Sperm cell capacitation status of ram semen after cooling

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time.

Use of cooled buffalo semen as a strategy to increase conception rates in fixed-time artificial insemination programs during unfavorable reproductive periods / [Uso de sêmen resfriado de búfalo como estratégia para aumentar as taxas de concepção em programas de inseminação artificial em tempo fixo, durante períodos reprodutivos desfavoráveis]

The objective of this study was to compare the reproductive efficiency of dairy buffaloes undergoing fixed-time artificial insemination (FTAI) protocols based on progesterone/estrogen (P4/E2) and eCG during unfavorable breeding season using cooled (CS) and frozen semen (FS). A total of 446 buffaloes (> 40 days postpartum) were randomly distributed into four blocks (years): B1-2014 (n = 143), B2-2015 (n = 34), B3-2016 (n = 90), and B4-2017 (n = 179). Each block was subdivided into two (AI with CS and FS using the same ejaculate of each bull). Thus, the block subdivision was as follows: B1 (CS = 71 and FS = 72); B2 (CS = 18 and FS = 16); B3 (CS = 47 and FS = 43); and B4 (CS = 90 and FS = 89). The ejaculates of eight Murrah bulls collected using an artificial vagina were divided into two aliquots: one aliquot was diluted in Botu-Bov® commercial extender and cooled (BB-CS), and the other was diluted in the same extender and frozen (BB-FS). BB-CS aliquots were cooled at 5 °C/24 h using a refrigerator. BB-FS group aliquots were also cooled, and after equilibrating at 5 °C for 4 h, were placed in a 21-L Styrofoam box, 5 cm above the surface of liquid nitrogen. In the afternoon (A) on D0 (2:00 p.m.) the animals received EB 2.0 mg IM (Estrogin®) and an ear implant (CRESTAR® 3.0 mg P4). At D9 (A), the implant was removed, and the animals received eCG 400 IU IM (Folligon® 5000) + Cloprostenol PGF2α 0.530 mg IM (Sincrocio®). At D10 (A), the animals received EB 1.0 mg IM (Estrogin®), and at D12 (8:00 a.m.), AI was performed. At D42, pregnancy was diagnosed via ultrasonography. Total CRs were 48.2% CS and 34.6% FS for years 2014 to 2017, with a significant difference of 13.7% (P<0.05). In conclusion, cooled semen resulted in higher CR than frozen semen in dairy buffaloes under the P4/E2 and eCG FTAI during the unfavorable reproductive season.(AU)

O objetivo deste estudo foi comparar a eficiência reprodutiva de búfalas leiteiras submetidas a protocolos de inseminação artificial em tempo fixo (IATF) à base de progesterona/estrogênio (P4/E2) e eCG, durante a estação reprodutiva desfavorável, usando-se sêmen resfriado (SR) e congelado (SC) Um total de 446 búfalas (> 40 dias após o parto) foi distribuído aleatoriamente em quatro blocos (anos): B1-2014 (n = 143), B2-2015 (n = 34), B3-2016 (n = 90) e B4-2017 (n = 179). Cada bloco foi subdividido em dois (IA com SR e SC utilizando-se a mesma ejaculação de cada touro). Assim, a subdivisão do bloco foi a seguinte: B1 (SR = 71 e SC = 72); B2 (SR = 18 e SC = 16); B3 (SR = 47 e SC = 43); e B4 (SR = 90 e SC = 89). Os ejaculados de oito touros Murrah coletados com vagina artificial foram divididos em duas alíquotas: uma alíquota diluída em diluente comercial Botu-Bov® e resfriada (BB-SR), e a outra diluída no mesmo diluente e congelada (BB-SC). As alíquotas de BB-SR foram resfriados a 5°C/24h usando-se um refrigerador. As alíquotas do grupo BB-SC também foram resfriadas e, após equilíbrio a 5°C por 4h, foram colocadas em uma caixa de isopor de 21L, 5 cm acima da superfície do nitrogênio líquido. À tarde (A), no D0 (14h), os animais receberam BE 2,0 mg IM (Estrogin®) e um implante auricular (Crestar® 3,0 mg P4). No D9 (A), o implante foi retirado e os animais receberam eCG 400 UI IM (Folligon® 5000) + cloprostenol PGF2α 0,530 mg IM (Sincrocio®). No D10 (A), os animais receberam BE 1,0mg IM (Estrogin®), e, no D12 (8h da manhã), foram realizadas as IAs. No D42, a gestação foi diagnosticada por ultrassonografia. As taxas de concepção (TC) totais foram 48,2% SR e 34,6% SC para os anos de 2014 a 2017, com uma diferença significativa de 13,7% (P<0,05). Em conclusão, o sêmen resfriado resultou em maior TC do que o sêmen congelado em bubalinos leiteiros sob P4/E2 e eCG FTAI durante a estação reprodutiva desfavorável.(AU)

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen / Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.(AU)

O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.(AU)

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen / Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.(AU)

O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.(AU)

Effects of L-Carnitine on Equine Semen Quality During Liquid Storage.

l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through ß-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In the second experiment, ejaculates (n = 16) from four Quarter Horses were used. Each ejaculate was split into four treatment groups with different LC concentrations: 0 (control), 0.5, 1.0, and 2.0 mM. Sperm motility, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species content, and plasma membrane stability were evaluated immediately after samples reached 5°C (0 hour) and after 24, 48, and 72 hours. There was a positive correlation (p < 0.05) between LC levels in seminal plasma with both sperm concentration and plasma and acrosomal membrane integrity. Furthermore, the addition of LC (1 and 2 mM) preserved the motility of equine sperm stored at 5°C. It was concluded that the concentrations of LC with seminal plasma present correlate to semen parameters and the addition of LC to skimmed milk-based extender preserves the motility of equine sperm stored at 5°C for up to 48 hours.

Comparison of extenders and storage temperature in chilling canine semen / Diferentes diluentes e temperaturas de armazenamento para a refrigeração de sêmen canino

This study aimed to compare the effects a commercial milk-based extender and a self-made egg yolk extender had on the quality of canine semen stored at two different temperatures, 5ºC or 15ºC. The ejaculate obtained was split into two aliquots of equal volume and diluted with the milk or egg yolk extender. The final concentration was 100×106 spermatozoa/mL. Diluted semen was placed in transport containers and maintained at final storage temperatures of 5ºC and 15ºC. The quality of the chilled semen was assessed 12, 24, and 36 hours after storage. Semen diluted with the milk extender had higher motility, vigour, and plasma membrane integrity (p<0.05) of the spermatozoa than that diluted with the egg yolk extender. No difference in the semen quality was observed between the stored temperatures in both the groups. The difference observed between the extenders could be due to the standard formulation of the commercial milk extender and the presence of glucose in the mixture. In conclusion, the milk extender was better than the egg yolk extender at preserving the motility, viability, and membrane integrity of chilled canine semen for up to 36 hours. The storage temperature did not seem to affect the semen quality, suggesting that canine semen can be refrigerated at 15ºC.

O objetivo desse estudo foi avaliar a influência de um diluente comercial à base de leite, comparado à um diluente preparado com de gema de ovo na qualidade do sêmen de cão, armazenado em duas diferentes temperaturas (5º C ou 15º C). O ejaculado obtido foi dividido em duas alíquotas de volumes iguais, que foram diluídas com o diluente leite ou o diluente gema de ovo, apresentando uma concentração final de 100x106 espermatozoides/mL. O sêmen diluído foi colocado em caixas de transporte, mantendo temperaturas finais de refrigeração de 5º C e 15º C. A qualidade do sêmen refrigerado foi avaliada após 12, 24 e 36h de armazenamento. O sêmen diluído com o diluente leite resultou em maior motilidade, vigor e integridade de membrana plasmática (p<0,05) dos espermatozoides que o diluído com o diluente gema de ovo. Dentro de cada grupo, não foi encontrada nenhuma influencia da temperatura de armazenamento em relação à qualidade do sêmen. A diferença observada entre os diluentes, pode ter ocorrido devido à formulação padrão do diluente comercial à base de leite, além da presença de glicose em sua composição. Em conclusão, o diluente leite apresentou superior preservação da motilidade, viabilidade e integridade de membrana plasmática do sêmen de cão refrigerado, por até 36h de armazenamento, que o diluente gema de ovo, e a temperatura de armazenamento não interferiu na qualidade seminal, sugerindo que 15º C também pode ser utilizado para a refrigeração do sêmen canino.

Comparison of extenders and storage temperature in chilling canine semen / Diferentes diluentes e temperaturas de armazenamento para a refrigeração de sêmen canino

This study aimed to compare the effects a commercial milk-based extender and a self-made egg yolk extender had on the quality of canine semen stored at two different temperatures, 5ºC or 15ºC. The ejaculate obtained was split into two aliquots of equal volume and diluted with the milk or egg yolk extender. The final concentration was 100×106 spermatozoa/mL. Diluted semen was placed in transport containers and maintained at final storage temperatures of 5ºC and 15ºC. The quality of the chilled semen was assessed 12, 24, and 36 hours after storage. Semen diluted with the milk extender had higher motility, vigour, and plasma membrane integrity (p<0.05) of the spermatozoa than that diluted with the egg yolk extender. No difference in the semen quality was observed between the stored temperatures in both the groups. The difference observed between the extenders could be due to the standard formulation of the commercial milk extender and the presence of glucose in the mixture. In conclusion, the milk extender was better than the egg yolk extender at preserving the motility, viability, and membrane integrity of chilled canine semen for up to 36 hours. The storage temperature did not seem to affect the semen quality, suggesting that canine semen can be refrigerated at 15ºC.(AU)

O objetivo desse estudo foi avaliar a influência de um diluente comercial à base de leite, comparado à um diluente preparado com de gema de ovo na qualidade do sêmen de cão, armazenado em duas diferentes temperaturas (5º C ou 15º C). O ejaculado obtido foi dividido em duas alíquotas de volumes iguais, que foram diluídas com o diluente leite ou o diluente gema de ovo, apresentando uma concentração final de 100x106 espermatozoides/mL. O sêmen diluído foi colocado em caixas de transporte, mantendo temperaturas finais de refrigeração de 5º C e 15º C. A qualidade do sêmen refrigerado foi avaliada após 12, 24 e 36h de armazenamento. O sêmen diluído com o diluente leite resultou em maior motilidade, vigor e integridade de membrana plasmática (p<0,05) dos espermatozoides que o diluído com o diluente gema de ovo. Dentro de cada grupo, não foi encontrada nenhuma influencia da temperatura de armazenamento em relação à qualidade do sêmen. A diferença observada entre os diluentes, pode ter ocorrido devido à formulação padrão do diluente comercial à base de leite, além da presença de glicose em sua composição. Em conclusão, o diluente leite apresentou superior preservação da motilidade, viabilidade e integridade de membrana plasmática do sêmen de cão refrigerado, por até 36h de armazenamento, que o diluente gema de ovo, e a temperatura de armazenamento não interferiu na qualidade seminal, sugerindo que 15º C também pode ser utilizado para a refrigeração do sêmen canino.(AU)

Effects of ascorbic acid 2-glucoside and alpha-tocopherol on the characteristics of equine spermatozoa stored at 5°C.

The aim of this experiment was to evaluate the effects of adding ascorbic acid 2-glucoside (AA2G), a water-soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat-soluble antioxidant α-tocopherol (α-Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/108 sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.

Physical characteristics and fertility of fractionated donkey semen cooled at 5°C / Características físicas e fertilidade do sêmen asinino fracionado e resfriado a 5ºC

The aim of this study was to evaluate the effect of two different extenders (Skimmed Milk Glucose - SMG or Lactose - Egg Yolk - LEY) on physical characteristics and fertility of fractionated donkey semen cooled at 5°C. For this, four Pêga donkeys were used as semen donors. The sperm rich fraction of the ejaculate was diluted preparing insemination doses containing 400 x 106 motile spermatozoa in a volume of 22 mL, cooled to 5°C and stored up to 48 hours in a container proposed by Palhares (1997). Sperm motility and vigor were assessed in fresh semen, after first semen dilution, before insemination, at 24 and 48 hours after storage. For the fertility evaluation, 44 mares were inseminated with semen stored for a period between 12 and 24 hours. The mares were inseminated on fixed days (Mondays, Wednesdays, and Fridays) after the detection of a follicle greater than a 30mm diameter in one of the ovaries through ovulation. Pregnancy diagnosis was performed on day 12 post-ovulation, using transrectal ultrasonography. Semen diluted in SMG showed superior sperm motility than LEY, at the Pre-AI evaluation (P<0.05). At 48 hours of storage, all donkeys had motility values between 45 and 53% for semen diluted in SMG, while only one donkey showed motility greater than 30% in the LEY treatment. The pregnancy rate/cycle for mares inseminated with semen diluted in SMG was superior than that obtained using LEY (56.52% vs 4.76%, respectively).(AU)

Objetivou-se com o presente experimento avaliar o efeito de dois diferentes diluidores (leite em pó desnatado glicose - SMG ou lactose gema de ovo - LEY) sobre as características físicas e a fertilidade do sêmen asinino coletado de forma fracionada e resfriado a 5ºC. Para isso, quatro jumentos da raça Pêga foram utilizados como doadores de sêmen. A fração espermática rica do ejaculado foi diluída preparando-se doses inseminantes contendo 400 x 106 espermatozoides móveis em um volume de 22 mL, resfriadas a 5ºC e armazenadas por até 48 horas em contêiner proposto por Palhares (1997). A motilidade e o vigor espermáticos foram avaliados no sêmen fresco, após a pré-diluição, antes das inseminações, às 24 e 48 horas de armazenamento. Para avaliação de fertilidade, 44 éguas foram inseminadas com sêmen armazenado por um período entre 12 e 24 horas, em dias fixos (segundas, quartas e sextas-feiras), após a detecção de um folículo de diâmetro maior ou igual a 30mm em um dos ovários, até a ovulação. O diagnóstico de gestação foi realizado a partir de 12 dias após a ovulação, por meio de ultrassonografia transretal. O sêmen diluído em SMG apresentou motilidade espermática superior à do LEY, já a partir do tempo pré-IA. Às 48 horas de armazenamento, todos os jumentos apresentaram valores de motilidade entre 45% e 53%, quando o sêmen foi diluído em SMG, enquanto apenas um jumento apresentou motilidade superior a 30% no tratamento utilizando LEY. A taxa de concepção/ciclo das éguas inseminadas também foi superior para o sêmen diluído em SMG em relação ao diluído em LEY (56,52% versus 4,76%, respectivamente).(AU)

Physical characteristics and fertility of fractionated donkey semen cooled at 5°C / Características físicas e fertilidade do sêmen asinino fracionado e resfriado a 5ºC

The aim of this study was to evaluate the effect of two different extenders (Skimmed Milk Glucose - SMG or Lactose - Egg Yolk - LEY) on physical characteristics and fertility of fractionated donkey semen cooled at 5°C. For this, four Pêga donkeys were used as semen donors. The sperm rich fraction of the ejaculate was diluted preparing insemination doses containing 400 x 106 motile spermatozoa in a volume of 22 mL, cooled to 5°C and stored up to 48 hours in a container proposed by Palhares (1997). Sperm motility and vigor were assessed in fresh semen, after first semen dilution, before insemination, at 24 and 48 hours after storage. For the fertility evaluation, 44 mares were inseminated with semen stored for a period between 12 and 24 hours. The mares were inseminated on fixed days (Mondays, Wednesdays, and Fridays) after the detection of a follicle greater than a 30mm diameter in one of the ovaries through ovulation. Pregnancy diagnosis was performed on day 12 post-ovulation, using transrectal ultrasonography. Semen diluted in SMG showed superior sperm motility than LEY, at the Pre-AI evaluation (P<0.05). At 48 hours of storage, all donkeys had motility values between 45 and 53% for semen diluted in SMG, while only one donkey showed motility greater than 30% in the LEY treatment. The pregnancy rate/cycle for mares inseminated with semen diluted in SMG was superior than that obtained using LEY (56.52% vs 4.76%, respectively).(AU)

Objetivou-se com o presente experimento avaliar o efeito de dois diferentes diluidores (leite em pó desnatado glicose - SMG ou lactose gema de ovo - LEY) sobre as características físicas e a fertilidade do sêmen asinino coletado de forma fracionada e resfriado a 5ºC. Para isso, quatro jumentos da raça Pêga foram utilizados como doadores de sêmen. A fração espermática rica do ejaculado foi diluída preparando-se doses inseminantes contendo 400 x 106 espermatozoides móveis em um volume de 22 mL, resfriadas a 5ºC e armazenadas por até 48 horas em contêiner proposto por Palhares (1997). A motilidade e o vigor espermáticos foram avaliados no sêmen fresco, após a pré-diluição, antes das inseminações, às 24 e 48 horas de armazenamento. Para avaliação de fertilidade, 44 éguas foram inseminadas com sêmen armazenado por um período entre 12 e 24 horas, em dias fixos (segundas, quartas e sextas-feiras), após a detecção de um folículo de diâmetro maior ou igual a 30mm em um dos ovários, até a ovulação. O diagnóstico de gestação foi realizado a partir de 12 dias após a ovulação, por meio de ultrassonografia transretal. O sêmen diluído em SMG apresentou motilidade espermática superior à do LEY, já a partir do tempo pré-IA. Às 48 horas de armazenamento, todos os jumentos apresentaram valores de motilidade entre 45% e 53%, quando o sêmen foi diluído em SMG, enquanto apenas um jumento apresentou motilidade superior a 30% no tratamento utilizando LEY. A taxa de concepção/ciclo das éguas inseminadas também foi superior para o sêmen diluído em SMG em relação ao diluído em LEY (56,52% versus 4,76%, respectivamente).(AU)

Viability of Equine Semen Stored in a Polyethylene System for Transport for Eight Hours

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen.Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequentl

Viability of Equine Semen Stored in a Polyethylene System for Transport for Eight Hours

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen.Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequentl

Viability of Equine Semen Stored in a Polyethylene System for Transport for Eight Hours

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen.Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequentl

Viability of Equine Semen Stored in a Polyethylene System for Transport for Eight Hours

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen.Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequentl

Viability of equine semen stored in a polyethylene system for transport for eight hours / Viabilidade do sêmen equino armazenado em sistema de polietileno para transporte por oito horas

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen. Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequently to this, the semen was diluted 1+1 (diluent+semen) with skim UHT milk, and divided into four aliquots of equal volume, yielding a total of four groups. In the control group (GC) the semen was analyzed immediately after dilution (zero h - 0-h). Samples from other groups were stored for eight h (8-h) in three different devices: Equitainer® (GE), BotuFLEX® (GB) or Polyethylene System - Cooled (SP) and non-Cooled (SPN). [...](AU)

Viability of equine semen stored in a polyethylene system for transport for eight hours / Viabilidade do sêmen equino armazenado em sistema de polietileno para transporte por oito horas

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen. Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequently to this, the semen was diluted 1+1 (diluent+semen) with skim UHT milk, and divided into four aliquots of equal volume, yielding a total of four groups. In the control group (GC) the semen was analyzed immediately after dilution (zero h - 0-h). Samples from other groups were stored for eight h (8-h) in three different devices: Equitainer® (GE), BotuFLEX® (GB) or Polyethylene System - Cooled (SP) and non-Cooled (SPN). [...]

Viability of Equine Semen Stored in a Polyethylene System for Transport for Eight Hours

Background: Equine semen storage and shipment, being it colled or frozen, allows the veterinarian to direct matings, providing the use of genetically superior stallions, which are mostly located in breeding stations or training centers. Achieving good pregnancy rates depends, beyond the moment of artificial insemination (AI), on factors related to the semen cooling, such as: system used for transport, cooling rate, final storage temperature, storage time and individual variation among stallions, such as age and resistance to cooling. Based on these aspects, this experiment was conducted in order to test a polyethylene system to ship equine semen.Materials, Methods & Results: A total of 87 ejaculates from five stallions with known fertility were used. The stallions aged between 6 to 14 years old, being three Thoroughbred and two Miniature Pony horse. The ejaculates were collected twice a week using a Hannover artificial vagina. After each collection, the semen sample was macroscopically evaluated for appearance, color and smell. A semen sample was used to evaluate the parameters of total motility, vigor and concentration, being these last three parameters assessed by counting 100 sperm cells for analysis. These analysis were performed using an optical microscope, being the concentration taken with a Neubauer chamber after dilution of 1:20 (semen: citrate formol). Subsequentl

Effect of month of conception on fertility of mares inseminated with jackass semen / Influência do mês da concepção sobre a eficiência reprodutiva de éguas inseminadas com sêmen asinino

Fertility obtained by cross-breeding mares (Equus caballus) with jackasses (Equus asinus) was evaluated. Two extenders, containing skim milk-glucose or egg yolk-glycine were used to study the fertility of mares inseminated with diluted jackass semen (T1 and T2) or diluted and cooled semen at 5°C for 12 hours (T3 and T4). A total of 272 cycles of 208 mares of undefined breeds were evaluated, being uniformly distributed between groups. The cycles were controlled by transrectal palpation and teasing, and mares were inseminated every Tuesday, Thursday and Saturday (three times/week), from the detection of a follicle with 3.0 to 3.5cm diameter in one of the ovaries until ovulation. Pregnancy was detected using transrectal palpation, teasing and ultrasound exams every 14 days. The extenders had no effect on fertility (P>0.05). Pregnancy rates for the first cycle were 64.52%, 61.11%, 50.72% and 54.17% and pregnancy rates/cycle were 63.64%, 54.55%, 52.69% and 47.06%, respectively, for T1, T2, T3 and T4. Differences in pregnancy loss rates between groups and effect of month of conception on fertility were found. Pregnancy loss rates were significantly higher (P<0.05) in January (38.46%) and in February and March (52.38%), with an average of 33.09%. The results indicate that mares conceiving at the end of the physiological reproduction time, carrying a mule embryo, are more susceptible to pregnancy loss.

Avaliou-se a eficiência reprodutiva de cruzamentos interespécies entre fêmeas equinas e reprodutores asininos da raça Pêga. Para tal, estudou-se o efeito de dois diluidores, à base de leite em pó desnatado-glicose ou glicina-gema de ovo, sobre a fertilidade de éguas inseminadas com sêmen fresco diluído (T1 e T2) ou diluído e resfriado a 5°C por 12 horas (T3 e T4). Foram utilizados 272 ciclos de 208 éguas mestiças, distribuídas uniformemente entre os tratamentos, após agrupamento por idade e categoria reprodutiva. Os ciclos foram acompanhados por palpação retal e rufiação, sendo as inseminações realizadas às terças, quintas e aos sábados (três vezes por semana), a partir da detecção de um folículo de 3,0 a 3,5cm de diâmetro, em um dos ovários, até a ovulação. As gestações foram acompanhadas por palpação transretal, rufiação e por ultrassonografia, realizada a cada 14 dias. As taxas de gestação obtidas, ao primeiro ciclo, não diferiram (P>0,05) entre os tratamentos, sendo de 64,52% para T1, 61,11% para T2, 50,72% para T3 e 54,17% para T4. Da mesma maneira, as taxas de gestação por ciclo também não foram influenciadas (P>0,05) pelos tratamentos, com valores de 63,64%, 54,55%, 52,69% e 47,06% para T1, T2, T3, e T4, respectivamente. No que diz respeito às taxas de perdas gestacionais, obtidas por meio das taxas de perdas e de perdas ajustadas, houve influência (P<0,05) dos tratamentos. Observou-se, ainda, efeito do mês da concepção, com taxas de perda superiores para os meses de janeiro (38,46%) e de fevereiro e março (52,38%), sendo a média de 33,09%. Sugere-se que a redução dos padrões de secreção do LH, ao final da estação de monta, responda pelas maiores taxas de perdas gestacionais do cruzamento interespécie, notadamente naquelas éguas que conceberam no final da estação de monta fisiológica dos equinos.

Effect of month of conception on fertility of mares inseminated with jackass semen / Influência do mês da concepção sobre a eficiência reprodutiva de éguas inseminadas com sêmen asinino

Fertility obtained by cross-breeding mares (Equus caballus) with jackasses (Equus asinus) was evaluated. Two extenders, containing skim milk-glucose or egg yolk-glycine were used to study the fertility of mares inseminated with diluted jackass semen (T1 and T2) or diluted and cooled semen at 5°C for 12 hours (T3 and T4). A total of 272 cycles of 208 mares of undefined breeds were evaluated, being uniformly distributed between groups. The cycles were controlled by transrectal palpation and teasing, and mares were inseminated every Tuesday, Thursday and Saturday (three times/week), from the detection of a follicle with 3.0 to 3.5cm diameter in one of the ovaries until ovulation. Pregnancy was detected using transrectal palpation, teasing and ultrasound exams every 14 days. The extenders had no effect on fertility (P>0.05). Pregnancy rates for the first cycle were 64.52%, 61.11%, 50.72% and 54.17% and pregnancy rates/cycle were 63.64%, 54.55%, 52.69% and 47.06%, respectively, for T1, T2, T3 and T4. Differences in pregnancy loss rates between groups and effect of month of conception on fertility were found. Pregnancy loss rates were significantly higher (P<0.05) in January (38.46%) and in February and March (52.38%), with an average of 33.09%. The results indicate that mares conceiving at the end of the physiological reproduction time, carrying a mule embryo, are more susceptible to pregnancy loss.(AU)

Avaliou-se a eficiência reprodutiva de cruzamentos interespécies entre fêmeas equinas e reprodutores asininos da raça Pêga. Para tal, estudou-se o efeito de dois diluidores, à base de leite em pó desnatado-glicose ou glicina-gema de ovo, sobre a fertilidade de éguas inseminadas com sêmen fresco diluído (T1 e T2) ou diluído e resfriado a 5°C por 12 horas (T3 e T4). Foram utilizados 272 ciclos de 208 éguas mestiças, distribuídas uniformemente entre os tratamentos, após agrupamento por idade e categoria reprodutiva. Os ciclos foram acompanhados por palpação retal e rufiação, sendo as inseminações realizadas às terças, quintas e aos sábados (três vezes por semana), a partir da detecção de um folículo de 3,0 a 3,5cm de diâmetro, em um dos ovários, até a ovulação. As gestações foram acompanhadas por palpação transretal, rufiação e por ultrassonografia, realizada a cada 14 dias. As taxas de gestação obtidas, ao primeiro ciclo, não diferiram (P>0,05) entre os tratamentos, sendo de 64,52% para T1, 61,11% para T2, 50,72% para T3 e 54,17% para T4. Da mesma maneira, as taxas de gestação por ciclo também não foram influenciadas (P>0,05) pelos tratamentos, com valores de 63,64%, 54,55%, 52,69% e 47,06% para T1, T2, T3, e T4, respectivamente. No que diz respeito às taxas de perdas gestacionais, obtidas por meio das taxas de perdas e de perdas ajustadas, houve influência (P<0,05) dos tratamentos. Observou-se, ainda, efeito do mês da concepção, com taxas de perda superiores para os meses de janeiro (38,46%) e de fevereiro e março (52,38%), sendo a média de 33,09%. Sugere-se que a redução dos padrões de secreção do LH, ao final da estação de monta, responda pelas maiores taxas de perdas gestacionais do cruzamento interespécie, notadamente naquelas éguas que conceberam no final da estação de monta fisiológica dos equinos.(AU)

Cooling of equine semen at 16C for 36 hours with addition of different glutathione concentrations / Refrigeração do sêmen equino a 16ºC por 36h com adição de glutationa em diferentes concentrações

Handling equine semen during the refrigeration process reduces sperm viability, and consequently causes membrane lipid peroxidation, among other challenges. The present study aimed to evaluate the in vitro effects of glutathione (control, 1. 0, 1. 5, and 2. 5 mM) on equine semen in a refrigeration protocol of 16ºC for 36 hours. The following variables were evaluated after 0, 12, 24, and 36 hours refrigeration: total sperm motility, vigor, viability, and plasma and acrosomal membrane integrity. Motility was higher with 2. 5mM of glutathione (57. 8 ± 7. 3) after 12 hours of refrigeration compared to the control (53. 2 ± 8. 3) (P 0. 05). After 36 hours of refrigeration, motility was higher with 1. 5 mM (43. 4 ± 12. 7) and 2. 5mM glutathione (45. 5 ± 6. 2), than it was with 1mM glutathione (38. 2 ± 9) and the control (35. 5 ± 18. 4) (P 0. 05), respectively. Vigor was highest with 1. 5mM glutathione (3. 7 ± 0. 3) after 36 hours compared to the control (3. 2 ± 1. 1), (P 0. 05). Viability differed between control and 1mM treatments (79. 5 ± 1. 8) only after 24 hours (75. 5 ± 9. 7) (P 0. 05). Throughout the investigation, no significant differences were noted in plasma and acrosomal membrane integrity (P > 0. 05). The 1. 5 and 2. 5mM glutathione levels were more efficient in protecting sperm cells and yielded higher total motility values after 36 hours of...(AU)

A manipulação do sêmen equino durante o processo de refrigeração reduz a viabilidade espermática em consequência, entre outros problemas, da peroxidação de lipídios da membrana. Objetivou-se avaliar o efeito in vitro da adição de glutationa (controle; 1,0; 1,5 e 2,5mM) em protocolo de refrigeração a 16ºC por 36h. As variáveis avaliadas foram motilidade total, vigor, viabilidade espermática e integridade das membranas plasmática e acrossomal em quatro momentos diferentes de refrigeração (0, 12, 24 e 36h). A motilidade foi superior com 2,5mM de glutationa (57,8±7,3) com 12h de refrigeração quando comparado ao controle (53,2±8,3), P 0,05. Com 36h de refrigeração a motilidade foi superior com 1,5mM (43,4±12,7) e 2,5mM (45,5±6,2) quando comparado com 1mM (38,2±9) e ao controle (35,5±18,4), P 0,05. Com relação ao vigor houve superioridade com 1,5mM de glutationa (3,7±0,3), na avaliação de 36h, em comparação ao controle (3,2±1,1), P 0,05. Para viabilidade só houve diferença em relação ao controle na avaliação de 24h (75,5±9,7) quando comparado ao tratamento 1mM (79,5±1,8), P 0,05. Não houve diferença em relação ao controle para as avaliações de integridade de membrana plasmática e acrosomal, em todos os momentos de avaliação, P>0,05. As concentrações de 1,5 e 2,5MM de glutationa foram mais eficientes para proteção da célula espermática com valores superiores de...(AU)