Characteristics of the puberty in hair ram lambs and its crosses in Colombia under low altitude conditions / Características de la pubertad en corderos de pelo y sus cruces en Colombia en condiciones de baja altitud

ABSTRACT Objective. Aimed to describe the characteristics of the onset of puberty in males in Colombian hair ram lambs (Ovino de Pelo Colombiano, OPC), and their crosses with Katahdin and Santa Inés sheep in a farm located in Villavicencio, Meta. Materials and methods. 15 lambs of three biotypes: OPC x OPC (OPC), Santa Ines x OPC (SO) and Katahdin X OPC (KO) from four until 12 months old. Ram lambs were grazing and they had supplementation with commercial salt and water ad libitum. Monthly body weight (BW), scrotal circumference (SC), testicular volume (TV) were measured and testosterone level were determined by Elisa test, and ultrasound of the two testicles was performed to determine the presence of the testicular mediastinum, also evaluating the presence and detachment of the urethral prolongation and then electro ejaculation was performed to determine the macroscopic and microscopic characteristics of the semen. Results. Genotype effect was significant for evolution of body BW, SC, TV over time. The SO and KO crosses presented the highest values in BW, whereas OPC lambs maintained a lower value until the end of the assay. At six months old, the three biotypes presented a minimum concentration of 150 million of sperm per ml with 30% of individual progressive motility. Conclusions. In non-seasonal tropical conditions in Colombia (Orinoquia), depending on the variables included, body weight, testicular development, pennis morphology, semen quality, sperm concentration and testosterone levels, it is postulated that around six months of age, the onset of puberty is displayed in the three biotypes.

RESUMEN Objetivo. Describir las características del inicio de la pubertad en corderos machos (Ovino de Pelo Colombiano, OPC) y sus cruces en una granja ubicada en Villavicencio, Meta. Materiales y métodos. Se evaluaron 15 corderos de tres razas: OPC x OPC (OPC), Santa Inés x OPC (SO) y Katahdin X OPC (KO), a partir de los cuatro meses de edad, cinco corderos por cruzamiento, manejados en pastoreo rotacional, con sal mineralizada comercial y agua a voluntad. Mensualmente hasta los doce meses de edad, se evaluó el peso corporal (PC), circunferencia escrotal (CE), volumen testicular (VT), y se determinó la concentración de testosterona en suero mediante la prueba de Elisa, se determinó la presencia del mediastino testicular mediante ecografía y se evalúo el desprendimiento de la prolongación uretral. Finalmente se determinaron las características macroscópicas y microscópicas del eyaculado. Resultados. El efecto racial o de cruzamiento fue significativo para los resultados del PC, CE y VT en el tiempo. Los cruces OPC y KO presentaron los valores más altos en PC, mientras que los corderos OPC mantuvieron un valor menor de PC hasta el final de la investigación. Se realizaron al menos cuatro evaluaciones seminales hasta la presentación de una concentración mínima de 150 millones de espermatozoides por ml con un 30% de motilidad progresiva individual. Conclusiones. En condiciones tropicales no estacionales en Colombia (Orinoquia), para las variables peso corporal, desarrollo testicular, morfología del pene, calidad del semen, concentración de espermatozoides y niveles de testosterona, se postula que alrededor de los seis meses de edad, se presenta el inicio de la pubertad en los tres biotipos.

Ram semen deterioration by short-term exposure to high altitude is prevented by improvement of antioxidant status.

Ovine reproduction efficiency in herds at high altitude (ha) is lower than that at low altitude (la). In ewes, ha effects are due to hypoxia and oxidative stress. Our aim was to establish the effect of antioxidant vitamin supplementation on semen traits and antioxidant status of rams exposed to short or long time ha. A total of 32 rams native to la (~500 m) were used, 16 were kept at la and the other 16 were brought to ha (~3600 m), where they were placed in the same flock as the ha native rams (n=16). Half of the animals in each group were supplemented daily with vitamins C 600 mg and E 450 IU per os, during the entire experimental period, starting the 4th day after animal's arrival at ha (day 0). At days 0, 30 and 60 of treatment, blood and semen samples were collected for evaluation of antioxidant status and semen standard characteristics. Data were compared within each experimental time by analysis of variance using a general linear model. Elevated concentrations of oxidative stress biomarkers were present in blood from animals maintained at ha. Ejaculates from ha exposed rams showed decreased sperm concentration, progressive motility and viability, in addition to decreased antioxidant status in seminal fluid. A total of 30 days of oral supplementation with vitamins C and E prevented some ha negative effects on semen characteristics, mainly in recently ha exposed rams. It is concluded that exposure of rams to ha negatively affects semen quality, where oxidative stress plays a predominant role. These effects are mainly prevented by oral supplementation of vitamins C and E, which constitutes a simple and cheap alternative to improve semen quality of rams when they are moved to ha.

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables.

Adição do látex de mangabeira (Hancornia speciosa) ao diluidor citrato-glicose-gema na motilidade do sêmen ovino resfriado por 24h: estudos preliminares / Addition of mangabeira latex (Hancornia speciosa) to citrate-glucose-egg yolk extender on motility of ram semen cooled for 24 hours: preliminary studies

This study aimed to evaluate the effect of different concentrations of mangabeira latex (Hancorniaspeciosa) added to ram semen extender on motility of cooled semen stored for 24 hours. The latex was added tocitrate-glucose-yolk extender in concentrations of 0% (control), 2%, 4% and 6% (v:v) and diluted with SantaInês ram semen and cooled to 4°C for 24h. The semen was evaluated for sperm motility and vigor. No statisticaldifferences were found for fresh semen and the treatments with the addition of latex. These results indicate thatno improvement in ram semen quality cooled and stored for 24 by the addition of mangabeira latex.(AU)

Adição do látex de mangabeira (Hancornia speciosa) ao diluidor citrato-glicose-gema na motilidade do sêmen ovino resfriado por 24h: estudos preliminares / Addition of mangabeira latex (Hancornia speciosa) to citrate-glucose-egg yolk extender on motility of ram semen cooled for 24 hours: preliminary studies

This study aimed to evaluate the effect of different concentrations of mangabeira latex (Hancorniaspeciosa) added to ram semen extender on motility of cooled semen stored for 24 hours. The latex was added tocitrate-glucose-yolk extender in concentrations of 0% (control), 2%, 4% and 6% (v:v) and diluted with SantaInês ram semen and cooled to 4°C for 24h. The semen was evaluated for sperm motility and vigor. No statisticaldifferences were found for fresh semen and the treatments with the addition of latex. These results indicate thatno improvement in ram semen quality cooled and stored for 24 by the addition of mangabeira latex.

Criopreservação do sêmen ovino em meio diluente à base de água de coco em pó (ACP-102c) / Cryopreservation of ram semen in powdered coconut water (ACP-102c) based extender

The aim of this study was to evaluate the ACP-102c extender in the cryopreservation of ram semen compared to tris-glucose-egg yolk (TRIS) extender and fresh semen. Forty-eight ejaculates were collected from four rams and two aliquots per ejaculate were taken for dilution and cryopreservation in ACP-102c or TRIS and a third aliquot used for the fresh semen analysis. Either the fresh semen and cryopreserved in both extenders were evaluated for viability, integrity of plasma and acrosomal membrane, hypoosmotic swelling test, DNA fragmentation and sperm motility. The extenders did not differ for sperm viability, acrosome and plasma membrane integrity, DNA fragmentation and quantitative and qualitative parameters of sperm velocity after thawing, but differed in hypoosmotic swelling test, total and progressive motility and lateral extent of the head as well as in all motility parameters evaluated (except linearity and straightness) after two hours of incubation at 37 oC. There was variability among rams in total and progressive motility of semen cryopreserved in ACP-102c after thawing. The ACP-102c extender showed less protection in the cryopreservation of ram sperm when compared to TRIS, but the improvement in its formulation and freezing protocols may make it an alternative to freezing ram semen.

O objetivo deste trabalho foi avaliar o diluente ACP-102c na criopreservação do sêmen ovino em comparação com o diluidor tris-glicose-gema (TRIS) e o sêmen fresco. Foram coletados 48 ejaculados de quatro ovinos, sendo tomadas duas alíquotas por ejaculado para diluição e criopreservação em ACP-102c ou TRIS e uma terceira alíquota utilizada para análise do sêmen fresco. O sêmen fresco e o criopreservado em ambos os diluidores foram avaliados para viabilidade, integridade de membrana plasmática e acrossomal, teste hiposmótico, fragmentação do DNA e de motilidade espermática. Após descongelamento, ambos os diluidores não diferiram para viabilidade espermática, integridade de membrana plasmática e acrossomal, fragmentação de DNA e nas variáveis quantitativas e qualitativas de velocidade espermática, mas diferiram no teste hiposmótico, motilidade total e progressiva e amplitude lateral da cabeça, bem como em todas as variáveis de motilidade avaliadas, exceto linearidade e progressividade, após duas horas de incubação à 37 oC. Houve variabilidade entre reprodutores na motilidade total e progressiva do sêmen criopreservado em ACP-102c após descongelamento. O diluidor ACP-102c conferiu menor proteção aos espermatozoides ovinos contra danos do congelamento quando comparado ao TRIS, mas o aprimoramento de sua formulação e protocolos mais adequados de congelação poderão torná-lo uma alternativa na congelação do sêmenovino.

Criopreservação do sêmen ovino em meio diluente à base de água de coco em pó (ACP-102c) / Cryopreservation of ram semen in powdered coconut water (ACP-102c) based extender

The aim of this study was to evaluate the ACP-102c extender in the cryopreservation of ram semen compared to tris-glucose-egg yolk (TRIS) extender and fresh semen. Forty-eight ejaculates were collected from four rams and two aliquots per ejaculate were taken for dilution and cryopreservation in ACP-102c or TRIS and a third aliquot used for the fresh semen analysis. Either the fresh semen and cryopreserved in both extenders were evaluated for viability, integrity of plasma and acrosomal membrane, hypoosmotic swelling test, DNA fragmentation and sperm motility. The extenders did not differ for sperm viability, acrosome and plasma membrane integrity, DNA fragmentation and quantitative and qualitative parameters of sperm velocity after thawing, but differed in hypoosmotic swelling test, total and progressive motility and lateral extent of the head as well as in all motility parameters evaluated (except linearity and straightness) after two hours of incubation at 37 oC. There was variability among rams in total and progressive motility of semen cryopreserved in ACP-102c after thawing. The ACP-102c extender showed less protection in the cryopreservation of ram sperm when compared to TRIS, but the improvement in its formulation and freezing protocols may make it an alternative to freezing ram semen.(AU)

O objetivo deste trabalho foi avaliar o diluente ACP-102c na criopreservação do sêmen ovino em comparação com o diluidor tris-glicose-gema (TRIS) e o sêmen fresco. Foram coletados 48 ejaculados de quatro ovinos, sendo tomadas duas alíquotas por ejaculado para diluição e criopreservação em ACP-102c ou TRIS e uma terceira alíquota utilizada para análise do sêmen fresco. O sêmen fresco e o criopreservado em ambos os diluidores foram avaliados para viabilidade, integridade de membrana plasmática e acrossomal, teste hiposmótico, fragmentação do DNA e de motilidade espermática. Após descongelamento, ambos os diluidores não diferiram para viabilidade espermática, integridade de membrana plasmática e acrossomal, fragmentação de DNA e nas variáveis quantitativas e qualitativas de velocidade espermática, mas diferiram no teste hiposmótico, motilidade total e progressiva e amplitude lateral da cabeça, bem como em todas as variáveis de motilidade avaliadas, exceto linearidade e progressividade, após duas horas de incubação à 37 oC. Houve variabilidade entre reprodutores na motilidade total e progressiva do sêmen criopreservado em ACP-102c após descongelamento. O diluidor ACP-102c conferiu menor proteção aos espermatozoides ovinos contra danos do congelamento quando comparado ao TRIS, mas o aprimoramento de sua formulação e protocolos mais adequados de congelação poderão torná-lo uma alternativa na congelação do sêmenovino.(AU)

Criopreservação do sêmen ovino em meio diluente à base de água de coco em pó (ACP-102c)

The aim of this study was to evaluate the ACP-102c extender in the cryopreservation of ram semen compared to tris-glucose-egg yolk (TRIS) extender and fresh semen. Forty-eight ejaculates were collected from four rams and two aliquots per ejaculate were taken for dilution and cryopreservation in ACP-102c or TRIS and a third aliquot used for the fresh semen analysis. Either the fresh semen and cryopreserved in both extenders were evaluated for viability, integrity of plasma and acrosomal membrane, hypoosmotic swelling test, DNA fragmentation and sperm motility. The extenders did not differ for sperm viability, acrosome and plasma membrane integrity, DNA fragmentation and quantitative and qualitative parameters of sperm velocity after thawing, but differed in hypoosmotic swelling test, total and progressive motility and lateral extent of the head as well as in all motility parameters evaluated (except linearity and straightness) after two hours of incubation at 37 ºC. There was variability among rams in total and progressive motility of semen cryopreserved in ACP-102c after thawing. The ACP-102c extender showed less protection in the cryopreservation of ram sperm when compared to TRIS, but the improvement in its formulation and freezing protocols may make it an alternative to freezing ram semen.

O objetivo deste trabalho foi avaliar o diluente ACP-102c na criopreservação do sêmen ovino em comparação com o diluidor tris-glicose-gema (TRIS) e o sêmen fresco. Foram coletados 48 ejaculados de quatro ovinos, sendo tomadas duas alíquotas por ejaculado para diluição e criopreservação em ACP-102c ou TRIS e uma terceira alíquota utilizada para análise do sêmen fresco. O sêmen fresco e o criopreservado em ambos os diluidores foram avaliados para viabilidade, integridade de membrana plasmática e acrossomal, teste hiposmótico, fragmentação do DNA e de motilidade espermática. Após descongelamento, ambos os diluidores não diferiram para viabilidade espermática, integridade de membrana plasmática e acrossomal, fragmentação de DNA e nas variáveis quantitativas e qualitativas de velocidade espermática, mas diferiram no teste hiposmótico, motilidade total e progressiva e amplitude lateral da cabeça, bem como em todas as variáveis de motilidade avaliadas, exceto linearidade e progressividade, após duas horas de incubação à 37 ºC. Houve variabilidade entre reprodutores na motilidade total e progressiva do sêmen criopreservado em ACP-102c após descongelamento. O diluidor ACP-102c conferiu menor proteção aos espermatozoides ovinos contra danos do congelamento quando comparado ao TRIS, mas o aprimoramento de sua formulação e protocolos mais adequados de congelação poderão torná-lo uma alternativa na congelação do sêmen ovino.

Effect of post-thaw incubation on semen characteristics of ram spermatozoa cryopreserved under controlled and uncontrolled rate of cooling

Exposure of frozen-thawed spermatozoa to a thermal resistance test reveals damages which are not apparent immediately after thawing but are useful to assess the fertilizing ability of ram spermatozoa. Our earlier study has shown that cryopreservation of ram spermatozoa under controlled rate of cooling piror yo controlled rate of freezing. Egg yolk-Test-glycerol extender and packaged in 0.25 ml straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25 to 5°C at the rate of 0.15°C per minute followed by a holding time of 2 h forequilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5 to –125°C at the rate of 25°C per minute. Thawing of straws was done at 50°C for 10 seconds and thawed spermatozoa were subjected to a thermal resistance test at 37°C for4 h. Samples were assessed immediately after thawing and at hourly interval for sperm motion characteristics by computer-aided semen analysis technique. Post-thaw incubated spermatozoa were also evaluated at 0, 1, 2, 3, and 4 h for acrosomal integrity after staining the dried semen smears with Giemsa stain. The % motility, % rapid moving spermatozoa, % linearity and % sperm with normal acrosome were significantly (P < 0.05) higher in Group 1 compared to Group 2. The effect of incubation time was also significant (P < 0.05) on % motility, fraction of rapid motile spermatozoa, % linearity, curvilinear velocity, average path velocity, straight line velocity, area of sperm head, lateral head displacement and % spermatozoa with normal acrosome. The % motility, % rapid motile spermatozoa, sperm velocity, lateral head displacement and % spermatozoa with normal acrosomeprogressively declined during 4 h of incubation but the decline in all the traits was less in Group 1 compared to Group 2. The results showed that controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa for post-thaw thermoresistance test compared to uncontrolled rate of cooling prior to programmable freezing.

Effect of post-thaw incubation on semen characteristics of ram spermatozoa cryopreserved under controlled and uncontrolled rate of cooling

Exposure of frozen-thawed spermatozoa to a thermal resistance test reveals damages which are not apparent immediately after thawing but are useful to assess the fertilizing ability of ram spermatozoa. Our earlier study has shown that cryopreservation of ram spermatozoa under controlled rate of cooling piror yo controlled rate of freezing. Egg yolk-Test-glycerol extender and packaged in 0.25 ml straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25 to 5°C at the rate of 0.15°C per minute followed by a holding time of 2 h forequilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5 to –125°C at the rate of 25°C per minute. Thawing of straws was done at 50°C for 10 seconds and thawed spermatozoa were subjected to a thermal resistance test at 37°C for4 h. Samples were assessed immediately after thawing and at hourly interval for sperm motion characteristics by computer-aided semen analysis technique. Post-thaw incubated spermatozoa were also evaluated at 0, 1, 2, 3, and 4 h for acrosomal integrity after staining the dried semen smears with Giemsa stain. The % motility, % rapid moving spermatozoa, % linearity and % sperm with normal acrosome were significantly (P < 0.05) higher in Group 1 compared to Group 2. The effect of incubation time was also significant (P < 0.05) on % motility, fraction of rapid motile spermatozoa, % linearity, curvilinear velocity, average path velocity, straight line velocity, area of sperm head, lateral head displacement and % spermatozoa with normal acrosome. The % motility, % rapid motile spermatozoa, sperm velocity, lateral head displacement and % spermatozoa with normal acrosomeprogressively declined during 4 h of incubation but the decline in all the traits was less in Group 1 compared to Group 2. The results showed that controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa for post-thaw thermoresistance test compared to uncontrolled rate of cooling prior to programmable freezing.(AU)