Viability of Pony Stallion Semen in Different Temperature and Dilution

Background: Artificial insemination and transport of cooled semen has been routinely used in equine industry in the past 20 years. However, more investigations are needed regarding the methods for long time storage in pony stallion semen. The effect of dilution and cooling temperature on pH, sperm motility, membrane integrity and mitochondrial activity were investigated before and after cooling of stallion semen.Materials, Methods & Results: Two ejaculates each from nine Brazilian ponies were diluted in a nonbuffered powder milk extender cooled at 5°C or 15°C for 48 h using three different dilutions (1:1, 1:2 or 1:3). Data were assessed by analysis of variance and the rate comparison was performed using the Duncan test. Samples diluted 1:1 at 5oC or 15°C showed higher pH values (7.63 ± 0.34 e 7.57 ± 0.27) and lower progressive motility (10.3 ± 11.05, 17.08 ± 9.95). All samples cooled at 15°C also showed lower incidence of morphologically altered spermatozoa (1:1 = 55.84%; 1:2 = 51.84%; 1:3 = 49.95%) [P < 0.01]. Mitochondrial activity was higher on the 1:3 dilution (0.86 ± 0.19 nm) at 5°C and on the 1:1 (0.89 ± 0.23 nm), 1:2 (0.93 ± 0.2 nm) and 1:3 (0.92 ± 0.2 nm) dilutions at 15°C. Progressive motility was higher when semen was diluted 1:3 and cooled at 15°C (42.22 ± 12.38; P < 0.05). Considering mitochondrial activity, similar results were observed when different dilutions of semen were used (P > 0.05) despite time and temperature. The pH, progressive motility, mitochondrial activity and membrane integrity remained similar (P > 0.05) on fresh semen samples independent of the dilution grade used. The best results were obtained when semen was diluted 1:3 and cooled at 15°C. All dilution grades were safe for fresh semen and pH wasincreased when semen was diluted and cooled for 48 h.Discussion: The methodology used to collect and process equine semen and semen from ponies is practically the same. Equine semen when sent for artificial insemination is usually cooled to 5°C. Our results showed that cooling reduces sperm viability, which has also been demonstrated by other studies. In contrast, the best cooling temperature was at 15°C. However, it is easier to keep the temperature at 5°C during transport, due to the large temperature oscillation that may occur during transportation. The semen of ponies can tolerate cooling at both 5 and 15°C. The 1:3 dilution cooled to 15°C provided better viability of pony sperm, and more stable pH during 48 h of cooling. Dilution 1:1 should not be used for cooling in powdered skim milk extender

Transcriptional profile of equine female endometrium with diferents grades of fibrosis

A proposta do experimento baseia-se na análise uterina de éguas distribuídas em três graus de fibrose, conforme estabelecido em 1978 por Kenney (grupo I – endométrio saudável; grupo II, endométrio saudável ou com poucas alterações; grupo III – endométrio com alterações severas). Portanto, o estudo tem por objetivo determinar o perfil da expressão gênica de possíveis genes causadores da fibrose, conhecidos pelo o envolvimento em outros órgãos e espécies, no endométrio de éguas classificadas com diferentes graus de saúde uterina. Uma amostra de tecido endometrial foi colhida por meio de biopsia uterina e seccionada em dois fragmentos para posterior análise. Para os exames laboratoriais, a primeira amostra foi destinada à avaliação histopatológica e a segunda à validação de genes-alvo por reação polimerase em tempo real (qPCR). No exame histopatológico, o grau de fibrose e fase do ciclo estral foram estimados para distribuir as fêmeas nos grupos, além disso, o histórico reprodutivo foi analisado e a partir dos dados do exame histopatológico e histórico foram selecionadas 20 fêmeas para realização do qPCR (grupo I = 11; grupo II = 6; grupo III = 3). A segunda amostra de tecido foi armazenada em nitrogênio líquido imediatamente após a coleta, sendo posteriormente armazenada em freezer -80°C. Após o processamento das amostras, a quantificação relativa da abundância dos transcritos de Fator de Transformação do Crescimento-β1 (TGF-β1), Fator de Crescimento do Tecido Conjuntivo (CTGF), Colágeno tipo I (COL-I), α actina de músculo liso (αSMA), Molécula de Adesão Intercelular-1 (ICAM-1), e Cluster de Diferenciação 147 (CD147) foi comparada entre os grupos. A expressão de αSMA (P = 0,78), COL-I (P = 0,57) e ICAM-1 (P = 0,98) não resultou em diferença entre os grupos; e a de TGF-β1 e CTGF foi maior no grupo I (P = 0,04). Nos casos de fibrose severa, a transcrição de mRNA de CD147 foi maior em comparação com o grupo I (P = 0,04). Desta forma, acredita-se que a partir da avaliação de genes expressos do endométrio de éguas com diferentes graus de fibrose pode ser possível identificar genes marcadores que aumentam a precisão da classificação previamente proposta.The purpose of the experiment was to analyze the uterus of mares distributed in three degrees of fibrosis, as established by Kenney in 1978 (group I - healthy endometrium; group II, healthy endometrium or with few alterations; group III - endometrium with severe changes). A sample of endometrial tissue was collected by biopsy which was sectioned into two fragments for further analysis. For the laboratory tests, the first sample was used for histopathological evaluation and the second for the validation of target genes by quantitative polymerase chain reaction (qPCR). In the histopathological examination, the degree of fibrosis and phase of the estrous cycle were estimated to distribute the females into groups; in addition, the reproductive history was analyzed and from the obtained data of histopathological evaluation and history 20 mares were selected for qPCR (group I = 11; Group II = 6, group III = 3). The second tissue sample was stored in liquid nitrogen immediately after collection and stored in a freezer at -80 ° C. After the samples were processed, the relative abundance quantification of Transforming Growth Factor-β1 (TGF-β1), Connective Growth Factor (CTGF), α Smooth Muscle Actin (αSMA), Intercellular Adhesion Molecule-1 (ICAM-1), Type I Collagen (COL-I), and Differentiation Cluster 147 (CD147) was carried out. The expression of αSMA (P = 0.78), COL-I (P = 0.57) and ICAM-1 (P = 0.98) did not resulted in difference between the groups; and TGF-β1 and CTGF were higher in group I (P = 0.04). In severe cases of fibrosis, CD147 mRNA transcription was higher in comparison to the group I (P = 0.04). Thus, it is believed that from the evaluation of expressed endometrial genes in the mare with different degrees of fibrosis it may be possible to identify marker genes that increase the accuracy of the previously proposed classification

Transcriptional profile of equine female endometrium with diferents grades of fibrosis

A proposta do experimento baseia-se na análise uterina de éguas distribuídas em três graus de fibrose, conforme estabelecido em 1978 por Kenney (grupo I – endométrio saudável; grupo II, endométrio saudável ou com poucas alterações; grupo III – endométrio com alterações severas). Portanto, o estudo tem por objetivo determinar o perfil da expressão gênica de possíveis genes causadores da fibrose, conhecidos pelo o envolvimento em outros órgãos e espécies, no endométrio de éguas classificadas com diferentes graus de saúde uterina. Uma amostra de tecido endometrial foi colhida por meio de biopsia uterina e seccionada em dois fragmentos para posterior análise. Para os exames laboratoriais, a primeira amostra foi destinada à avaliação histopatológica e a segunda à validação de genes-alvo por reação polimerase em tempo real (qPCR). No exame histopatológico, o grau de fibrose e fase do ciclo estral foram estimados para distribuir as fêmeas nos grupos, além disso, o histórico reprodutivo foi analisado e a partir dos dados do exame histopatológico e histórico foram selecionadas 20 fêmeas para realização do qPCR (grupo I = 11; grupo II = 6; grupo III = 3). A segunda amostra de tecido foi armazenada em nitrogênio líquido imediatamente após a coleta, sendo posteriormente armazenada em freezer -80°C. Após o processamento das amostras, a quantificação relativa da abundância dos transcritos de Fator de Transformação do Crescimento-β1 (TGF-β1), Fator de Crescimento do Tecido Conjuntivo (CTGF), Colágeno tipo I (COL-I), α actina de músculo liso (αSMA), Molécula de Adesão Intercelular-1 (ICAM-1), e Cluster de Diferenciação 147 (CD147) foi comparada entre os grupos. A expressão de αSMA (P = 0,78), COL-I (P = 0,57) e ICAM-1 (P = 0,98) não resultou em diferença entre os grupos; e a de TGF-β1 e CTGF foi maior no grupo I (P = 0,04). Nos casos de fibrose severa, a transcrição de mRNA de CD147 foi maior em comparação com o grupo I (P = 0,04). Desta forma, acredita-se que a partir da avaliação de genes expressos do endométrio de éguas com diferentes graus de fibrose pode ser possível identificar genes marcadores que aumentam a precisão da classificação previamente proposta.The purpose of the experiment was to analyze the uterus of mares distributed in three degrees of fibrosis, as established by Kenney in 1978 (group I - healthy endometrium; group II, healthy endometrium or with few alterations; group III - endometrium with severe changes). A sample of endometrial tissue was collected by biopsy which was sectioned into two fragments for further analysis. For the laboratory tests, the first sample was used for histopathological evaluation and the second for the validation of target genes by quantitative polymerase chain reaction (qPCR). In the histopathological examination, the degree of fibrosis and phase of the estrous cycle were estimated to distribute the females into groups; in addition, the reproductive history was analyzed and from the obtained data of histopathological evaluation and history 20 mares were selected for qPCR (group I = 11; Group II = 6, group III = 3). The second tissue sample was stored in liquid nitrogen immediately after collection and stored in a freezer at -80 ° C. After the samples were processed, the relative abundance quantification of Transforming Growth Factor-β1 (TGF-β1), Connective Growth Factor (CTGF), α Smooth Muscle Actin (αSMA), Intercellular Adhesion Molecule-1 (ICAM-1), Type I Collagen (COL-I), and Differentiation Cluster 147 (CD147) was carried out. The expression of αSMA (P = 0.78), COL-I (P = 0.57) and ICAM-1 (P = 0.98) did not resulted in difference between the groups; and TGF-β1 and CTGF were higher in group I (P = 0.04). In severe cases of fibrosis, CD147 mRNA transcription was higher in comparison to the group I (P = 0.04). Thus, it is believed that from the evaluation of expressed endometrial genes in the mare with different degrees of fibrosis it may be possible to identify marker genes that increase the accuracy of the previously proposed classification

Viability of Pony Stallion Semen in Different Temperature and Dilution

Background: Artificial insemination and transport of cooled semen has been routinely used in equine industry in the past 20 years. However, more investigations are needed regarding the methods for long time storage in pony stallion semen. The effect of dilution and cooling temperature on pH, sperm motility, membrane integrity and mitochondrial activity were investigated before and after cooling of stallion semen.Materials, Methods & Results: Two ejaculates each from nine Brazilian ponies were diluted in a nonbuffered powder milk extender cooled at 5°C or 15°C for 48 h using three different dilutions (1:1, 1:2 or 1:3). Data were assessed by analysis of variance and the rate comparison was performed using the Duncan test. Samples diluted 1:1 at 5oC or 15°C showed higher pH values (7.63 ± 0.34 e 7.57 ± 0.27) and lower progressive motility (10.3 ± 11.05, 17.08 ± 9.95). All samples cooled at 15°C also showed lower incidence of morphologically altered spermatozoa (1:1 = 55.84%; 1:2 = 51.84%; 1:3 = 49.95%) [P < 0.01]. Mitochondrial activity was higher on the 1:3 dilution (0.86 ± 0.19 nm) at 5°C and on the 1:1 (0.89 ± 0.23 nm), 1:2 (0.93 ± 0.2 nm) and 1:3 (0.92 ± 0.2 nm) dilutions at 15°C. Progressive motility was higher when semen was diluted 1:3 and cooled at 15°C (42.22 ± 12.38; P < 0.05). Considering mitochondrial activity, similar results were observed when different dilutions of semen were used (P > 0.05) despite time and temperature. The pH, progressive motility, mitochondrial activity and membrane integrity remained similar (P > 0.05) on fresh semen samples independent of the dilution grade used. The best results were obtained when semen was diluted 1:3 and cooled at 15°C. All dilution grades were safe for fresh semen and pH wasincreased when semen was diluted and cooled for 48 h.Discussion: The methodology used to collect and process equine semen and semen from ponies is practically the same. Equine semen when sent for artificial insemination is usually cooled to 5°C. Our results showed that cooling reduces sperm viability, which has also been demonstrated by other studies. In contrast, the best cooling temperature was at 15°C. However, it is easier to keep the temperature at 5°C during transport, due to the large temperature oscillation that may occur during transportation. The semen of ponies can tolerate cooling at both 5 and 15°C. The 1:3 dilution cooled to 15°C provided better viability of pony sperm, and more stable pH during 48 h of cooling. Dilution 1:1 should not be used for cooling in powdered skim milk extender