. 80.9% were following antiretroviral treatment. The viral load was measured in 66.9% of the patients, 58.5% of whom showed undetectable levels of RNA copies. Semen quality of HIV-1-seropositive menHIV-1-seropositive patients showed a significant reduction in semen volume (P=0.04), percentage of translative motility (P<0.001) and percentage of normal spermatozoa (P<0.001) compared to a group of semen-donor candidates. A significant reduction in semen volume (P=0.04) and percentage of normal spermatozoa (P=0.03) was observed in the group of patients that had received antiretroviral therapy compared to those who had not. Semen washing and detection of HIV-1 using PCRThe double-washing technique used in this study presented a low rate of sperm retrieval (6.8%). 96.4% of the samples tested HIV-1 negative after washing. Artificial-insemination techniques (AI) and in vitro fertilisation by microinjection (IVF-ICSI)High pregnancy rates per cycle (26.3%) were achieved with AI and the probability of infecting the partner was reduced in relation to the practice of unprotected intercourse. When AI techniques were applied, the probability of the men in the more advanced stages of the disease (groups A3, B3, C1, C2 and C3 of the CDC classification) impregnating their partners was significantly lower compared to patients in the A1, A2, B1 and B2 groups (30.2% vs. 18.5, P<0.01). The IVF-ICSI technique using washed, frozen and tested semen is a useful technique that helps solve problems of sterility in male HIV-1-seropositive patients. It can also be applied in patients that do not respond successfully to AI and to women with tubular obstructions. The pregnancy rate after applying IVF-ICSI in these patients is high (49.2%/cycle).

Onset of puberty was observed in 12 bison bulls by the collection of semen at monthly intervals for 12 months beginning at 13 months of age. Onset of puberty was defined as the time in development when an ejaculate contained a minimum of 50x106 sperm showing at least 10% progressive motility. At each collection, data were recorded on body weight, semen quality, fecal testosterone concentration, and physical development. Semen was evaluated for gross motility, individual progressive motility, sperm morphology, sperm concentration and volume. From these data bison bulls attained onset of puberty at an average age of 16.5 months and an average body weight of 353 ± 52.8 kg. Age was the greatest determining factor for onset of puberty in this group of bulls. <p> Monthly abattoir collections of epididymal sperm (n=288) and testicular tissue (n=120) were evaluated to determine if bison bulls undergo seasonal changes in sperm production. Although epididymal sperm morphology did not give any indication of seasonal variation, the histological study of testicular tissue showed greater seminiferous tubule diameter (27.0 ± 4.3 ìm) during the breeding months (July, August and September) than during any other seasons. Semen collected at 4 different occasions during the year (June, November, January, and April) from live mature breeding bulls (n=21) was used to verify data collected from abattoir samples. Semen from mature bulls showed a significantly greater proportion of normal sperm in June than in November (73.8 ± 9.1%; 44.1 ± 24.3%), respectively. There was little improvement in sperm morphology at the January sampling but in April morphology improved to a level close to that observed in June. Fecal testosterone concentrations were highest in June (128.6 ± 67.4 ng/g) and lowest in April (48.5 ± 33.3 ng/g). Although there was no clear seasonal trend in sperm morphology from bulls sampled at the abattoir, mature bulls showed slight seasonal variations in semen quality.

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep™, or Cushion Fluid™. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥ 90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

Somatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.

Density gradient centrifugation of sperm is a common assisted-reproduction procedure in humans used to improve semen quality. The technique allows sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, leading to their retention above centrifuged density gradients, with structurally normal sperm passing through the gradient. Three experiments were conducted to evaluate the effects of tube size, sperm number following centrifugation, and density gradient volume (height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, one-mL aliquots were layered over EquiPure? Bottom Layer (1-Layer) or over-tiered EquiPure? Top and Bottom Layers (2-Layer). For Experiment 2, one-mL aliquots were layered over three different heights of EquiPure? Bottom Layer in 15-mL or 50-mL conical-bottom tubes. For Experiment 3, four different aliquots containing a sperm load of 1-4x were layered over a constant volume of EquiPure? Bottom Layer in 15-mL or 50-mL conical bottom tubes. The tubes were then centrifuged. Resulting sperm pellets were evaluated for morphologic quality, DNA integrity, motility and recovery rate. Sperm-EquiPure? centrifugation yielded improvements in motility, morphology and DNA integrity parameters (P<0.05), as compared to controls. The 1-Layer method resulted in a higher recovery rate than the 2-Layer method (P<0.05). Sperm processed in the 15-mL tubes yielded higher velocity and higher recovery rates than sperm processed in the 50-mL tubes (P<0.05). Within tube type, gradient volume did not impact parameters of semen quality or recovery rate. An increase in sperm number for density gradient centrifugation resulted in a decreased recovery rate (P<0.05) when 15-mL tubes were used.